Article

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma

Authors:
  • Selma-Lagerlöf Sekundarschule
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Abstract

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing for an accurate and early tumor diagnosis. To identify potential markers with such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent non-tumorous tissue samples as controls) using two independent proteomic approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), Myristoylated alanine-rich C-kinase substrate (MARCKS) and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as potential biomarker for early-HCC diagnosis.

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... 8,9 Increased expression of VCAN has been reported in several malignancies, including leukemias as well as brain, colon, liver, prostate, breast, ovarian, oral squamous cell, and lung cancers, and is associated with adverse outcomes. [9][10][11][12][13][14][15] A recent bioinformatic analysis revealed that VCAN may have an oncogenic role in GC, while another study suggested that VCAN expression predicts favorable outcomes in patients with GC. 16,17 However, the clinical significance and prognostic value of VCAN in GC remains largely unclear. Therefore, it is vital to identify reliable biomarkers for GC diagnosis and prognosis. ...
... A previous study reported that relative expression of VCAN in different hepatocellular carcinoma grades and stages showed progressive upregulation in more aggressive cancers, consistent with our results in GC patients. 15 Kaplan-Meier curves for overall survival revealed that high expression of VCAN was associated with poor outcomes in GC patients. Univariate and multivariate Cox analyses of both TCGA and GEO databases indicated the VCAN expression was a potential independent marker for poor prognosis in GC, and ROC analysis confirmed the diagnostic value of VCAN expression in gastric cancer. ...
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Objective Versican (VCAN) has been reported as a potential biomarker in some cancers. However, its role in gastric cancer (GC) is poorly understood. Methods Associations between clinical variables and VCAN were assessed. The diagnostic value of VCAN expression in GC patients was determined through receiver operating characteristic (ROC) curve analysis. Cox regression and the Kaplan–Meier method were used to explore clinicopathologic factors related to overall survival in GC patients. The Gene Expression Omnibus and the Human Protein Atlas were used for further validation. Gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas dataset. Results High expression of VCAN was associated with high stage and T classification in GC. The area under the ROC curve was 0.853. Patients with high VCAN expression had worse prognoses than those with low VCAN expression. Multivariate analysis showed that VCAN was an independent risk factor for overall survival in both cohorts. GSEA identified pathways involved in cancer, ECM-receptor interaction, Wnt signaling, T cell receptor signaling, and chemokine signaling as differentially enriched in GCs with high VCAN expression. Conclusion We demonstrated that VCAN is expressed at high levels in GC, and represents a potential independent molecular marker for diagnosis and prognosis of GC.
... Early diagnosis of HCC is of the utmost importance. Successful screening for HCC at early stage is challenging due to the lack of well characterized and specific biomarkers [67,68] . Data of previous studies have confirmed that some Wnt signalings could modify HCC growth and invasive ability. ...
... (A) Wnt5a in HCC; (B) Wnt3a in HCC. HCC: hepatocellular carcinoma; Wnt3a: wingless-type MMTV integration site family member 3a; Wnt5a: wingless-type MMTV integration site family member 5a Table 2. Comparative analysis of circulating Wnt3a, AFP, HS-GGT, and GPC-3 detection in diagnosis of HCC Wnt3a [19] (> 800 ng/L) AFP [19] (> 50 ng/mL) HS-GGT [17] (> 5.5 U/L) GPC-3 [67] (positive) Wnt3a [19] + AFP Sensitivity (%) 92. 50 ...
Article
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Hepatocellular carcinoma (HCC) is still one of the most common and rapidly fatal malignancies worldwide with a multi-factorial, multi-step, complex process, and poor prognosis. Early discovery and effective therapy of HCC are of utmost importance. Recent studies demonstrated that Wnt/β-catenin pathway play important roles in occurrence and development of HCC including hepatocytes malignant transformation, metastasis, chemoresistance and liver cancer stem cells. Oncogenic wingless-type MMTV integration site family member 3a (Wnt3a) signaling is a promising biomarker in diagnosis and prognosis for HCC. This review presents current data on mechanisms of hepatocarcinogenesis involving participation of the Wnt canonical pathway, and focuses on the Wnt3a expression in HCC progression and its clinical application.
... Analysis of 19 paired HCC tumor and surrounding nontumoral tissues lead to the identification of a panel of HCCassociated proteins. Among them, overexpression of DDX39, fibulin-5, myristoylated alanine-rich C-kinase substrate (MARCKS) and serpin H1 was confirmed by SRM in additional 19 cases and Versican core protein was significantly associated with well-differentiated and low-stage HCC [36]. These studies provide specific proteins and proteoform fingerprints that configure a molecular framework to explain liver tumor heterogeneity and might well complement other clinical tests to enhance the sensitivity and specificity of current diagnostics and prognostics strategies. ...
... One of the above-mentioned B/D HPP initiatives is the Human Liver Proteome Project [88] a large-scale international initiative launched in 2002 to investigate the human liver proteome in health and disease. Coordinated efforts within the initiative are leading to in depth descriptions of the liver proteome (including different liver cell types) [89], identification of primary liver cancer-related proteins and protein post-translational modification (PTMs) [31,49,73,74], discovery and development of targeted methods to validate HCC biomarker candidates [36], definition of metabolomics-specific patterns of NASH subtypes [20]. Mass spectrometry-based imaging is also emerging as a powerful tool to assess NASH microheterogeneity [90,91]. ...
Article
Introduction: Hepatocellular carcinoma (HCC) is recognized as the fifth most common neoplasm and currently represents the second leading form of cancer-related death worldwide. Despite great progress has been done in the understanding of its pathogenesis, HCC represents a heavy societal and economic burden as most patients are still diagnosed at advanced stages and the 5-year survival rate remain below 20%. Early detection and revolutionary therapies that rely on the discovery of new molecular biomarkers and therapeutic targets are therefore urgently needed to develop precision medicine strategies for a more efficient management of patients. Areas covered: This review intends to comprehensively analyse the proteomics-based research conducted in the last few years to address some of the principal still open riddles in HCC biology, based on the identification of molecular drivers of tumor progression and metastasis. Expert commentary: The technical advances in mass spectrometry experienced in the last decade have significantly improved the analytical capacity of proteome wide studies. Large-scale protein and protein variant (posttranslational modifications) identification and quantification have allowed detailed dissections of molecular mechanisms underlying HCC progression and are already paving the way for the identification of clinically relevant proteins and the development of their use on patient care.
... In HCC, versican expression was significantly upregulated compared with adjacent nontumor tissues and mainly localized in the cytoplasm [104,105]. Quantitative tissue proteomics comparing HCC and corresponding tissue samples revealed that versican core protein was significantly abundant in well-differentiated and early-stage HCC, suggesting that versican is a potential biomarker for early-stage HCC [106]. ...
... Cont.Versican Tissue↑ CLD with fibrosis[102,103,105] ↑ HCC with poor prognosis[104,105] ↑ early-stage HCC[106] BCLC: Barcelona clinic liver cancer, CLD: Chronic liver disease, N/A: Not available, *: Applied for diagnosis, ↑: increased, ↓: decreased. ...
Article
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Proteoglycans, which consist of a protein core and glycosaminoglycan chains, are major components of the extracellular matrix and play physiological roles in maintaining tissue homeostasis. In the carcinogenic tissue microenvironment, proteoglycan expression changes dramatically. Altered proteoglycan expression on tumor and stromal cells affects cancer cell signaling pathways, which alters growth, migration, and angiogenesis and could facilitate tumorigenesis. This dysregulation of proteoglycans has been implicated in the pathogenesis of diseases such as hepatocellular carcinoma (HCC) and the underlying mechanism has been studied extensively. This review summarizes the current knowledge of the roles of proteoglycans in the genesis and progression of HCC. It focuses on well-investigated proteoglycans such as serglycin, syndecan-1, glypican 3, agrin, collagen XVIII/endostatin, versican, and decorin, with particular emphasis on the potential of these factors as biomarkers and therapeutic targets in HCC regarding the future perspective of precision medicine toward the “cure of HCC”.
... Annexin A2 (ANXA2) was found to be significantly higher abundant in HCC compared to both opposed experimental conditions (Fig. 2D). We compared the proteins differentially abundant in "HCC vs. Healthy" with HCC biomarker candidates that were identified in previous tissue-based studies [10,16,17]. Ten proteins (highlighted in Fig. 2) were found to overlap with one or several of these studies with a consistent elevation of abundance in HCC (Supplementary Table S4). ...
... VCAN showed an elevated abundance in cirrhotic patients with a further increase in HCC. VCAN is an extracellular matrix component and has been described in relation to various types of cancer [16]. VCAN is also known to interact with hyaluronan which has been proposed as a non-invasive biomarker for liver fibrosis and cirrhosis [18,19]. ...
