Description

Publications in this journal

• Toxic effect of using twenty percent alcohol on corneal epithelial tight junctions during LASEK.

  Authors: Peng Zhang, Min Liu, Rongfeng Liao

  Molecular medicine reports. 6(1):33-38.

  The aim of this study was to determine the effect of using 20% alcohol on corneal epithelial tight junctions during laser epithelial keratomileusis (LASEK). Sixty Sprague-Dawley rats were randomlyThe aim of this study was to determine the effect of using 20% alcohol on corneal epithelial tight junctions during laser epithelial keratomileusis (LASEK). Sixty Sprague-Dawley rats were randomly divided into two equal groups. The central area of the rat corneas in one group were demarcated with a 3-mm trephine, treated with 20% alcohol for 45 sec and washed with sterile balanced salt solution. The epithelium was removed by an epithelial microhoe used in LASEK. In the other group, the rat corneal epithelium in the central area was mechanically scraped. The experimental animals were sacrificed at 24, 48, 72, 96 and 120 h after surgery. The levels of the tight junction proteins, claudin-1 and ZO-1, were determined by immunofluorescence and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. We found that at approximately 48 h after surgery, the wounded corneas were replaced by corneals with regenerated epithelium. Immunofluorescence analysis demonstrated that the expressions of claudin-1 and ZO-1 in the corneal epithelium of the alcohol-treated group were weaker compared to the mechanical group at the 24 and 48 h time-points. Semi-quantitative RT-PCR analysis showed that the mRNA levels of claudin-1 and ZO-1 in the central cornea after alcohol treatment were lower compared to those in the mechanical group from 24 to 48 h, with no significant difference after 72 h. Thus, we conclude that the treatment with 20% alcohol during LASEK results in damage to the corneal epitheleal tight junctions and prolongs normal recovery time.

• HOXA10 expression is decreased by testosterone in luteinized granulosa cells in vitro.

  Authors: Hong He, Tian Li, Delong Yin, Rong Liu, Qionghua Chen, Ji Wang, Gang Zhong, Demin Pu

  Molecular medicine reports. 6(1):51-56.

  Polycystic ovary syndrome (PCOS) is perhaps the most prevalent endocrine disorder in women of reproductive age, characterized by elevated levels of circulating androgens or clinical manifestations ofPolycystic ovary syndrome (PCOS) is perhaps the most prevalent endocrine disorder in women of reproductive age, characterized by elevated levels of circulating androgens or clinical manifestations of androgen excess. The specific cytokine profile of PCOS patients is probably related to the lower implantation rate, since follicular fluid appears to function as an embryotrophic agent. For a better understanding of the local regulation of human follicles, the present study investigated the protein expression levels and cellular localization of HOXA10 in granulosa cells (GCs) from women with normal ovarian function undergoing IVF due to their husbands suffering from azoospermia. We demonstrated by immunohistochemical studies that the expression of HOXA10 was mainly localized in the cytoplasm of GCs. Our data indicate that these alterations were associated with changes in the expression of ovarian transcription factors of HOXA10. GC dose-responsive decreases in HOXA10 protein were observed in response to physiological or supraphysiological concentrations (10-4 to 10-7 M) of testosterone. These data reveal that testosterone may be involved in HOXA10 gene regulation in GCs. Decreased HOXA10 expression in GCs treated with testosterone suggest that this androgen is responsible for the decreased expression of HOXA10 in PCOS patients.

• Purification of HRSV F protein from a eukaryotic expression vector and establishment of a sandwich ELISA method.

  Authors: Hong Bao, Ting Yu, Yufen Jin, Chune Wang, Yanlei Li

  Molecular medicine reports. 6(1):111-114.

  In order to increase the expression of the fusion (F) protein and lay a foundation for the construction of a genetically engineered vaccine and rapid clinical detection, the F protein of the humanIn order to increase the expression of the fusion (F) protein and lay a foundation for the construction of a genetically engineered vaccine and rapid clinical detection, the F protein of the human respiratory syncytial virus (HRSV) was expressed and purified, and a sandwich enzyme-linked immunosorbent assay (ELISA) method was established. The F1 fragment of the HRSV F protein was amplified following reverse transcription, and was then combined with the vector and transformed into eukaryotic cells. The recombinant protein was induced and purified. The purified protein was used to immunize mice to produce antiserum and establish indirect ELISA. The established method was tested and verified by analyzing 100 samples using gold immunochromatography (GICA). The F1 fragment of the F gene was successfully amplified, the DNA (+) recombinant was selected, and a protein of molecular weight approximately 45,000 was obtained after the induction. The optimal reaction conditions and working concentration of ELISA were determined. The optimal concentration of mice anti-F1 IgG is 3.2 µg/ml, the best reaction time of the samples is 70 min at 37˚C, and the working concentration of the rabbit anti‑mouse IgG is 1:6,000. Compared with the GICA method, the sample's positive co-efficient of variation was 3.2-8.6%, and the negative co-efficient of variation was 5.1-8.3%. These were <10%, indicating that the ELISA method was reproducible. The F1 protein can be greatly expressed in transfected eukaryotic cells, and the purified F1 protein has good immunogenicity. The antiserum produced by the purified recombinant protein can be precisely detected using the ELISA detection method described in this study.

• Phosphoproteomic analysis of electroacupuncture analgesia in an inflammatory pain rat model.

  Authors: Si-Hyoung Lee, Sun-Young Kim, Ji-Hwan Kim, Hye-Yun Jung, Jeong-Hee Moon, Kwang-Hee Bae, Byung-Tae Choi

  Molecular medicine reports. 6(1):157-162.

