[Show abstract][Hide abstract] ABSTRACT: A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.
BioMed research international. 01/2014; 2014:458457.
[Show abstract][Hide abstract] ABSTRACT: Oral squamous cell carcinoma (OSCC) is a common and lethal malignancy. Thus, improvement in current knowledge of molecular changes associated with OSCC is urgently needed to explore novel avenues of diagnostics and treatment of this disease. While aberrant expression of long non‑coding RNAs (lncRNAs) has been functionally associated with certain types of cancer, including lung, breast and prostate carcinomas, their expression pattern and biological relevance in OSCC is currently unknown. In the present study, the relative abundance of a collection of lncRNAs in tissue or saliva samples from OSCC patients was investigated. It was shown that subsets of lncRNAs are expressed across non‑tumor, tumor and metastatic tissue samples. Some detected lncRNAs were shown to be aberrantly expressed in cases of oral cancer and metastasis. Moreover, whole saliva contained a detectable amount of some lncRNAs, which appeared to be potential markers for OSCC. These findings suggest that the detection of lncRNAs in saliva may be used as a noninvasive and rapid diagnostic tool for the diagnosis of oral cancer.
Molecular Medicine Reports 12/2012; · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The expression of AKAP12 (A Kinase anchoring protein 12) is markedly reduced in a variety of cancers. The purpose of this study was to establish a methylation-sensitive high resolution melting (MS-HRM) assay for the quantitative detection of AKAP12 promoter methylation and expression and the association with clinicopathological variables in human colorectal cancer. We also assessed the effect of AKAP12 re-expression on cell growth and colony formation.
Downregulation or loss of AKAP12 mRNA expression was detected in 31 of 45 tissue samples (68.9%). No significant correlation was observed between the reduced expression levels and patient age, gender, Duke's stage or tumor differentiation. Methylation (>1%) of the AKAP12 promoter region was present in 35 of 45 (77.8%) carcinoma tissue samples and 6 of 45 (13.3%) adjacent tissue samples. AKAP12 methylation was significantly higher in the colorectal cancer tissues exhibiting advanced Duke's stages. Treatment of the three colorectal carcinoma cell lines (LoVo, COLO320 and SW480) with completely methylated AKAP12 with inhibitors of DNA methyltransferase (5-Aza-2'-deoxycytidine) markedly increased expression of AKAP12 and decreased methylation levels. Ectopic expression of AKAP12 in the LoVo cell line suppressed cell growth and inhibited colony formation.
The AKAP12 gene was examined by quantitative RT-PCR, MS-HRM analysis and bisulfite sequencing in 45 paired tissue samples obtained from primary colorectal carcinomas and the corresponding adjacent tissues. In addition, five colorectal carcinoma cell lines (LoVo, COLO205, SW480, LS174T and COLO320) were investigated and western blot analysis was used to investigate changes in protein expression. A proliferation assay and soft agar assay were performed after overexpression of AKAP12.
Our study demonstrated that MS-HRM is a robust, fast and sensitive method for AKAP12 methylation analysis. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of this cancer.
Cancer biology & therapy 06/2010; 9(11):862-71. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers.
We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines.
In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p=0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation.
We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer.
Clinica chimica acta; international journal of clinical chemistry 03/2010; 411(13-14):940-6. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Promoter methylation is an important pathway in transcriptional silencing of tumor suppressor genes (TSGs) in brain tumors. The identified 3p21.3 tumor suppressor gene RAS association domain family protein 1A (RASSF1A) is highly methylated in primary lung, breast and other tumors. We investigated the promoter methylation and gene expression of RASSF1A in gliomas.
The methylation status of the promoter region of RASSF1A, p16INK4A and death-associated protein kinase (DAPK) genes was analyzed by methylation-specific PCR (MSP) in 41 surgically resected gliomas. RASSF1A expression was also detected by reverse-transcription polymerase chain reaction (RT-PCR) in 28 glioma tissues.
The frequencies of RASSF1A, p16INK4A and DAPK promoter methylation were 13/41 (31.7%), 3/41 (7.3%) and 6/41 (14.6%) respectively. However, the methylations of those genes were not correlated with the clinical characteristics of patients (tumor grade, tumor types and sex). Among 28 glioma tissues, 6 showed the loss of the gene (21.4%). Promoter hypermethylation of RASSF1A is associated with loss of gene expression in glioma tissues. (p=0.022).
Our results suggested that the RASSF1A gene might play an important role in glioma carcinogenesis. It also gives us an insight for future glioma medical therapy with a demethylating agent.
[Show abstract][Hide abstract] ABSTRACT: To investigate the changes of sugar chain structures of alkaline phosphatase (ALP) in hepatoma tissue and its relation to the invasiveness of hepatocellular carcinoma (HCC).
The binding ratios of ALP from 9 normal liver tissues, 16 hepatoma tissues and 16 noncancerous tissues surrounding hepatoma were analysed by affinity chromatography on various lectin columns including leukoagglutinating phytohemagglutinin (L-PHA), lentil lectin (LCA), Datura stramonium agglutinin (DSA), erythroagglutinating phytohemagglutinin (E-PHA) and Sambucus nigra bark agglutinin (SNA).