Article
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Purpose: Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. Cirrhosis induced by hepatitis-C virus (HCV) infection is the most critical risk factor for HCC. However, the mechanism of HCV-induced carcinogenesis is not fully understood. Plasma microparticles (PMP) contribute to numerous physiological and pathological processes and contain proteins whose composition correlates to the respective pathophysiological conditions. Experimental design: We analyzed PMP from 22 HCV-induced cirrhosis patients, 16 HCV-positive HCC patients with underlying cirrhosis and 18 healthy controls. PMP were isolated using ultracentrifugation and analyzed via label-free LC-MS/MS. Results: We identified 840 protein groups and quantified 507 proteins. 159 proteins were found differentially abundant between the three experimental groups. PMP in both disease entities displayed remarkable differences in the proteome composition compared to healthy controls. Conversely, the proteome difference between both diseases was minimal. GO analysis revealed that PMP isolated from both diseases were significantly enriched in proteins involved in complement activation, while endopeptidase activity was downregulated exclusively in HCC patients. Conclusion: This study reports for the first time a quantitative proteome analysis for PMP from patients with HCV-induced cirrhosis and HCC. Data are available via ProteomeXchange with identifier PXD005777.
... 17 Emerging evidence has suggested that MARCKS is capable of specifically promoting cancer migration and metastasis but its function in tumor growth has not been worked out. [18][19][20][21][22][23][24][25][26][27][28][29][30][31] MARCKS has also been implicated in lung, breast and pancreatic cancer progression. 22,23,[26][27][28][29][30][31] Intriguingly, MARCKS has been reported to be prometastatic, 18,19,24 but also a tumor suppressor 20,21,32 in glioma, melanoma and colon cancer. ...
... [18][19][20][21][22][23][24][25][26][27][28][29][30][31] MARCKS has also been implicated in lung, breast and pancreatic cancer progression. 22,23,[26][27][28][29][30][31] Intriguingly, MARCKS has been reported to be prometastatic, 18,19,24 but also a tumor suppressor 20,21,32 in glioma, melanoma and colon cancer. Despite these findings in various neoplastic diseases, there has been no information regarding the role of MARCKS in kidney cancer. ...
Article
Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/vascular endothelial growth factor signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high-grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably vascular endothelial growth factor-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multikinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS (MARCKS phosphorylation site domain) peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multikinase inhibitors.
... WNT1-inducible signaling pathway protein 2 was more highly expressed in gastric cancer tissues compared to adjacent normal tissues and might be a favorable biomarker for the early stage prediction and prognosis of gastric cancer (Li et al., 2017). Fibulin-5 was reported to be a potential biomarker for hepatic fibrosis, early-stage hepatocellular carcinoma and calcified thoracic aortic aneurysms (Bracht et al., 2015;Matsumoto et al., 2012;Naboulsi et al., 2016). Plastin-2 may have potential as a biomarker for the prediction of type 2 diabetes mellitus (Chan et al., 2012). ...
... Previous studies have demonstrated that self-factors and environmental factors can cause differences in the urine proteome (Guo et Ethnic group differences may be an influencing factor in urine proteome studies and should be considered when human urine samples are used for biomarker discovery. (Reszka, 2012) Pro-epidermal growth factor P01133 Type 2 diabetic nephropathy (Guo et al., 2015a) WNT1-inducible-signaling pathway protein 2 O76076 Gastric cancer (Li et al., 2017) Fibulin-5 Q9UBX5 Hepatic fibrosis (Bracht et al., 2015) Hepatocellular carcinoma (Naboulsi et al., 2016) Calcified thoracic aortic aneurysms (Matsumoto et al., 2012) Plastin-2 P13796 Type 2 diabetes mellitus (Chan et al., 2012) Neutrophil gelatinase-associated lipocalin Thrombospondin-4 P35443 S-Adenosylhomocysteine hydrolase (AHCY) deficiency (Sedic et al., 2011) Colorectal cancer (Greco et al., 2010) Coactosin-like protein Q14019 Small cell lung cancer (Jeong et al., 2011) Peptidoglycan recognition protein 1 O75594 Oral Inflammation in Kidney Disease (Nylund et al., 2017) Atherosclerosis (Rohatgi et al., 2009) Putative elongation factor 1-alpha-like 3 Q5VTE0 Hepatocellular carcinoma (Chen et al., 2018) Schistosomiasis (Onile et al., 2017) Pancreatic cancer (Buanes, 2016) Dipeptidase 1 P16444 Colorectal cancer (Toiyama et al., 2011;Eisenach et al., 2013;Tachibana et al., 2017) Gastrointestinal cancer (Arner et al., 2015) Neurodegenerative diseases (Yin et al., 2009) Lamin A/C Q5TCI8 Cervical cancer (Capo-chichi et al., 2016) Gastric carcinoma (Wu et al., 2009) Colorectal cancer (Willis et al., 2008) Carbonic anhydrase 1 P00915 Schistosoma mansoni infection (Ward et al., 2017) Type 2 diabetic (Bellei et al., 2008) Non-small cell lung cancer L-xylulose reductase Q7Z4W1 Prostate adenocarcinoma (Cho-Vega et al., 2007) ...
Article
Biomarkers indicate changes associated with disease. Blood is relatively stable due to the homeostatic mechanisms of the body; however, urine accumulates metabolites from changes in the body, making it a better source for early biomarker discovery. The Li ethnic group is a unique minority ethnic group that has only lived on Hainan Island for approximately 5,000 years. Studies have shown that various specific genetic variations are different between the Li and Han ethnic groups. However, whether the urinary proteome between these two ethnic groups is significantly different remains unknown. In this study, differential urinary proteins were identified in the Li and Han ethnic groups using liquid chromatography tandem mass spectrometry (LC-MS/MS). In total, 1,555 urinary proteins were identified. Twenty-five of the urinary proteins were statistically significantly different, 16 of which have been previously reported to be biomarkers of many diseases, and that these significantly different proteins were caused by ethnic differences rather than random differences. Ethnic group differences may be an influencing factor in urine proteome studies and should be considered when human urine samples are used for biomarker discovery.
... Indeed, a diagnostic model was determined using a learning algorithm of radial basis function neural network which identified a serglycin-derived peptide as one of seven peptides which may be utilized as a diagnosis tool for HCC metastasis to bone (23). Furthermore, the usage of quantitative tissue proteomics analysis indicated versican as a promising biomarker for the detection of HCC at an early stage (58). ...
Article
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Proteoglycans (PGs), important constituents of the extracellular matrix, have been associated with cancer pathogenesis. Their unique structure consisting of a protein core and glycosaminoglycan chains endowed with fine modifications constitutes these molecules as capable cellular effectors important for homeostasis and contributing to disease progression. Indeed, differential expression of PGs and their interacting proteins has been characterized as specific for disease evolvement in various cancer types. Importantly, PGs to a large extent regulate the bioavailability of hormones, growth factors, and cytokines as well as the activation of their respective receptors which regulate phenotypic diversibility, gene expression and rates of recurrence in specific tumor types. Defining and targeting these effectors on an individual patient basis offers ground for the development of newer therapeutic approaches which may act as either supportive or a substitute treatment to the standard therapy protocols. This review discusses the roles of PGs in cancer progression, developing technologies utilized for the defining of the PG “signature” in disease, and how this may facilitate the generation of tailor-made cancer strategies.
... It inhibits invasion of bladder cancer and is an unfavorable prognostic factor for bladder cancer 10 . DDX39 is a putative biomarker of breast cancer and early-stage hepatocellular carcinoma 11,12 . DDX39 also upregulates in malignant pleural mesothelioma cells and pancreatic cancer cells acquired gemcitabine resistance 13,14 . ...
Article
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Hepatocellular carcinoma (HCC) is the third leading cause of cancer related death worldwide; however, the molecular mechanisms regulating HCC progression remain largely unknown. In this study, we determined the role of DDX39 which a DEAD-box RNA helicase in HCC progression, and found DDX39 was upregulated in HCC tissues and cells, DDX39 expression was positive correlated with advanced clinical stage, survival analysis showed patients with high-DDX39 levels had poor outcome, it was an independent prognostic factor. Functional analysis revealed that DDX39 overexpression promoted HCC cell migration, invasion, growth, and metastasis, DDX39 knockdown inhibited HCC cell migration, invasion, growth, and metastasis. Mechanism analysis suggested DDX39 overexpression increased β-catenin expression in nucleus and increased Wnt/β-catenin pathway target genes levels, while DDX39 knockdown reduced this effect. Knockdown of Wnt/β-catenin pathway co-activators TCF4 and LEF1 in DDX39 overexpressing HCC cells inhibited Wnt/β-catenin pathway target genes. The invasion ability was also reduced, confirming DDX39 regulates HCC progression by activating Wnt/β-catenin pathway. In conclusion, we found DDX39 is a target and prognostic factor for HCC, and promotes HCC migration, invasion, growth, and metastasis by activating Wnt/β-catenin pathway.