  The phosphorylation changes of nociceptive signaling proteins in the spinal cord dorsal horn (SCDH) are important in creating exaggerated pain following peripheral inflammation. ElectroacupunctureThe phosphorylation changes of nociceptive signaling proteins in the spinal cord dorsal horn (SCDH) are important in creating exaggerated pain following peripheral inflammation. Electroacupuncture (EA) has been widely used to relieve acute and chronic inflammatory pain in human and experimental pain models. In the present study, we performed a phosphoproteomic analysis to investigate whether EA alters protein phosphorylation in SCDH to attenuate pain development. Inflammatory hyperalgesia was induced by intraplantar injection of complete Freund's adjuvant (CFA) into the rat hind paw. EA treatment at ST36 and SP6 acupoints alleviated thermal hyperalgesia of the CFA-induced inflammatory pain model rats. The SCDH proteins from the control, inflammatory pain model and EA treatment rats were separated by 2-dimensional gel electrophoresis and the alterations in phosphoproteins were detected by Pro-Q Diamond staining. Eight proteins were differentially phosphorylated following EA treatment in the inflammatory pain model. Aldolase C, nascent polypeptide-associated complex α, stress-induced phosphoprotein 1 and heat shock protein 90 were identified as phosphoproteins whose expression was increased, whereas GDP dissociation inhibitor 1, thiamine triphosphatase, phosphoglycerate kinase 1 and 14-3-3 γ were phosphoproteins whose expression was decreased. This is the first phosphoproteomic screening study to elucidate the working mechanisms of EA analgesia. The results suggest that the regulation of cellular pathways in which the identified proteins are involved may be associated with an EA analgesic mechanism.

• Foxp3 expression in A549 cells is regulated by Toll-like receptor 4 through nuclear factor-κB.

  Authors: Ting Jia, Haiying Fu, Jitong Sun, Yinglin Zhang, Wei Yang, Yi Li

  Molecular medicine reports. 6(1):167-172.

  Foxp3 is a master regulator of the development and function of CD4+CD25+ regulatory T cells (Tregs). Previous studies have reported that Foxp3 is also expressed in tumour cells and promotes tumourFoxp3 is a master regulator of the development and function of CD4+CD25+ regulatory T cells (Tregs). Previous studies have reported that Foxp3 is also expressed in tumour cells and promotes tumour immune evasion. However, the regulation of the expression of Foxp3 in tumour cells remains unclear. Toll-like receptor 4 (TLR4), a member of the pattern recognition receptor family, is also expressed in tumour cells. Previous studies have found that the TLR4 signaling pathway is involved in tumour immune evasion in lung cancer cells, and that the transcription factor, nuclear factor-κB (NF-κB), plays a key role in the TLR4 signaling pathway. Moreover, recent studies found that NF-κB promotes the transcription of Foxp3. We hypothesised that TLR4 may also be involved in the regulation of Foxp3 in A549 cells through NF-κB. Therefore, we examined the effect of TLR4 and NF-κB on the expression of Foxp3 in the A549 lung cancer cell line. The results showed that Foxp3 and TLR4 are expressed in A549 cells; the expression of Foxp3 increased after lipopolysaccharide (LPS) stimulation and decreased after blocking the TLR4 signaling pathway. In addition, the expression of NF-κB (p65) increased after LPS stimulation and the expression of Foxp3 decreased after blocking NF-κB. These results suggest that TLR4 is involved in the regulation of Foxp3 in A549 cells through NF-κB.

• A novel bFGF antagonist peptide inhibits breast cancer cell growth.

  Authors: Quchou Li, Susu Gao, Yonglin Yu, Wenhui Wang, Xilei Chen, Ruixue Wang, Tao Li, Cong Wang, Xiaokun Li, Xiaoping Wu

  Molecular medicine reports. 6(1):210-214.

  Breast cancer is the most common type of cancer in women worldwide. Elevated expression of the basic fibroblast growth factor (bFGF) has been found in patients suffering from breast cancer. WeBreast cancer is the most common type of cancer in women worldwide. Elevated expression of the basic fibroblast growth factor (bFGF) has been found in patients suffering from breast cancer. We previously obtained a high-affinity bFGF-binding peptide (named P7) from a phage-display random heptapeptide library. In this study, we show that P7 peptides significantly inhibits the proliferation of the bFGF-stimulated MDA-MB-231 breast cancer cell line. Additional experiments revealed that the mechanisms of the P7 peptide inhibition of the cell proliferation of breast cancer cells stimulated with bFGF in vitro involved cell cycle arrest at the G0/G1 phase, blockade of the activation of Erk and P38 cascades and the upregulation of the expression of the growth inhibitor, proliferation-associated protein 2G4. These results suggest that the bFGF-binding peptide may have therapeutic potential in breast cancer therapy.

• RNA interference therapeutics for cancer: Challenges and opportunities (Review).

  Authors: Roop Singh Bora, Dikshi Gupta, Trilochan Kanwaljit Singh Mukkur, Kulvinder Singh Saini

  Molecular medicine reports. 6(1):9-15.

  RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism in animals and plants, which is mediated by double-stranded RNA (dsRNA). There has recently been anRNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism in animals and plants, which is mediated by double-stranded RNA (dsRNA). There has recently been an increasing interest in harnessing the gene silencing activity of dsRNA to develop novel drugs for the treatment of various diseases, such as cancer, neurological disorders, age-related macular degeneration and viral infections. Small interfering RNA (siRNA)-based drugs have distinct advantages over conventional small molecule or protein-based drugs, including high specificity, higher potency and reduced toxicity. However, there are several technical obstacles to overcome before siRNA-based drugs reach the clinic. Delivery of siRNA to the target tissues and stability in the serum remain a major challenge and are the main focus of current research and development efforts. This review focused primarily on the progress made in developing RNAi as therapeutics for cancer and the challenges associated with its clinical development. Use of ligands recognizing cell-specific receptors to achieve tumor-specific delivery of siRNA, methods for enhanced siRNA delivery, improving the bioavailability and pharmacokinetic properties of siRNA and reducing the off-target effects and non-specific gene silencing are discussed in the light of current evidence.

• Generation of fibroblasts overexpressing liver-specific PEPCK in a miniature pig model of human type 2 diabetes mellitus.

  Authors: Yu-Kyung Kim, Geun-Shik Lee, Eui-Man Jung, Sang-Hwan Hyun, Woo-Suk Hwang, Eui-Bae Jeung

  Molecular medicine reports. 6(1):45-50.

  Type 2 diabetes mellitus (T2DM) is one of the most common complex metabolic disorders in humans, and is characterized by hyperglycemia and metabolic alterations. In T2DM, fasting hyperglycemia isType 2 diabetes mellitus (T2DM) is one of the most common complex metabolic disorders in humans, and is characterized by hyperglycemia and metabolic alterations. In T2DM, fasting hyperglycemia is attributed to excessive hepatic glucose production, and increased gluconeogenesis has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK). In this study, we analyzed porcine PEPCK promoter activities to generate liver-specific expression vectors. We generated miniature pig fibroblasts overexpressing PEPCK via transgenes to provide an animal model of human T2DM. Various regions of the promoter showed high levels of activity in the presence of glucocorticoids, a PEPCK gene inducer, in liver cells compared to a positive control promoter. The selected promoter region for a liver-specific expression system was adopted based on the current targeting vector containing two selection markers, green fluorescence protein and a neomycin-resistance gene. The linearized vector was introduced into pig fibroblasts which facilitated liver-specific PEPCK overexpression and screening according to the two selection markers. In the present study, we used a liposome-mediated transfection protocol rather than a virus-mediated gene delivery system, since the virus may cause side effects. Following transfection, 46 colonies out of 33 transfection trials had positively integrated the overexpression vector, indicating that a relatively high transfection efficiency rate was obtained by the liposomal-mediated system. Thus, we recommend the optimal liver-specific expression system for safe and effective transfection of pig cells. We plan to use these cells for somatic cell nuclear transfer to produce piglets that overexpress liver-specific PEPCK as an animal model for human T2DM.

• Metabolic characteristics of acute necrotizing pancreatitis and chronic pancreatitis.

  Authors: Chao Ma, Bing Tian, Jian Wang, Gen-Jin Yang, Chun-Shu Pan, Jian-Ping Lu

  Molecular medicine reports. 6(1):57-62.

  The etiology and pathogenesis of pancreatitis remains unclear. In the presence of pancreatic inflammation, metabolite abnormalities appear before transformation of tissue structure and changes inThe etiology and pathogenesis of pancreatitis remains unclear. In the presence of pancreatic inflammation, metabolite abnormalities appear before transformation of tissue structure and changes in functions occur. Detection of abnormalities in metabolite levels facilitates a greater understanding of the pathophysiological events and aids in the early diagnosis of the disease. In this study, metabolic profiles from the pancreas of Wistar rats were examined using high-resolution proton magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy to investigate the metabolite indicator(s) of acute necrotizing pancreatitis (ANP) and chronic pancreatitis (CP). The animals were divided into three groups: those with histologically confirmed ANP (n=7), those with CP (n=6) and a control group (n=9). The processed NMR spectra were analyzed using principal component analysis (PCA) to extract characteristic metabolites of ANP and CP. Levels of leucine, isoleucine and valine were increased in the ANP group, whereas an opposite trend was observed in the CP group. Increases in phosphocholine, glycerophosphocholine and choline levels, and decreases in fatty acids, lactate, betaine and glycine levels were observed in both the ANP and CP groups. Additionally, the lipid content in the CP group was higher than that observed in the ANP group. An increase in taurine levels was observed only in the CP group. In conclusion, pancreatitis causes a disruption of the metabolism in the pancreas at a molecular level, with increased taurine levels being a potential metabolite indicator for those with CP.

• Regulation of HRG-β1-induced proliferation, migration and invasion of MCF-7 cells by upregulation of GPR30 expression.

  Authors: Shu-Qin Ruan, Shan-Wei Wang, Zhan-Huai Wang, Su-Zhan Zhang

  Molecular medicine reports. 6(1):131-138.

  The cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There isThe cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There is evidence of crosstalk between the G protein-coupled receptor 30 (GPR30), the newly discovered estrogen receptor (ER), and the ErbB family. Heregulin (HRG)-β1, the ligand for ErbB3 and ErbB4, upregulates GPR30 expression in MCF-7, T-47D and BT-474 breast cancer cell lines that express ERα. In the present study, recombinant human HRG-β1 was used to investigate the upregulation of GPR30 expression by HRGs in MCF-7 breast cancer cells which were ERα-positive. In MCF-7 cells, the ErbB2 inhibitor, AG825, the MAPK inhibitor, PD98059, and the MEK1/2 inhibitor, U0126, blocked the HRG-β1-induced GPR30 expression. 17-β-estradiol (E2) boosted the HRG-β1-induced proliferation, migration and invasion of MCF-7 cells. Similar to E2, the specific GPR30 agonist, G-1, promoted HRG-β1-induced migration and invasion, but inhibited growth. Using the specific GPR30 antagonist, G-15, or the small interfering RNA for GPR30, the functions of GPR30 after treatment with HRG-β1 were further investegated. The results from our study indicate that the interruption between GPR30 signaling and the ErbB family system may serve as a promising therapeutic strategy for breast cancer.