... The above relationships were validated using several independent cohorts at gene expression protein and metabolite levels. First, the antagonistic behavior observed among co-expressed redox genes (Fig. 1) was also displayed at the gene expression level in 61 HCC tumors (Appendix Fig. S9A, Ref. [14]) and 91 HCC tumors [42,43] (Appendix Fig. S2A), and at the proteomic level in 19 HCC tumors [44] (Dataset 17). ...
... The above relationships were validated using several independent cohorts at gene expression protein and metabolite levels. First, the antagonistic behavior observed among co-expressed redox genes (Fig. 1) was also displayed at the gene expression level in 61 HCC tumors (Appendix Fig. S9A, Ref. [14]) and 91 HCC tumors [42,43] (Appendix Fig. S2A), and at the proteomic level in 19 HCC tumors [44] (Dataset 17). ...
Article
Background: Redox metabolism is often considered a potential target for cancer treatment, but a systematic examination of redox responses in hepatocellular carcinoma (HCC) is missing. Methods: Here, we employed systems biology and biological network analyses to reveal key roles of genes associated with redox metabolism in HCC by integrating multi-omics data. Findings: We found that several redox genes, including 25 novel potential prognostic genes, are significantly co-expressed with liver-specific genes and genes associated with immunity and inflammation. Based on an integrative analysis, we found that HCC tumors display antagonistic behaviors in redox responses. The two HCC groups are associated with altered fatty acid, amino acid, drug and hormone metabolism, differentiation, proliferation, and NADPH-independent vs -dependent antioxidant defenses. Redox behavior varies with known tumor subtypes and progression, affecting patient survival. These antagonistic responses are also displayed at the protein and metabolite level and were validated in several independent cohorts. We finally showed the differential redox behavior using mice transcriptomics in HCC and noncancerous tissues and associated with hypoxic features of the two redox gene groups. Interpretation: Our integrative approaches highlighted mechanistic differences among tumors and allowed the identification of a survival signature and several potential therapeutic targets for the treatment of HCC.
... mdpi.com/article/10.3390/cancers14071704/s1. References [4,15,[23][24][25]34,[49][50][51][52][53][54][55][56][57][58][59][60][61] were included in the supplementary materials. File S1: Supplementary Materials and Methods, File S2: Original Blots; Figure S1: TIA1 mRNA level in HCC, ICC and hepatoblastoma and impact of its silencing on HCC cells proliferation; Figure S2: TIA1 is a reliable marker of stress granules in sorafenib-treated HCC cells; Figure S3: RNAseq on polysomes from HepG2 cells following TIA1 silencing; Figure S4: TIA1 mRNA level in various chronic liver diseases; Figure S5: TIA1 mRNA level in mouse models of NAFLD/NASH.; Figure S6: Impact of TIA1 loss on NAFLD and NASH development; Figure S7: Impact of TIA1 loss on hepatic carcinogenesis in LPTENKO mice; Figure S8: Immunostaining of TIA1 in HCC cells and human tissue microarrays of HCC patients; Figure S9: TIA1 mRNA level in HCC and liver development; Figure S10: Relative mRNA expression of TIA1 exon 4 and exon 11 in livers of TIA1KO vs. wild type (WT) mice at 3 months (GSE54118); Figure S11: Potential regulatory mechanisms of TIA1 expression; Figure S12: MicroRNAs-dependent regulation of TIA1 expression; Figure S13: Potential TIA1 mRNA targets involved in hepatocarcinogenesis; Figure S14: TIA1 controls the splicing of several transcripts involved in HCC; Figure S15: Deregulation of oncogenes and tumor suppressors in the liver of TIA1KO mice; Figure S16: TIA1 is part of a network of RNA-Binding proteins involved in HCC; Figure S17: TIA1 binds and regulates the expression of long-non-coding RNA involved in HCC; Figure S18: Expression of stress granules assembly factors in human HCC; Table S1: Human GEO datasets used in the article; Table S2: Rodent GEO datasets used in the article; Table S3: RNA-sequencing on polysomes from HepG2 after TIA1 silencing; Table S4: Role in HCC and presence of AUUUA sequence of potential TIA1 targets identified in the trans-latomics performed in HepG2 siTIA1 cells, that were annotated by CancerMine ( Figure 2); Table S5: Roles of TIA1 in cancer.; ...
Article
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Alterations in specific RNA-binding protein expression/activity importantly contribute to the development of fatty liver disease (FLD) and hepatocellular carcinoma (HCC). In particular, adenylate–uridylate-rich element binding proteins (AUBPs) were reported to control the post-transcriptional regulation of genes involved in both metabolic and cancerous processes. Herein, we investigated the pathophysiological functions of the AUBP, T-cell-restricted intracellular antigen-1 (TIA1) in the development of FLD and HCC. Analysis of TIA1 expression in mouse and human models of FLD and HCC indicated that TIA1 is downregulated in human HCC. In vivo silencing of TIA1 using AAV8-delivered shRNAs in mice worsens hepatic steatosis and fibrosis induced by a methionine and choline-deficient diet and increases the hepatic tumor burden in liver-specific PTEN knockout (LPTENKO) mice. In contrast, our in vitro data indicated that TIA1 expression promoted proliferation and migration in HCC cell lines, thus suggesting a dual and context-dependent role for TIA1 in tumor initiation versus progression. Consistent with a dual function of TIA1 in tumorigenesis, translatome analysis revealed that TIA1 appears to control the expression of both pro- and anti-tumorigenic factors in hepatic cancer cells. This duality of TIA1′s function in hepatocarcinogenesis calls for cautiousness when considering TIA1 as a therapeutic target or biomarker in HCC.
... SERPINH1 is located at 11q13.5 and encodes heat shock protein 47 (HSP47), which is a 418-amino-acid protein and belongs to the serpin superfamily [17,18]. The overexpression of SERPINH1 in HCC was presented by Naboulsi et al. for the first time [19]. Although the expression of HSP47 in different types of cancer has different effects, in some types of cancer, such as glioblastoma and breast cancer, HSP47 is associated with invasiveness [20]. ...
Article
We aimed to assess the roles of small nucleolar RNA host gene 6 (SNHG6) in hepatocellular carcinoma (HCC) progression, and establish the lncRNA-miRNA-mRNA regulation mechanism for HCC therapy. SNHG6 is one of the host genes in small nucleolar RNAs (snoRNAs), which make a difference in the development of human cancers. SERPINH1 is a gene encoding a member of the serpin superfamily of serine proteinase inhibitors with miRNA predicted by TargetScan and DIANA Tools. SNHG6, serpin family H member 1 (SERPINH1) and miR-139-5p expression levels in HCC tissues and cells were determined by quantitative real-time PCR (qRT-PCR). Migration and invasion of HCC cells were measured by transwell assay. Cell cycle analysis was determined by using flow cytometry. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay were performed for cell viability analysis. The expression of SERPINH1 was detected by qRT-PCR and western blot. Dual-luciferase reporter gene assay was conducted to identify the targeted relationship between miR-139-5p and SNHG6, as well as SERPINH1 and miR-139-5p. The positive regulation between SNHG6 and SERPINH1 was demonstrated in this study. In contrast, miR-139-5p was significantly down-regulated in HCC cells, the inhibition of miR-139-5p promotes the proliferation of HCC cells, and accelerated the cell cycle of HCC cells. Our study demonstrated the co-expression of SNHG6 and SERPINH1 in HCC cells for the first time, which revealed that SNHG6 could serve as a novel oncogene for the HCC therapy by its regulation.
... To demonstrate the workflow, we analyzed a bottom-up proteomics dataset where 19 samples of cancerous liver tissue from HCC patients and 19 samples of neighboring healthy tissue obtained from the same patients were measured with LC-MS/MS. In the original publication (Naboulsi et al., 2016), these samples and the clinical background are described. Furthermore, the high-throughput proteomics data from this study is publicly available from the PRIDE repository (accession number: PXD002171) and the targeted proteomics data generated using SRM from this study can be downloaded from the PASSEL repository (accession number: PASS00691). ...