• Retinoblastoma and p53 protein expression in pre-malignant oral lesions and oral squamous cell carcinoma.

  Authors: Marília Gerhardt de Oliveira, Luciana Maria Pereira Ramalho, Leonilson Gaião, Daniel Humberto Pozza, Ramon Andrade de Mello

  Molecular medicine reports. 6(1):163-166.

  The retinoblastoma (Rb) and p53 genes play a fundamental role in cell cycle mechanisms, and their deregulation is related to many steps of oral cancer carcinogenesis. This study was conducted toThe retinoblastoma (Rb) and p53 genes play a fundamental role in cell cycle mechanisms, and their deregulation is related to many steps of oral cancer carcinogenesis. This study was conducted to evaluate the expression of the p53 and Rb proteins in malignant and pre-malignant oral cavity lesions. This retrospective study was conducted at the Federal University of Bahia, Salvador, Brazil, and the Molecular Biology Laboratory of the Otorhinolaryngology Department at the University of Heidelberg, Heidelberg, Germany. Excisional biopsy samples of oral cavities were collected from patients with suspected oral lesions. The samples were processed by immunohistochemistry to be classified by a semi-quantitative score: samples with a ≤10% positivity were considered to have weak/negative expression (-); those with 11-50% positivity, moderate expression (+); and those with >50% positivity, high expression (++). Seventy-one patients were studied (75% male; median age, 52 years; range, 24-84). Of the samples studied, 59.4% were oral squamous cell carcinoma (OSCC) and 40.6% were pre-neoplastic lesions (leukoplakia and actinic cheilitis). OSCC presented higher expression of Rb protein compared to pre-malignant lesions: 75 vs. 25% (p<0.001). Pre-neoplastic lesions presented higher expression of p53 protein compared to OSCC lesions: 55.2 vs. 44.8% (p=0.030). Despite the small number of samples, the expression of these cell cycle biomarkers (p53 and Rb protein) in excisional biopsies suggests that molecular lesion assessment can determine pre-malignant lesions, and that its use may improve the clinical and surgical treatment of early lesions. Thus, p53 protein expression may be related to the early steps of carcinogenesis in OSCC. Finally, a higher Rb expression was also observed in malignant lesions.

• Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris.

  Authors: Jing-Tu Wei, Cun-Duo Tang, Min-Chen Wu, Gao-Lei Liu, Hong-Ling Shi, Jian-Fang Li

  Molecular medicine reports. 6(1):173-178.

  hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLYhly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacⅠ and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His+, Mut+) with multiple hly copies. One transformant resistant to 4.0 mg/ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol.

• In vitro labeling of endothelial progenitor cells isolated from peripheral blood with superparamagnetic iron oxide nanoparticles.

  Authors: Jun-Hui Sun, Yue-Lin Zhang, Chun-Hui Nie, Su-Ping Qian, Xiao-Bo Yu, Hai-Yang Xie, Lin Zhou, Shu-Sen Zheng

  Molecular medicine reports.

  The transplantation of endothelial progenitor cells (EPCs) provides a novel method for the treatment of human tumors or vascular diseases. Magnetic resonance imaging (MRI) has proven to be effectiveThe transplantation of endothelial progenitor cells (EPCs) provides a novel method for the treatment of human tumors or vascular diseases. Magnetic resonance imaging (MRI) has proven to be effective in tracking transplanted stem cells by labeling the cells with superparamagnetic iron oxide (SPIO) nanoparticles. The SPIO has been used to label and track the EPCs; however, the effect of SPIO upon EPCs remains unclear on a cellular level. In the present study, EPCs were labeled with home-synthesized SPIO nanoparticles in vitro and the biological characteristics of the labeled EPCs were evaluated. The EPCs were isolated from the peripheral blood of New Zealand rabbits and cultured in fibronectin-coated culture flasks. The EPCs were labeled with home-synthesized SPIO nanoparticles at a final iron concentration of 20 µg/ml. Labeled EPCs were confirmed with transmission electron microscopy and Prussian blue staining. The quantity of iron/cell was detected by atomic absorption spectrometry. The membranous antigens of EPCs were detected by cytofluorimetric analysis. Cell viability and proliferative capability between the labeled and unlabeled EPCs were compared. The rabbit EPCs were effectively labeled and the labeling efficiency was approximately 95%. The SPIO nanoparticles were localized in the endosomal vesicles of the EPCs, which were confirmed by transmission electron microscopy. No significant differences were found in cell viability and proliferative capability between labeled and unlabeled EPCs (P>0.05). In conclusion, rabbit peripheral blood EPCs were effectively labeled by home-synthesized SPIO nanoparticles, without influencing their main biological characteristics.

• Expression of podoplanin in salivary gland adenoid cystic carcinoma and its association with distant metastasis and clinical outcomes.

  Authors: He-Ming Wu, Guo-Xin Ren, Li-Zhen Wang, Chun-Ye Zhang, Wan-Tao Chen, Wei Guo

  Molecular medicine reports.

  Distant metastasis is a common cause of mortality in patients with salivary gland adenoid cystic carcinoma (SACC). However, presently, the development of distant metastasis is unable to be predictedDistant metastasis is a common cause of mortality in patients with salivary gland adenoid cystic carcinoma (SACC). However, presently, the development of distant metastasis is unable to be predicted in clinical practice. Recent studies have shown that overexpression of podoplanin is associated with metastasis and survival in patients with several cancer types. The purpose of the present study was to determine whether podoplanin is overexpressed in SACC and whether such overexpression is associated with distant metastasis and survival. Podoplanin expression was determined using immunohistochemistry (IHC) in tumors from 40 SACC patients. The expression status was analyzed in regards to patient clinicopatholo-gical parameters and survival rates. Overexpression of podoplanin was detected in 13 (32.5%) of the 40 tumors. Overexpression was significantly associated with disease-free survival (P=0.025) and distant metastasis (P=0.015), although it was not associated with recurrence and overall survival. In conclusion, podoplanin is overexpressed in a subset of SACCs and may be a biomarker predicting distant metastasis in patients with SACC.

• Effects of acupuncture on the gene expression profile of lung tissue from normal rats.

  Authors: Lei-Miao Yin, Yu Wang, Yan Wang, Yu-Dong Xu, Yan-Yan Liu, Wei-Rong Jin, Qing-Hua Zhang, Yong-Qing Yang

  Molecular medicine reports.

  Acupuncture has been demonstrated to be an effective treatment for various diseases. However, little attention has been paid to its physiological influences, especially on the changes in protein andAcupuncture has been demonstrated to be an effective treatment for various diseases. However, little attention has been paid to its physiological influences, especially on the changes in protein and mRNA levels following acupuncture treatment under normal conditions. In this study, we investigated the gene expression profile of lung tissue from acupuncture-treated normal rats and attempted to characterize the underlying mechanisms of the changes in expression. Three common acupoints, Dazhui (GV14), fengmen (BL12) and feishu (BL13) were selected for analysis, and 2 serial analyses of gene expression (SAGE) tag libraries of the lung tissues that were derived from the normal and acupuncture-treated rats were established. Bioinformatic analyses were carried out using the functional annotation tools of the database for annotation, visualization and integrated discovery (DAVID), the gene ontology (GO) Tree Machine and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. In total, 144 tags were differentially expressed (P<0.05), and the DAVID functional classification of genes demonstrated that the genes were divided into 6 types. Furthermore, GO Tree Machine analysis of the gene categories indicated that 10 enriched GO categories had become enriched after acupuncture, and that 15 KEGG pathways matched the differentially expressed tags of the 2 SAGE libraries. Our results show that the essential effects of acupuncture on normal rats include the regulation of macromolecular biosynthesis, transportation and metabolism. Cellular biosynthesis and cellular lipid metabolism are the common biological processes that occur in response to acupuncture under normal and morbid conditions, which may be the general physiological effects of acupuncture.

• Contactin 1 (CNTN1) expression associates with regional lymph node metastasis and is a novel predictor of prognosis in patients with oral squamous cell carcinoma.

  Authors: He-Ming Wu, Wei Cao, Dongxia Ye, Guo-Xin Ren, Yu-Nong Wu, Wei Guo

  Molecular medicine reports.

  The contactin 1 (CNTN1) gene exerts oncogene‑like activities and its expression has been linked to several human malignancies. In this study, a possible association between CNTN1 expression andThe contactin 1 (CNTN1) gene exerts oncogene‑like activities and its expression has been linked to several human malignancies. In this study, a possible association between CNTN1 expression and clinicopathological parameters and clinical outcomes in patients with oral squamous cell carcinoma (OSCC) was examined. CNTN1 protein expression was evaluated by immunohistochemistry in OSCC tissues of 45 patients. For the immunohistochemical assessment of CNTN1 expression, the cytoplasmic staining labeling index was analyzed using a semiquantitative score. The association between CNTN1 protein levels and clinicopathological factors was analyzed using the Mann-Whitney U test for categorical variables and the Kruskal-Wallis test for continuous variables. The effects of CNTN1 expression on overall and disease-free survival were assessed by using univariate survival analysis. The transcript levels of CNTN1 were detected in OSCC cell lines. In addition, specific siRNA against CNTN1 was applied to investigate the effect exerted by CNTN1 ablation on OSCC cell lines by proliferation and invasion assays in vitro. During follow-up, 16 patients (35.56%) had succumbed to OSCC; the median follow-up of patients was 5.0 years (range, 0.2-8.3). A high expression of CNTN1 was markedly associated with the regional lymph node metastasis of patients with OSCC (P=0.006). CNTN1 expression was significantly associated with overall survival of patients with OSCC (P=0.032; log-rank test) and disease-free survival of patients with OSCC (P=0.038; log-rank test). In addition, CNTN1 ablation notably suppressed the invasion potential of OSCC cell lines, but there was no significant change in the proliferation of OSCC cell lines by CNTN1 knockdown in vitro. The study supports CNTN1 as a novel predictor of regional lymph node metastasis in patients with OSCC and a prognostic marker for OSCC in patients.

• Lentivirally overexpressed T-bet regulates T-helper cell lineage commitment in chronic hepatitis B patients.

  Authors: Xueni Liu, Zhenghao Tang, Yi Zhang, Jianjun Hu, Dan Li, Guoqing Zang, Yongsheng Yu

  Molecular medicine reports.