Article
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The analysis of high-throughput mass spectrometry-based proteomics data must address the specific challenges of this technology. To this end, the comprehensive proteomics workflow offered by the de.NBI service center BioInfra.Prot provides indispensable components for the computational and statistical analysis of this kind of data. These components include tools and methods for spectrum identification and protein inference, protein quantification, expression analysis as well as data standardization and data publication. All particular methods of the workflow which address these tasks are state-of-the-art or cutting edge.
... Further details regarding patient characteristics, sample preparation, mass spectrometry, and proteomic data analysis have been extensively described in our prior publications. 37,38 Pathway Analysis ...
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Background & Aims Cancer cells rely on metabolic alterations to enhance proliferation and survival. Metabolic gene alterations that repeatedly occur in liver cancer are largely unknown. We aimed to identify metabolic genes that are consistently deregulated, and are of potential clinical significance in human hepatocellular carcinoma (HCC). Methods We studied the expression of 2,761 metabolic genes in 8 microarray datasets comprising 521 human HCC tissues. Genes exclusively up-regulated or down-regulated in 6 or more datasets were defined as consistently deregulated. The consistent genes that correlated with tumor progression markers (ECM2 and MMP9) (Pearson correlation P < .05) were used for Kaplan-Meier overall survival analysis in a patient cohort. We further compared proteomic expression of metabolic genes in 19 tumors vs adjacent normal liver tissues. Results We identified 634 consistent metabolic genes, ∼60% of which are not yet described in HCC. The down-regulated genes (n = 350) are mostly involved in physiologic hepatocyte metabolic functions (eg, xenobiotic, fatty acid, and amino acid metabolism). In contrast, among consistently up-regulated metabolic genes (n = 284) are those involved in glycolysis, pentose phosphate pathway, nucleotide biosynthesis, tricarboxylic acid cycle, oxidative phosphorylation, proton transport, membrane lipid, and glycan metabolism. Several metabolic genes (n = 434) correlated with progression markers, and of these, 201 predicted overall survival outcome in the patient cohort analyzed. Over 90% of the metabolic targets significantly altered at the protein level were similarly up- or down-regulated as in genomic profile. Conclusions We provide the first exposition of the consistently altered metabolic genes in HCC and show that these genes are potentially relevant targets for onward studies in preclinical and clinical contexts.
... Versican has been shown to be a potential biomarker in different cancers such as hepatocellular carcinoma (142), colon cancer (143), and recently in ovarian cancer (144). Hope et al. (145) provide a rational for testing versican proteolysis as a predictive and/or prognostic immune biomarker. ...
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The tumor microenvironment (TME) is composed of various cell types embedded in an altered extracellular matrix (ECM). ECM not only serves as a support for tumor cell but also regulates cell–cell or cell–matrix cross-talks. Alterations in ECM may be induced by hypoxia and acidosis, by oxygen free radicals generated by infiltrating inflammatory cells or by tumor- or stromal cell-secreted proteases. A poorer diagnosis for patients is often associated with ECM alterations. Tumor ECM proteome, also named cancer matrisome, is strongly altered, and different ECM protein signatures may be defined to serve as prognostic biomarkers. Collagen network reorganization facilitates tumor cell invasion. Proteoglycan expression and location are modified in the TME and affect cell invasion and metastatic dissemination. ECM macromolecule degradation by proteases may induce the release of angiogenic growth factors but also the release of proteoglycan-derived or ECM protein fragments, named matrikines or matricryptins. This review will focus on current knowledge and new insights in ECM alterations, degradation, and reticulation through cross-linking enzymes and on the role of ECM fragments in the control of cancer progression and their potential use as biomarkers in cancer diagnosis and prognosis.
... Studies have shown differentially expressed levels of MARCKS between healthy and tumor tissues, but different studies show inconsistent results as to the direction of the effect of MARCKS on cancer cell growth. MARCKS has been implicated in oncogenesis in lung cancer [58][59][60][61][62], cholangiocarcinoma [44], breast cancer [48][49][50], renal cell carcinoma (RCC) [56], pancreatic cancer [63], and hepatocellular carcinoma (HCC) [57]. In contrast, an early review connected MARCKS with suppression of proliferation; indeed, this has been the case in glioma [46], colorectal cancer [52], small-intestine adenocarcinoma [55], and melanoma [54]. ...
Article
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Emerging evidence implicates myristoylated alanine-rich C-kinase substrate (MARCKS), a major substrate of protein kinase C (PKC), in a critical role for cancer development and progression. MARCKS is tethered to the plasma membrane but can shuttle between the cytosol and plasma membrane via the myristoyl-electrostatic switch. Phosphorylation of MARCKS by PKC leads to its translocation from the plasma membrane to the cytosol where it functions in actin cytoskeletal remodeling, Ca²⁺ signaling through binding to calmodulin, and regulation of exocytic vesicle release in secretory cells such as neurons and airway goblet cells. Although the contribution of MARCKS to various cellular processes has been extensively studied, its roles in neoplastic disease have been conflicting. This review highlights the molecular and functional differences of MARCKS that exist between normal and tumor cells. We also discuss the recent advances in the potential roles of MARCKS in tumorigenesis, metastasis, and resistance to anti-cancer therapies, with a focus on addressing the inconsistent results regarding the function of MARCKS as a promoter or inhibitor of oncogenesis.
... Various targeted proteomic approaches have been used to identify biomarker candidates for disease treatment from a variety of clinical samples, including cerebrospinal fluid (CSF) [14,15], serum or plasma [16][17][18], saliva [19], urine [20], and tissue [21][22][23]. Biomarkers that have been identified using targeted proteomics approaches can then be used to develop multi-marker panels, which can then be used to determine diagnosis and prognosis, with high sensitivity and specificity [24]. ...
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... "/" indicates that the interaction signals or pathways are not shown in these studies. Blood (n = 30) Diagnosis for multiple myeloma [76] Tissues (n = 134/104) Prognosis for gastric cancer [77,78] Tissues (n = 142/212) Prognosis for non-small cell lung cancer [79,80] Tissues (n = 50, 19, 31) Diagnosis for hepatocellular carcinoma [81] Tissues (n = 139) Prognosis for oral squamous cell carcinoma [82] Tissues (n = 80/58) Prognosis for breast cancer [83,84] Tissues (n = 111/111) Prognosis for ovarian cancer [85,86] Tissues (n = 167) Prognosis for endometrial cancer [87] Tissues (n = 43) Prognosis for prostate cancer [88] Biglycan Tissues (n = 12,427) Prognosis for prostate cancer [89] ...
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The extracellular matrix (ECM) spatiotemporally controls cell fate; however, dysregulation of ECM remodeling can lead to tumorigenesis and cancer development by providing favorable conditions for tumor cells. Proteoglycans (PGs) and glycosaminoglycans (GAGs) are the major macromolecules composing ECM. They influence both cell behavior and matrix properties through direct and indirect interactions with various cytokines, growth factors, cell surface receptors, adhesion molecules, enzymes, and glycoproteins within the ECM. The classical features of PGs/GAGs play well-known roles in cancer angiogenesis, proliferation, invasion, and metastasis. Several lines of evidence suggest that PGs/GAGs critically affect broader aspects in cancer initiation and the progression process, including regulation of cell metabolism, serving as a sensor of ECM’s mechanical properties, affecting immune supervision, and participating in therapeutic resistance to various forms of treatment. These functions may be implemented through the characteristics of PGs/GAGs as molecular bridges linking ECM and cells in cell-specific and context-specific manners within the tumor microenvironment (TME). In this review, we intend to present a comprehensive illustration of the ways in which PGs/GAGs participate in and regulate several aspects of tumorigenesis; we put forward a perspective regarding their effects as biomarkers or targets for diagnoses and therapeutic interventions.
... ; https://doi.org/10.1101/300673 doi: bioRxiv preprint adjacent non-tumorous tissue using the label-free shotgun proteomics over a 98-min LC gradient 15 . Gao et al 16 applied SWATH-MS to study 14 pairs of HCC and non-HCC tissues and quantified 4,216 proteins in at least one biological replicates and 1,903 proteins in at least three out of five biological replicates using a 120-min LC gradient. ...
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In this study, we optimized the pressure-cycling technology (PCT) and SWATH mass spectrometry workflow to analyze biopsy-level tissue samples (2 mg wet weight) from 19 hepatocellular carcinoma (HCC) patients. Using OpenSWATH and pan-human spectral library, we quantified 11,787 proteotypic peptides from 2,579 SwissProt proteins in 76 HCC tissue samples within about 9 working days (from receiving tissue to SWATH data). The coefficient of variation (CV) of peptide yield using PCT was 32.9%, and the R ² of peptide quantification was 0.9729. We identified protein changes in malignant tissues compared to matched control samples in HCC patients, and further stratified patient samples into groups with high α-fetoprotein (AFP) expression or HBV infection. In aggregate, the data identified 23 upregulated pathways and 13 ones. We observed enhanced biomolecule synthesis and suppressed small molecular metabolism in liver tumor tissues. 16 proteins of high documented relevance to HCC are highlighted in our data. We also identified changes of virus-infection-related proteins including PKM, CTPS1 and ALDOB in the HBV ⁺ HCC subcohort. In conclusion, we demonstrate the practicality of performing proteomic analysis of biopsy-level tissue samples with PCT-SWATH methodology with moderate effort and within a relatively short timeframe.