  Chronic hepatitis B virus (HBV) infection is commonly considered to occur as a result of disturbance of the immune system. T-box expressed in T cells (T-bet) is an essential transcription factor forChronic hepatitis B virus (HBV) infection is commonly considered to occur as a result of disturbance of the immune system. T-box expressed in T cells (T-bet) is an essential transcription factor for T helper (Th) cell differentiation and function. The aim of this study was to investigate the effect of T-bet overexpression on Th cell differentiation and the possible mechanism in chronic hepatitis B (CHB) patients. CD4+ T cells from the peripheral blood of 23 CHB patients, 8 acute hepatitis B (AHB) patients and 10 healthy controls were isolated. T-bet mRNA expression of CD4+ T cells was detected by quantitative real-time polymerase chain reaction (PCR). The T-bet DNA fragment was subcloned into the pGC-FU vector containing GFP to generate a recombinant lentiviral vector, pGC-FU-T-bet, while a no-load pGC-FU vector was used as the negative control. After transduction into CD4+ T cells from another 22 CHB patients, the induction of Th1- and Th2-type cytokines was assayed by an enzyme-linked immunosorbent assay (ELISA), and RT-PCR and western blot analysis were used to measure the mRNA and transcription levels of H2.0-like homeobox (HLX1), GATA-3 and STAT-6. T-bet mRNA expression in CD4+ T cells from AHB patients was enhanced compared with CHB patients and healthy controls. Th1-type cytokines and HLX1 expression was upregulated, while Th2-type cytokines and GATA-3 and STAT-6 expression was repressed after lentiviral introduction of T-bet. In conclusion, lentivirally overexpressed T-bet regulates Th cell lineage commitment in CHB patients, which may be mediated by regulating HLX1, GATA-3 and STAT-6 expression.

• PirB restricts neuronal regeneration in developing rat brain following hypoxia-ischemia.

  Authors: Hua Wang, Ying Xiong, Dezhi Mu

  Molecular medicine reports.

  The inhibitors of axonal regeneration in the myelin sheath are considered to be major contributors to the lack of regeneration in the central nervous system (CNS) following hypoxic-ischemic (HI)The inhibitors of axonal regeneration in the myelin sheath are considered to be major contributors to the lack of regeneration in the central nervous system (CNS) following hypoxic-ischemic (HI) brain damage. As well as the Nogo receptor (NgR), the paired-immunoglobulin-like receptor B (PirB) is a functional receptor for the myelin inhibitors of axonal regeneration. The inhibition of PirB and NgR activities may block most of the inhibitory effects of myelin inhibitors on nerve regeneration. We observed the PirB protein and mRNA expression in HI-damaged rat cortical neurons using immunohistochemistry and reverse transcription-polymerase chain reaction assays. In addition, we treated the HI-damaged rat cortical neurons using PirB antibodies to observe the regeneration of injured neurons. Moreover, the Rock II activity in HI-damaged rat cortical neurons treated with PirB antibodies was observed using western blot analysis. The mRNA and protein levels of PirB increased in newborn rat cortical neurons following HI damage. Treatment with PirB antibodies is able to improve axonal regeneration following HI damage compared with normal axonal growth. Rock II activity also increased in the HI-damaged rat brain. The inhibition of PirB is therefore a potential therapeutic method to promote the regeneration of HI-damaged axons and the inhibitory signal may be transduced through the Rho-ROCK signaling pathway.

• Dihydroartemisinin-induced inhibition of proliferation in BEL-7402 cells: An analysis of the mitochondrial proteome.

  Authors: Jin-Jian Lu, Zhen Yang, De-Zhao Lu, Xing-De Wo, Jia-Jie Shi, Tao-Qi Lin, Mi-Mi Wang, Yi Li, Li-Hua Tang

  Molecular medicine reports.

  Artemisinin, the active ingredient of the Chinese medicinal herb Artemisia annua L., and its derivatives (ARTs) are currently widely used as anti-malarial drugs around the world. In this study, weArtemisinin, the active ingredient of the Chinese medicinal herb Artemisia annua L., and its derivatives (ARTs) are currently widely used as anti-malarial drugs around the world. In this study, we found that dihydroartemisinin (DHA), one of the main active metabolites of ARTs, inhibited the proliferation of human hepatocarcinoma BEL-7402 cells in a concentration-dependent manner. To interpret the mechanisms involved, an analysis of the mitochondrial proteome was performed employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Seven mitochondrial proteins including fumarate hydratase, 60 kDa heat shock protein, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, two subunits of ATP synthase and NADPH:adrenodoxin oxidoreductase were identified to be differentially expressed between the control and DHA-treated groups. Our results indicate that the imbalance of energy metabolism induced by DHA may contribute, at least in part, to its anti-cancer potential in BEL-7402 cells.

• Differential effects of mechanical strain on osteoclastogenesis and osteoclast-related gene expression in RAW264.7 cells.

  Authors: Xiao-Ying Xu, Chun Guo, Yu-Xian Yan, Yong Guo, Rui-Xin Li, Mei Song, Xi-Zheng Zhang

  Molecular medicine reports.

  Mechanical strain plays a critical role in the formation, proliferation and maturation of bone cells. However, little is known about the direct effects of different magnitudes of mechanical strain onMechanical strain plays a critical role in the formation, proliferation and maturation of bone cells. However, little is known about the direct effects of different magnitudes of mechanical strain on osteoclast differentiation. The aim of the present study was to investigate how the fusion and activation of osteoclasts can be regulated by mechanical strain magnitude using the RAW264.7 mouse monocyte/macrophage cell line as an osteoclast precursor. Mechanical strain (substrate stretching) was applied via a 4-point bending system when RAW cells were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL) for an indicated period of time. The numbers of tartrate-resistant acid phosphatase-positive (TRAP+) and apoptotic cells were counted. The expression of TRAP, matrix metalloproteinase-9 (MMP-9), RANK, cathepsin K and carbonic anhydrase II (CAII) was measured by semi-quantitative RT-PCR, and immunocytochemistry staining for RANK was performed. We found that the number of nuclei per osteoclast derived from RAW cells decreased under low magnitude mechanical strain and increased under high magnitude strain within physiological load with an enhanced fusion of TRAP+ osteoclasts, compared to the control with no mechanical strain. The expression of RANK mRNA was downregulated by low magnitude strain and beyond physiological load, while it was upregulated by high magnitude strain within physiological load, correlating with the increased expression of RANK examined by immunocytochemistry, suggesting the mechanical regulation of RANK expression. There was also an increase in the expression of MMP-9 mRNA in the groups subjected to a mechanical strain of 2,000 and 2,500 µε. No significant differences were detected in the expression of TRAP mRNA, cathepsin K and CAII under mechanical strain compared to the control under no strain (0 µε). These findings indicate that low-magnitude strain suppresses osteoclast fusion and activation, while high-magnitude strain within physiological load promotes osteoclast fusion and activation related to a mechanical magnitude-dependent response of RANK expression. These data, therefore, provide a deeper understanding of how different magnitudes of mechanical strains exert their effects on osteoclastogenesis.

• Protection against acute radiation-induced lung injury: A novel role for the anti-angiogenic agent Endostar.

  Authors: Kai Zhang, Shenghui Yang, Ying Zhu, Anwei Mo, Dan Zhang, Li Liu

  Molecular medicine reports.

  Radiotherapy is commonly used to treat thoracic malignancies, but often causes severe lung injury. Currently there are no effective protective strategies against radiation‑induced lung injury (RILI).Radiotherapy is commonly used to treat thoracic malignancies, but often causes severe lung injury. Currently there are no effective protective strategies against radiation‑induced lung injury (RILI). This study aimed to evaluate the ability of an angiogenesis antagonist, Endostar, against RILI, and the underlying mechanism in a mouse model. A total of 108 C57BL/6 female mice were randomized into 4 groups (n=27): i) control group; ii) Endostar group, animals were administered 7.75 ml/kg Endostar through intraperitoneal injection; iii) irradiation group, RILI was induced by exposing the animals to a single external irradiation on the thoraces (6 MV X-ray, 12 Gy); and iv) irradiation plus Endostar group, animals were subjected to Endostar treatment and irradiation as in groups 2 and 3. A total of 3 animals from each of the 4 groups were sacrificed at 1, 6, 12, 24 and 72 h and at 2, 4, 8 and 24 weeks following treatment. Clinical signs and pathology of RILI were examined. The expression of transforming growth factor-β 1 (TGF-β1) in lungs was analyzed by real-time quantitative polymerase chain reaction (RT-QPCR) and immunohistochemistry. Compared with the control group, irradiation induced evident interstitial edema and a significant increase in inflammatory cells in the lungs (P<0.05). Correlated with these changes, a notable increase in TGF-β1 mRNA level and a robust increase in TGF-β1 immunoreactivity were observed in lung tissues in a time-dependent manner following irradiation (P<0.05). Endostar administration effectively attenuated the magnitude of the increase in inflammatory cells as well as the elevation of TGF-β1 expression in lung tissues after RILI (P<0.05). In conclusion, radiation induced an increased expression of the inflammatory mediator TGF-β1 and the associated pathogenesis in the lung, while Endostar was able to at least partially attenuate RILI through downregulating the expression of TGF-β1 in mice. Our findings suggest that Endostar may be a novel protective agent against RILI.

• Correlation between serum H2S and pulmonary function in children with bronchial asthma.

  Authors: Man Tian, Yu Wang, Yue-Qing Lu, Ming Yan, Yan-He Jiang, De-Yu Zhao

  Molecular medicine reports.

  Endogenous hydrogen sulfide (H2S) has generated recent research interest because of its potential function as an inflammatory mediator. Despite its apparent functions in vascular smooth muscle, anEndogenous hydrogen sulfide (H2S) has generated recent research interest because of its potential function as an inflammatory mediator. Despite its apparent functions in vascular smooth muscle, an important player in airway remodeling in asthma, little research has been done to assess the role of H2S in the pathogenesis of asthma. To determine whether serum H2S concentration is correlated with pulmonary function in children with asthma, we measured serum H2S concentration and pulmonary function indices (FVC, FEV1, PEF, FEF25-75, MEF50 and MEF25) in 64 children with asthma and 60 healthy children. Pearson's correlation was used to determine the relationship between serum H2S concentration and lung function parameters. Compared to healthy children, both serum H2S concentration and all lung function parameters were significantly decreased in children with asthma (P<0.05). Furthermore, serum H2S concentration was positively correlated with lung function indices (P<0.05). Thus, decreasing levels of H2S in the serum may be used to indicate decreasing lung function. Further investigation into the causality behind these findings is required.

• SLCO1B1 *15 haplotype is associated with rifampin-induced liver injury.

  Authors: Ling-Min Li, Lei Chen, Guo-Hong Deng, Wen-Ting Tan, Yun-Jie Dan, Rong-Quan Wang, Wen-Sheng Chen

  Molecular medicine reports.