... Mass spectrometric analyses were performed using an Orbitrap Elite instrument coupled to an Ultimate 3000 RSLCnano system (Thermo Scientific, Waltham, MA, USA) as described before [15]. Briefly, 350 ng tryptic peptides were injected and concentrated on a trap column (Acclaim ...
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Background Asthma is an inflammatory disease of the respiratory system and a major factor of increasing health care costs worldwide. The molecular actors leading to the development of chronic asthma are not fully understood and require further investigation. Objective The aim of this study was to monitor the proteome dynamics during asthma development from early inflammatory to late fibrotic stages. Methods A mouse asthma model was used to analyse the lung proteome at four time points during asthma development (0 weeks = control, 5, 8 and 12 weeks of treatment, n=6 each). The model was analysed using lung function tests, immune cell counting and histology. Furthermore, a multi‐fraction mass spectrometry‐based proteome analysis was performed to achieve a comprehensive coverage and quantification of the lung proteome. Results At early stages, the mice showed predominant eosinophilic inflammation of the airways, which disappeared at later stages and was replaced by marked airway hyperreactivity and fibrosis of the airways. 3325 proteins were quantified with 435 proteins found to be significantly differentially abundant between the experimental groups (ANOVA p‐value ≤ 0.05, maximum fold change ≥ 1.5). We applied hierarchical clustering to identify common protein abundance profiles along the asthma development and analysed these clusters using gene ontology annotation and enrichment analysis. We demonstrate the correlation of protein clusters with the course of asthma development i.e. eosinophilic inflammation and fibrotic remodelling of the airways. Conclusions & Clinical Relevance Proteome analysis revealed proteins that were previously described to be important during asthma chronification. Moreover, we identified additional proteins previously not described in the context of asthma. We provide a comprehensive data set of a long‐term mouse model of asthma that may contribute to a better understanding and allow new insights into the progression and development of chronic asthma. Data are available via ProteomeXchange with identifier PXD011159.
... In recent years, the use of 'omic' methods, especially quantitative proteomics analysis, to search for and identify novel HCC biomarkers has attracted extensive attention. For instance, Naboulsi et al. demonstrated that versican core protein (VCAN) is a potential biomarker for early HCC diagnosis by label-free discovery analysis: selected (multiple) reaction monitoring (SRM/MRM) 20 . It was also reported that protein S100A9, which was dramatically up-regulated in sera from HCC patients, is a candidate HCC diagnostic biomarker 21 . ...
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The accuracy of current biomarkers for the diagnosis of hepatocellular carcinoma (HCC), especially chronic Hepatitis B Virus (HBV)-related HCC, is limited. Recent progress in glycoproteomics has provided a novel platform for screening novel serological biomarkers of HCC. In this study, lectin affinity chromatography by Maackia amurensis lectin (MAL) and iTRAQ combined with mass spectrometric analysis were performed to enrich and identify the glycoprotein fractions in serum samples from HBV-related HCC patients and from healthy controls. Seventeen differential MAL-associated glycoproteins were identified. Among them, Galectin 3 binding protein (Gal-3BP) was selected for further evaluated by ELISA analysis and showed a high diagnostic potential of HBV-related HCC, with the AUC of 0.898 and a sensitivity, specificity and accuracy of 80.00%, 93.75% and 86.88%, respectively. Moreover, we constructed a predictive model through the combined use of serum Gal-3BP and Alpha Fetoprotein (AFP), which improved the sensitivity (from 87.5% to 95%), specificity (from 93.75% to 95%) and accuracy (from 90.63% to 95%) of diagnosing early HCC. These data suggested serum Gal-3BP level is a promising biomarker to identify HBV-related HCC and the combined use of serum Gal-3BP and AFP improves the diagnostic potential of HBV-HCC compared with AFP alone in current clinical practice.
... For example, LYVE1, a down-regulated gene in HCC, may constitute as an early biomarker of postoperative survival in HCC patients [29]. And VCAN, an up-regulated gene in HCC, could serve as a potential biomarker for early-HCC diagnosis [30]. Besides, target genes of DEMs were mainly enriched in the processes of cancer, such as glioma, hippo signaling pathway, and signaling pathways regulating pluripotency of stem cells ( Figure S1C). ...
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Background Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, but its regulatory mechanism remains unclear. Although many TFs and miRNAs are reported to be important in HCC, their co-regulation and FFL modules in HCC development are needed to be investigated. Methods The feed-forward loop (FFL) regulatory module was identified by analyzing the miRNA and transcription factor co-regulatory network for differentially expressed genes between tumors and matched adjacent tissue samples. Gene expression and regulatory role of HCC development by key FFL in vitro and in vivo were validated by qPCR, Western blotting, cell proliferation assay, migration and invasion assay and experiments in nude mice with hepatoma xenografts. Results Here, by bioinformatics analysis, we identified FFL regulatory module miR-9-5p/FOXO1/CPEB3 may play critical roles in HCC progression. Gain- and loss-of-function studies demonstrated that miR-9-5p promote hepatocarcinogenesis, while FOXO1 and CPEB3 inhibit hepatocarcinoma growth. Furthermore, CPEB3 was firstly identified as a direct downstream target of miR-9-5p and FOXO1 by luciferase reporter assay and ChIP-Seq data, which was negatively regulated by miR-9-5p and activated by FOXO1. Following, the miR-9-5p/FOXO1/CPEB3 FFL was associated with poor prognosis and promoted cell growth and tumorigenesis of HCC in both in vitro and in vivo experiments. Conclusion Our study newly identified the existence of miR-9-5p/FOXO1/CPEB3 FFL and revealed its regulatory role in HCC progression, which may represent a new potential therapeutic target for cancer treatment.
... A very original finding of our study is the demonstration of meprin α being a mediator explaining the effects of Reptin on cell migration. Reptin has been consistently shown overexpressed in human HCC [2,[31][32][33] where a high level of expression is correlated with a poor prognosis [2]. We and others have shown that Reptin is a potent mediator of oncogenesis and is considered as an attractive target in cancer [2,3,[5][6][7]34]. ...
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Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects. MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers. Silencing Reptin decreased Mep1A expression and activity, without affecting meprin β. Mep1A, but not meprin β, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted. Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.
... Serpin H1 (also known as heat shock protein 47, Hsp 47) is a 47 kDa endoplasmic reticulum (ER) protein which can specifically bind to collagen and works as a chaperone in collagen synthesis (Ito and Nagata 2017). Hsp 47 (encoded by SERPINH1), a member from serpin family, participated in progression of cancer such as hepatocellular carcinoma (HCC) (Naboulsi et al. 2016) and cholangiocellular carcinoma (Padden et al. 2014). Overexpression of Hsp 47 was also reported in micro-vessels from invasive ductal carcinoma (IDC) of breast cancer (vs. ...
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Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N-glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identified with database search engine GPSeeker, and the differentially expressed intact N-glycopeptides (DEGPs) were quantified with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR ≤ 1%, 322 intact N-glycopeptides were identified; these intact N-glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N-glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N-glycosites). The sequence structures of 165 IDs were confirmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates, ≥ 1.5-fold change and p value < 0.05, 20 DEGPs were quantified, where five of them were up-regulated and 15 of them were down-regulated; the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.
... Previously, researchers have proposed that Hsp27 is upregulated and confers aggressiveness in HCC to facilitate HCC malignancy [23][24][25]. Similarly, DDX39A is upregulated in HCC and promotes HCC growth and metastasis [26,27]. Recently, Dang et al. reported NELFE as an oncogenic protein that induces a transcriptome perturbation by selectively regulating MYC signaling [28]. ...