  The organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) plays an important role in the transport of endogenous and xenobiotic compounds, such as bile acids and rifampin. In thisThe organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) plays an important role in the transport of endogenous and xenobiotic compounds, such as bile acids and rifampin. In this study, the association between OATP1B1 polymorphisms and rifampin hepatotoxicity was investigated using integrated population genetic analysis and functional studies. A total of 273 unrelated patients treated with rifampin were recruited. The allele frequencies were examined in patients with drug (rifampin)-induced liver injury (DILI) (n=118) and without (non-DILI) (n=155). Functional analyses were conducted to determine whether the inhibition of bile acids by rifampin was associated with OATP1B1 variants. In the present study, 24 single nucleotide polymorphisms (SNPs) in OATP1B1 were detected in a Chinese population, with two of them causing an amino acid change (rs2306283 and rs4149056). The haplotypes constructed by these two SNPs were OATP1B1 *1a, *1b, *5 and *15, with their respective frequencies being 23.44, 66.30, 0.73 and 9.52% in a total of 273 individuals. The logistic regression analysis indicated that the *15 haplotype was associated with susceptibility to DILI (p=0.03, OR=2.04, 95% CI 1.05-3.96). The frequency of the *15 haplotype in DILI patients was significantly higher than that in non-DILI patients (p=0.03). In the subgroup analysis, the *15 haplotype was associated with susceptibility to cholestatic/mixed injury (p=0.03, OR=2.31, 95% CI 1.06-5.02). Functional assessment of the OATP1B1 *15 haplotype revealed that the activity of bile acid uptake was markedly reduced compared to the three other haplotypes. In the inhibition study, the inhibition by rifampin in the *15 haplotype was greater compared to that in the other haplotypes. These results suggest that the OATP1B1 *15 haplotype is an important predisposing factor for rifampin-induced liver injury.

• Apoptosis-inducing effect of the DR5 monoclonal antibody, D-6, alone or in combination with cisplatin, on A2780 ovarian cancer cells.

  Authors: Qin Jiang, Hong Zhu, Baoquan Liang, Yan Huang, Chunmei Li

  Molecular medicine reports.

  Death receptor 5 (DR5) antibody (D-6) is a monoclonal antibody directed against DR5. The aim of this study was to explore the apoptosis-inducing effects of DR5 (D-6), alone or in combination withDeath receptor 5 (DR5) antibody (D-6) is a monoclonal antibody directed against DR5. The aim of this study was to explore the apoptosis-inducing effects of DR5 (D-6), alone or in combination with cisplatin, on A2780 ovarian cancer cells. The cells were treated with various concentrations of cisplatin and/or DR5 (D-6), or a combination of both. For the control group, the cells were treated only with culture medium. Twenty-four hours after the culture, the morphological changes of each group were observed under an inverted microscope. The cell growth inhibition rates were also analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assays, and the apoptosis was analyzed by flow cytometry. Western blotting analysis was used to detect the intracellular reaction. Our results showed that DR5 monoclonal antibody (D-6) was able to induce the apoptosis and increase the cell growth inhibition rates of ovarian cancer cells in a dose-dependent manner, and the effect was enhanced by cisplatin. There were significant morphological changes, a higher cell growth inhibition rate and apoptosis rate and lesser expression of caspase-3, 8, 9 precursors in the cells treated with both cisplatin and DR5 (D-6). The combination treatment of the DR5 monoclonal antibody (D-6) with cisplatin may be a promising treatment for ovarian cancer.

• Impact of intracerebroventricular obestatin on plasma acyl ghrelin, des-acyl ghrelin and nesfatin-1 levels, and on gastric emptying in rats.

  Authors: Chih-Yen Chen, Wei-Jei Lee, Keong Chong, Shou-Dong Lee, You-Di Liao

  Molecular medicine reports.

  Obestatin, which is a putative 23-amino-acid peptide, is derived from the C-terminal part of the mammalian preproghrelin gene. Nesfatin-1 mRNA is co-expressed with ghrelin in gastric endocrineObestatin, which is a putative 23-amino-acid peptide, is derived from the C-terminal part of the mammalian preproghrelin gene. Nesfatin-1 mRNA is co-expressed with ghrelin in gastric endocrine X/A-like cells; therefore, nesfatin-1 may also interact with preproghrelin gene products in the stomach. In this study, we investigated the impact of obestatin on the plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, and on the gastric emptying of a solid nutrient meal 2 h after an intracerebroventricular (ICV) injection in conscious, fasted rats. The rats were implanted with ICV catheters. Plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, expected to be co-expressed with obestatin, were measured, whereas the human/rat corticotropin-releasing factor (h/rCRF) was applied as an inhibitor of gastric emptying. The ICV administration of obestatin (0.1, 0.3 and 1.0 nmol/rat) did not modify the plasma acyl ghrelin and des-acyl ghrelin levels, the acyl ghrelin/des-acyl ghrelin ratio and nesfatin-1 concentrations. The ICV acute administration of obestatin had no influence on the 2-h rate of gastric emptying of a solid nutrient meal, but the ICV h/rCRF injection delayed it. The weight of food ingested 1 h before ICV injection significantly, but negatively correlated with the gastric emptying of a solid nutrient meal. Our study indicates that the ICV injection of obestatin does not change the 2-h rate of gastric emptying of a solid nutrient meal and the relatively weak interrelationships between ghrelin gene products and nesfatin-1. However, the weight of the ingested food negatively affects the gastric emptying of a solid nutrient meal in conscious, fasted rats.

Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.