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Splicing factors (SFs) have been increasingly documented to perturb the genome of cancers. However, little is known about the alterations of SFs in hepatocellular carcinoma (HCC). This study comprehensively delineated the genomic and epigenomic characteristics of 404 SFs in HCC based on the multi-omics data from the Cancer Genome Atlas database. The analysis revealed several clinically relevant SFs that could be effective biomarkers for monitoring the onset and prognosis of HCC (such as, HSPB1, DDX39A, and NELFE, which were the three most significant clinically relevant SFs). Functional enrichment analysis of these indicators showed the enrichment of pathways related to splicing and mRNA processes. Furthermore, the study found that SF copy number variation is common in HCC and could be a typical characteristic of hepato-carcinogenesis; the complex expression regulation of SFs was significantly affected by copy number variant and methylation. Several SFs with significant mutation patterns were identified (such as, RNF213, SF3B1, SPEN, NOVA1, and EEF1A1), and the potential regulatory network of SFs was constructed to identify their potential mechanisms for regulating clinically relevant alternative splicing events. Therefore, this study established a foundation to uncover the broad molecular spectrum of SFs for future functional and therapeutic studies of HCC.
... Abnormal MARCKS signals may participate in malignant deformation, uncontrolled proliferation, migration, and invasion of malignant tumor cells. Recently, some studies showed that as a proto-oncogene, MARCKS takes part in the tumorigenesis and development of several cancers such as lung cancer (Rohrbach et al., 2015), breast cancer (Chen et al., 2015), renal cell carcinoma (Chen et al., 2017), pancreatic cancer (Brandi et al., 2016), and HCC (Naboulsi et al., 2016) and plays an important role in participation in signaling pathways of many cancers. In addition, MARCKS also has an effect on the sensitivity of cancer cells to chemotherapy drugs and targeted radiation therapy. ...
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Metformin is a kind of widely used antidiabetic drug that regulates glucose homeostasis by inhibiting liver glucose production and increasing muscle glucose uptake. Recently, some studies showed that metformin exhibits anticancer properties in a variety of cancers. Although several antitumor mechanisms have been proposed for metformin action, its mode of action in human liver cancer remains not elucidated. In our study, we investigated the underlying molecular mechanisms of metformin's antitumor effect on Huh-7 cells of hepatocellular carcinoma (HCC) in vitro. RNA sequencing was performed to explore the effect of metformin on the transcriptome of Huh-7 cells. The results revealed that 4,518 genes (with log2 fold change > 1 or < −1, adjusted p-value < 0.05) were differentially expressed in Huh-7 cells with treatment of 25-mM metformin compared with 0-mM metformin, including 1,812 upregulated and 2,706 downregulated genes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses identified 54 classical pathways that were significantly enriched, and 16 pathways are closely associated with cancer, such as cell cycle, DNA replication, extracellular matrix–receptor interaction, and so on. We selected 11 differentially expressed genes, which are closely associated with HCC, to validate their differential expressions through a quantitative real-time reverse transcription-polymerase chain reaction. The result exhibited that the genes of fatty acid synthase, mini-chromosome maintenance complex components 6 and 5, myristoylated alanine-rich C-kinase substrate, fatty acid desaturase 2, C-X-C motif chemokine ligand 1, bone morphogenetic protein 4, S-phase kinase-associated protein 2, kininogen 1, and proliferating cell nuclear antigen were downregulated, and Dual-specificity phosphatase-1 is significantly upregulated in Huh-7 cells with treatment of 25-mM metformin. These differentially expressed genes and pathways might play a crucial part in the antitumor effect of metformin and might be potential targets of metformin treating HCC. Further investigations are required to evaluate the metformin mechanisms of anticancer action in vivo.
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There is a critical need for the discovery of novel biomarkers for early detection and targeted therapy of cancer, a major cause of deaths worldwide. In this respect, proteomic technologies, such as mass spectrometry (MS), enable the identification of pathologically significant proteins in various types of samples. MS is capable of high-throughput profiling of complex biological samples including blood, tissues, urine, milk, and cells. MS-assisted proteomics has contributed to the development of cancer biomarkers that may form the foundation for new clinical tests. It can also aid in elucidating the molecular mechanisms underlying cancer. In this review, we discuss MS principles and instrumentation as well as approaches in MS-based proteomics, which have been employed in the development of potential biomarkers. Furthermore, the challenges in validation of MS biomarkers for their use in clinical practice are also reviewed.
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Biological significance: Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity. In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies.
Chapter
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Dynamic liver function assessment by the 13C-methacetin maximal liver function capacity (LiMAx) test reflects the overall hepatic CYP1A2 activity. One proven strategy for preoperative risk assessement in liver surgery includes the combined assessment of the dynamic liver function by the LiMAx test, the volumetric analysis of the liver and calculation of future liver remnant function. This so-called volume-function analysis assumes that the remaining CYP1A2 activity in any tumor lesion is zero. The here presented study aims to assess the remaining CYP1A2 activities in different hepatic tumor lesions and its consequences for the preoperative volume-function analysis in patients undergoing liver surgery. The CYP1A2 activity analysis of neoplastic lesions and adjacent non-tumor liver tissue from resected tumor specimens revealed a significantly higher CYP1A2 activity (median, interquartile range) in non-tumor tissues (35.5, 15.9-54.4 µU/mg) as compared to hepatocellular adenomas (7.35, 1.2-32.5 µU/mg), hepatocellular carcinomas (0.18, 0.0-2.0 µU/mg) or colorectal liver metastasis (0.17, 0.0-2.1 µU/mg), respectively. In non-tumor liver tissue a gradual decline in CYP1A2 activity with exacerbating fibrosis was observed. The CYP1A2 activity differences were also reflected in CYP1A2 protein signals in the assessed hepatic tissues. Volume-function analysis showed a minimal deviation compared to the current standard calculation for hepatocellular carcinomas or colorectal liver metastasis (<1% difference), while a difference of 11.9% was observed for hepatocellular adenomas. These findings are important for a refined preoperative volume-function analysis and improved surgical risk assessment in hepatocellular adenoma cases with low LiMAx values.
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RNA helicases are members of a large family of enzymes that function in unwinding RNA duplexes and modulating interactions between RNAs and proteins. RNA helicases participate in numerous cellular processes, including transcription, translation, mRNA export, nonsense-mediated mRNA decay, and telomere maintenance. In this review, we discuss the recent discoveries on several RNA helicases that play key roles in aging and lifespan regulation. We first describe the roles of several mammalian RNA helicases implicated in cellular senescence. In addition, our previous reports show that three RNA helicases, HEL-1/DDX39, SACY-1/DDX41 and SMG-2/UPF1, promote longevity in C. elegans through distinct mechanisms. These findings reveal the critical functions of many RNA helicases in regulating aging and may lead to insights into the importance of RNA homeostasis in aging and longevity.
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Purpose To rapidly identify protein abundance changes in biopsy‐level fresh‐frozen hepatocellular carcinoma (HCC). Experimental design We applied the pressure‐cycling technology (PCT) and optimized sequential window acquisition of all theoretical mass spectra (SWATH‐MS) workflow to analyze 38 biopsy‐level tissue samples from 19 HCC patients. Each proteome was analyzed with 45 min liquid chromatography (LC) gradient. MCM7 was validated using immunohistochemistry (IHC). Results We quantified 11,787 proteotypic peptides from 2,579 SwissProt proteins with high confidence. The coefficient of variation (CV) of peptide yield using PCT was 32.9%, and the R² of peptide quantification was 0.9729. 541 proteins showed significant abundance change between the tumor area and its adjacent benign area. From 24 upregulated pathways and 13 suppressed ones, we observed enhanced biomolecule synthesis and suppressed small molecular metabolism in liver tumor tissues. We further analyzed protein changes based on α‐fetoprotein expression and HBV infection. Our data altogether highlighted 16 promising tumor marker candidates. The up‐regulation of minichromosome maintenance complex component 7 (MCM7) was further observed in multiple HCC tumor tissues by IHC. Conclusions and clinical relevance We demonstrated the practicality of rapid proteomic analysis of biopsy‐level fresh‐frozen HCC tissue samples with PCT‐SWATH and identified promising tumor marker candidates including MCM7. This article is protected by copyright. All rights reserved
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Myristoylated alanine‐rich C kinase substrate (MARCKS), a protein kinase C (PKC) substrate, facilitates mucus production and neutrophil migration. However, the effects of therapeutic procedures targeting the phosphorylation site of MARCKS on steroid‐resistant asthma and the mechanisms underlying such effects have not yet been investigated. We designed a peptide that targets the MARCKS phosphorylation site (MPS peptide) and assessed its therapeutic potential against steroid‐resistant asthma. Mice were sensitized with ovalbumin (OVA), alum, and challenged with aerosolized OVA 5 times a week for one month. The mice were intratracheally administered MPS peptides 3 times a week, 1 h before OVA challenge. Asthma symptoms and cell profiles in the bronchoalveolar lavage (BAL) were assessed, and key proteins were analyzed using western blotting. Phosphorylated (p)–MARCKS was highly expressed in inflammatory and bronchial epithelial cells in OVA‐immunized mice. MPS peptide reduced eosinophils, neutrophils, mucus production, collagen deposition, and airway hyper‐responsiveness (AHR). Dexamethasone (Dexa) did not alleviate steroid‐resistant asthma symptoms. MPS peptide caused a decrease in p‐MARCKS, nitrotyrosine and the expression of oxidative stress enzymes, NADPH oxidase dual oxidase 1 (Duox‐1) and iNOS, in lung tissues. Compared to Dexa, MPS peptides inhibited C5a production and attenuated interleukin‐17A (IL‐17A) and KC production in the airway more effectively, thus suppressing asthma symptoms. Our findings indicate that targeting MARCKS phosphorylation through MPS treatment may inhibit neutrophilic inflammation and relieves asthma symptoms, thereby highlighting its potential as a therapeutic agent for steroid‐resistant asthma.
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Human hepatocellular carcinoma (HCC) is the most prevalent and recurrent type of primary adult liver cancer without any effective therapy. Thus, there is an increase demands for finding new drugs and treatment strategies with selective and potent effects towards HCC. Plant-derived compounds acting as anti-cancer agents can induce apoptosis through targeting several signaling pathways. Thymoquinone (TQ), the major biologically active compound of the black seed oil (Nigella sativa) has demonstrated inhibitory activities on various cancers by targeting several pathways. In the present study, we have evaluated the molecular mechanisms that underlie the anti-proliferative, anti-metastatic, and pro-apoptotic activities exerted by TQ on liver cancer cell lineHepG2, a well-documented HCC in vitro model. Cell proliferation was determined by WST-1 assay, apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining, wound healing assay to investigate the metastasis, and the expression of target genes was assessed by Real-time RT–PCR analysis. We found that TQ significantly reduced HepG2 cell viability and induced apoptosis in a dose-dependent manner. Migration of HepG2 cells was suppressed in response to TQ. Moreover, TQ decreased the expression of several angiogenesis-related genes including versican (VCAN), growth factor receptor-bound protein 2 (Grb2), and the histone methyltransferase for lysine 27 of histone 3 (EZH2). The findings suggest that TQ exerts inhibitory effects on HCC most likely through targeting key genes involved in the invasiveness and
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Background and aims: The eradication of Hepatitis C (HCV) infection by direct-acting antiviral agents (DAAs) has been linked to an amelioration of liver synthesis and regression of fibrosis. Although changes in number and type of circulating microvesicles (MVs) have been reported in cirrhosis, conclusive data on the effect of DAAs treatment on MVs profile in HCV cirrhotic patients remain scarce. Methods: We measured levels of endothelial, platelet and hepatocyte MVs, as well as MVs expressing versican core protein (VCAN+) in patients with HCV-related cirrhosis at baseline, end of therapy (EOT), at 12, 24 and 48 weeks (W) after EOT by new generation flow-cytometry. Results: Fifty-eight patients were enrolled (86% Child's A). MVs were increased at EOT versus baseline, though only platelet MVs revealed a statistically significant difference (p< 0.01). MVs levels did not change significantly after EOT notwithstanding a steady downward trend towards baseline levels. Conversely, VCAN+MVs dropped significantly at EOT (p <0.001) and remained low throughout the follow-up. Hepatocyte MVs significantly correlated with liver stiffness (r 0.40, p 0.0021). Eight composite outcomes occurred during the 1-year follow-up: 3 portal vein thromboses, 2 hepatocellular carcinomas and 3 liver decompensation. Child's B, the presence of F2 oesophageal varices (OR for interaction 19.2 [95%CI 1.45-253.7], p 0.023) and platelet MVs (OR 1.026 [95%CI 1.00-1.05, p 0.023) correlated significantly with clinical outcomes. Conclusions: VCAN+MVs appear to mirror the profibrotic status of the cirrhotic disease; hepatocyte MVs correlate with liver stiffness and increased platelet MVs levels could be associated with a worse clinical outcome. This article is protected by copyright. All rights reserved.
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More efficient biomarkers are needed to facilitate the early detection of hepatocellular carcinoma (HCC). We aimed to identify candidate biomarkers for HCC detection by proteomic analysis. First, we performed a global proteomic analysis of 10 paired HCC and non-tumor tissues. Then, we validated the top-ranked proteins by targeted proteomic analyses in another tissue cohort. At last, we used enzyme-linked immunosorbent assays to validate the candidate biomarkers in multiple serum cohorts including HCC cases (HCCs), cirrhosis cases (LCs), and normal controls (NCs). We identified and validated 33 up-regulated proteins in HCC tissues. Among them, eight secretory or membrane proteins were further evaluated in serum, revealing that aldo-keto reductase family 1 member B10 (AKR1B10) and cathepsin A (CTSA) can distinguish HCCs from LCs and NCs. The area under the curves (AUCs) were 0.891 and 0.894 for AKR1B10 and CTSA, respectively, greater than that of alpha-fetoprotein (AFP; 0.831). Notably, combining the three proteins reached an AUC of 0.969, which outperformed AFP alone (P < 0.05). Furthermore, the serum AKR1B10 levels dramatically decreased after surgery. AKR1B10 and CTSA are potential serum biomarkers for HCC detection. The combination of AKR1B10, CTSA, and AFP may improve the HCC diagnostic efficacy.
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Introduction and hypothesis: Pelvic organ prolapse (POP) in women is associated with deficiency of elastic fibers, and fibulin-5 is known to be a critical protein in the synthesis of elastin. The purpose of this study is to investigate the related pathway for the synthesis of elastin via fibulin-5 using fibulin-5 knockout mice. Methods: Fibulin-5 knockout mice were generated using the CRISPR/Cas9 system, and vaginal dilatation was used to mimic vaginal delivery. We divided the mice into three groups: Fbln5+/+ mice immediately after dilatation (Fbln5+/+ day0), Fbln5+/+ mice 3 days after dilatation (Fbln5+/+ day3) and Fbln5-/- mice 3 days after dilatation (Fbln5-/- day3). Proteins related to elastogenesis in the vaginal wall were measured by liquid chromatography mass spectrometry (LC-MS/MS) analysis, and differences in the expression of these proteins between the Fbln5-/- mice and the Fbln5+/+ mice were analyzed using western blotting. Results: In the LC-MS/MS analysis, protein tyrosine kinase 7 (PTK7) was not detected in the Fbln5-/- day3 group, although the expression increased by > 1.5 times between the Fbln5+/+ day0 and day3 groups. PTK7 and β-catenin are known to act in the Wnt/β-catenin pathway, and both were upregulated after dilatation in the Fbln5+/+ mice, though not in the Fbln5-/- mice. Conclusion: Our findings suggest that these proteins are involved in elastogenesis via fibulin-5, and the impairment of these proteins might be the underlying cause of POP manifestation.
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Mutations affecting phosphorylation sites in the b-catenin gene have been implicated in the development of human and rodent hepatocellular carcinomas (HCCs). To further investigate the involvement of this gene in hepatocarcinogenesis, we used several transgenic mouse models of hepatic tumors induced by overexpression of c-myc in the liver either alone or in combination with transforming growth factor (TGF) a or TGF-b1. Acti- vation of b-catenin, as judged by the presence of mutations and/or nuclear translocation of the protein, was most frequent in liver tumors from c-myc (4/17; 23.5%) and c-myc/TGF-b1 (6/18; 33.3%) transgenic mice. How- ever, it was very rare in faster growing and histologically more aggressive HCCs developed in c-myc/TGF-a mice (1/20; 5%). Administration of diethylnitrosamine, phenobarbital, or 2-amino-3,8-diethylimidazo(4,5- f)quinoxaline did not significantly affect the occurrence of b-catenin mutations. Notably, nuclear accumulation of b-catenin was observed only in adenomas and highly differentiated carcinomas with eosinophilic phe- notype. Furthermore, preneoplastic lesions with eosinophilic phenotype frequently displayed focal nuclear positivity, colocalized with areas of high proliferation. In contrast, basophilic and clear-cell foci, as well as pseudo- glandular and poorly differentiated HCCs, exhibited a normal or reduced membranous immunoreactivity for b-catenin. These studies suggest that nuclear translocation of b-catenin and activation of Wingless/Wnt signal- ing may represent an early event in liver carcinogenesis, providing a growth advantage in a subset of hepatic tumors with a more differentiated phenotype.
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Background Diagnosis of early-stage hepatocellular carcinoma (HCC) followed by curative resection or liver transplantation offers the best chance for long-term patient survival. Clinically, ultrasonography has suboptimal sensitivity for detecting early-stage HCC. Several serological tests including alpha-fetoprotein (AFP), the ratio of lens culinaris agglutinin-reactive alpha-fetoprotein to total AFP (AFP-L3/AFP), des-gamma carboxyprothrombin (DCP), and glypican-3 (GPC-3) have been widely investigated as diagnostic biomarkers for early-stage HCC in at-risk populations. However, these tests are not recommended for routine HCC screening. Our objective is to determine the diagnostic performance of AFP, AFP-L3/AFP, DCP, and GPC-3 for the detection of HCC, particularly early-stage tumors meeting the Milan criteria. Methods/design We will include cross-sectional studies that consecutively or randomly recruit target populations. We will search the Cochrane Library, Medline, Embase, Science Citation Index, and the Chinese National Knowledge Infrastructure. We will also search the MEDION and ARIF databases to identify diagnostic systematic reviews that include primary studies. Reference lists of relevant reviews will be searched for additional trials. Language restrictions will not be applied. Two reviewers will independently screen study eligibility and extract data. Methodological quality will be assessed according to the revised tool for the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). Two authors will apply the QUADAS-2 assessment to all the included studies, and any discrepancies will be resolved by the third author. The following test characteristics will be extracted into 2 × 2 tables for all included studies: true positives, false positives, true negatives, and false negatives. Study-specific estimates of sensitivity and specificity with 95% confidence intervals will be displayed in forest plots. When possible, we will use the bivariate random-effects model or the Rutter and Gatsonis hierarchical summary receiver operating characteristic model for statistical analysis. To investigate heterogeneity, we will include study designs, population characteristics, test characteristics, and types of reference standard as the study-level variables. Discussion Our systematic review will allow patients, clinicians, and researchers to determine the diagnostic performance of AFP, AFP-L3/AFP, DCP, and GPC-3 for the detection of early-stage HCC and the potential roles of these diagnostic biomarkers in the existing diagnostic pathways. Systematic Review Registration: PROSPERO 2013; CRD42013003879
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Background Differential diagnosis of high-grade dysplastic nodules (HGDN) and well-differentiated hepatocellular carcinoma (WDHCC) represents a challenge to experienced hepatic clinicians, radiologists and hepatopathologists. Methods The expression profiles of aminoacylase-1 (ACY1), sequestosome-1 (SQSTM1) and glypican-3 (GPC3) in low-grade dysplastic nodules (LGDN), HGDN and WDHCC were assessed by immunohistochemistry. The differential diagnostic performances of these three markers alone and in combination for HGDN and WDHCC were investigated by logistic regression models (HGDN = 21; WDHCC = 32) and validated in an independent test set (HGDN, n = 21; WDHCC n = 24). Postoperative overall survival and time to recurrence were evaluated by univariate and multivariate analyses in an independent set of 500 patients. Results ACY1, SQSTM1 and GPC3 were differentially expressed in each group. For the differential diagnosis of WDHCC from HGDN, the sensitivity and specificity of the combination of ACY1 + SQSTM1 + GPC3 for detecting WDHCC were 93.8% and 95.2% respectively in the training set, which were higher than any of the three two-marker combinations. The validities of the four diagnostic models were further confirmed in an independent test set, and corresponding good sensitivity and specificity were observed. Interestingly, GPC3 expression in HCC tissues combined with serum α-fetoprotein (AFP) was found to be an independent predictor for overall survival and time to recurrence. Conclusions ACY1 + SQSTM1 + GPC3 combination represents a potentially valuable biomarker for distinguishing between WDHCC and HGDN using immunohistochemistry. Meanwhile, low GPC3 staining combined with positive serum AFP may play a practical role in predicting poor postoperative outcome and high tumor recurrence risk.
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Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-β-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. We have examined the status of the β-catenin gene in different transgenic mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the β-catenin gene similar to those found in colon cancers and melanomas. These alterations in the β-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of β-catenin and thus to carcinogenesis. We then analyzed human HCCs and found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations led to the accumulation of β-catenin in the nucleus. Thus alterations in the β-catenin gene frequently are selected for during liver tumorigenesis and suggest that disregulation of the Wnt-β-catenin pathway is a major event in the development of HCC in humans and mice.
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This study was designed to explore the role of versican in the development of hepatocellular carcinoma (HCC). Ectopic expression of the versican 3'-untranslated region (3'-UTR) was studied as a competitive endogenous RNA for regulating miRNA functions. We used this approach to modulate the expression of versican and its related proteins in 3'-UTR transgenic mice and in the liver cancer cell line HepG2, stably transfected with the 3'-UTR or a control vector. We demonstrated that transgenic mice expressing the versican 3'-UTR developed HCC and increased expression of versican isoforms V0 and V1. HepG2 cells transfected with versican 3'-UTR displayed increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis. We found that versican 3'-UTR could bind to miRNAs miR-133a, miR-199a*, miR-144, and miR-431 and also interacted with CD34 and fibronectin. As a consequence, expression of versican, CD34, and fibronectin was up-regulated by ectopic transfection of the versican 3'-UTR, which was confirmed in HepG2 cells and in transgenic mice as compared with wild-type controls. Transfection with siRNAs targeting the versican 3'-UTR abolished the effects of the 3'-UTR. Taken together, these results demonstrate that versican V0 and V1 isoforms play important roles in HCC development and that versican mRNAs compete with endogenous RNAs in regulating miRNA functions.-Fang, L., Du, W. W., Yang, X., Chen, K., Ghanekar, A., Levy, G., Yang, W., Yee, A. J., Lu, W.-Y., Xuan, J. W., Gao, Z., Xie, F., He, C., Deng, Z., Yang, B. B. Versican 3'-untranslated region (3'-UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity.
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Carboxypeptidase D (CPD), a membrane-bound metallocarboxypeptidase that functions as a docking receptor for duck hepatitis B virus, is frequently overexpressed in human cancers. We have explored its expression pattern, clinical significance, and biological function of CPD in hepatocellular carcinoma (HCC). CPD expression was markedly elevated in HCCs relative to adjacent non-tumor liver tissues, as determined by quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis. Immunohistochemistry showed that 164 of 400 (41%) HCCs had high expression of CPD. CPD overexpression was significantly associated with serum levels of hepatitis B surface antigen and hepatitis B e antigen, liver cirrhosis, pathological grade, and intrahepatic metastasis. Knockdown of endogenous CPD expression in Huh7 HCC cells by RNA interference reduced cell proliferation, blocked the cell cycle at G1 phase, and increased apoptosis. Many genes implicated in cell-cycle regulation, including P21waf1, P27 Kip1, SKP2, and CDC2, were deregulated by CPD downregulation. Thus CPD is frequently upregulated in HCC, and targeting CPD inhibits HCC cell proliferation through induction of G1 cell-cycle arrest and apoptosis, thereby providing a potential therapeutic target for this malignancy.
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Objectives: Chitinase 3-like 1 (CHI3L1) is associated with poor prognosis of various human cancers. However, the clinical and prognostic significance of CHI3L1 in hepatocellular carcinoma (HCC) is largely unknown. The aim of the present study is to investigate the expression of CHI3L1 in human HCC cell lines, clinical HCC specimens and its association with expressions of phosphorylated-Akt (p-Akt), E-cadherin and prognostic significance. Methods: The protein level of CHI3L1 in HCC cell lines was evaluated by western blot. The mRNA and protein levels of CHI3L1 in 19 self-paired HCC specimens were assessed by RT-PCR and western blot assays. The clinical and prognostic significance of CHI3L1 in 70 cases of HCC patients was determined by immunohistochemistry. In addition, expressions of p-Akt and E-cadherin were also assessed. Results: The protein level of CHI3L1 paralleled with increased malignant potential of HCC cell lines (P < 0.05). The mRNA and protein levels of CHI3L1 in HCC tissues were up-regulated compared with those in adjacent peritumoral tissues and further increased in tumors with metastasis (P < 0.05). Clinicopathological analysis showed that positive CHI3L1 expression was significantly associated with larger tumor size, capsular invasion, advanced TNM stages and status of metastasis (P = 0.035, 0.003, 0.023 and 0.003, respectively). Furthermore, CHI3L1 expression was positively correlated with high level of p-Akt (r = 0.293, P = 0.014), but inversely correlated with expression of E-cadherin (r = -0.267, P = 0.026). Additionally, Kaplan-Meier survival analysis showed that HCC patients with positive CHI3L1 expression had a worse overall survival and disease-free survival compared with those with negative CHI3L1 expression (P < 0.001, respectively). Multivariate analysis identified CHI3L1 as an independent prognostic predictor for overall survival and disease-free survival of HCC patients (P = 0.044 and 0.031, respectively). Conclusions: CHI3L1 plays an essential role in HCC malignancies and may be served as a valuable prognostic biomarker for HCC patients.
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