The binding ratios of ALP on L-PHA (22.94%+/-5.30%), DSA (55.97%+/-13.72%), LCA (38.16%+/-8.87%), E-PHA (11.56%+/-4.81%) and SNA (69.80%+/-13.71%) in HCC tissues were significantly increased (P<0.01) compared with that in normal liver tissues (L-PHA 5.89%+/-2.75%, DSA 36.20%+/-11.58%, LCA 17.90%+/-6.71%, E-PHA 5.38%+/-2.20%, SNA 57.32%+/-11.27%), respectively. t values between the two groups were 8.94, 3.64, 5.94, 3.62 and 2.32, respectively. L-PHA-binding ratio (25.84%+/-4.67%) of ALP in HCC with invasiveness was significantly higher than that (18.10%+/-3.64%) without invasiveness (t=3.71, P<0.01).
The changes of ALP sugar chain structures occur in HCC tissue. b1-6 branching sugar chain structure of ALP is related to the invasiveness of HCC.
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology 12/2003; 11(12):739-41.
[Show abstract][Hide abstract] ABSTRACT: Pigment epithelium derived factor (PEDF) was first isolated from medium conditioned by human fetal retinal pigment epithelial cells. PEDF was detected in a broad range of human fetal and adult tissues including almost all brain areas. It can also inhibit the proliferation of cultured rat astrocytes. Recent studies have implicated PEDF in activities that are inhibitory to angiogenesis.
To investigate the expression of PEDF in gliomas to assess its "gliastatic" effects and its role in anti-angiogenesis.
PEDF mRNA values were measured by quantitative real time reverse transcription polymerase chain reaction (RT-PCR) analysis of normal brain tissue and tumour specimens from both low and high grade gliomas. In addition, immunohistochemical staining for PEDF and vascular endothelial growth factor (VEGF) was performed on 32 paraffin wax embedded glioma samples, 10 of them grade IV, 10 grade III, seven grade II, and five grade I.
RT-PCR showed that PEDF mRNA values were 5.0 (p < 0.001) and 15.4 (p < 0.001) times higher in normal human brain specimens (n = 5) than in tumour tissue specimens of low grade glioma (grades I and II; n = 15) and high grade glioma (grades III and IV; n = 10), respectively. VEGF was strongly positive in 90% of grade IV, 70% of grade III, 43% of grade II, and 20% of grade I cases. In contrast, PEDF was positive in none of grade IV, 20% of grade III, 43% of grade II, and 60% of grade I tumours. There was an inverse correlation between VEGF and PEDF expression, and a lack of PEDF in advanced grade gliomas.
It is possible that the absence of PEDF expression is a potent factor for the enhancement of angiogenesis in glioma.
Journal of Clinical Pathology 04/2003; 56(4):277-82. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipocalin-type prostaglandin D synthase (LPGDS; PGH(2)D-isomerase; EC 184.108.40.206) is a bifunctional protein first identified in the mammalian brain. It acts as a PGD(2)-producing enzyme and a retinoid transporter. Recent studies have shown that LPGDS is anomalously expressed in ovarian tumors and that retinoid may have a role as an ovarian cancer chemotherapeutic agent. To determine whether there is a relationship between retinoid and LPGDS in ovarian tumors, we examined the regulation of the gene encoding LPGDS by all-trans retinoic acid (RA). Real-time quantitative RT-PCR analysis showed that RA strongly induced the accumulation of LPGDS mRNA in human 3AO ovarian cancer cells. Furthermore, treatment of the cells with RA induced the synthesis and secretion of LPGDS into the culture medium. This increased expression of LPGDS was accompanied by an inhibition of cell proliferation in the ovarian cancer cells. Prostaglandin D synthase, ovarian cancer, retinoic acid, real-time quantitative RT-PCR.
Cell Biology International 02/2003; 27(7):587-92. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.
Protein Expression and Purification 12/2002; 26(2):229-34. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF expression appears to be a marker for malignant angiogenesis in human solid tumors. However TF expression and its relationship to angiogenesis and tumor progression in human glioma are still unclear.
Quantitative reverse transcription PCR and immunofluorescence were performed on the U251 glioma cell line, 5 normal brain specimens, and 34 glioma surgical specimens. Of the gliomas, 10 were grade IV, 12 grade III, 7 grade II, and 5 grade I. Microvessels were highlighted using a monoclonal antibody specific to human von Willebrand factor.
TF was strongly positive in 90% of the grade IV cases, 58% of grade III, 43% of grade II, and 20% of grade I. TF staining was not present in any normal brain specimens. The mean level of TF mRNA in normal brain tissue was 5.0 x 10(3) copies/microg RNA. Among the gliomas, TF mRNA ranged from 1.7 x 10(5) to 6.8 x 10(7) copies/microg, with a mean of 4.6 x 10(6). TF expression was highest in glioblastomas that showed greatest vascularity.
These findings support a role for TF in angiogenesis in glioma. TF is expressed in glioma and the level of expression correlates with the histologic grade of malignancy and vascularity.
[Show abstract][Hide abstract] ABSTRACT: Initiation of the coagulation serine protease cascade in mammalian cells is mediated by tissue factor (TF), which is a cell surface receptor and cofactor for coagulation factor VII (FVII) and its activated form FVII (FVIIa). Increasing evidence suggests that TF is expressed in a wide range of cancer cells and plays important roles in cancer progression and metastasis. In this study, we investigated the association between the expression level of TF transcript and histologic features of glioma.
RNA was extracted from normal brain tissues and glioma tissues. We developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify TF gene expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the mRNA level in human glioma.
The dynamic range of the assay was 10(3)-10(8) copy/microg RNA. The relationship between Ct and log starting concentration was linear (r2 > or = 0.99). The mean expression of TF in healthy brain tissue was 6.2 x 10(3) copy/microg RNA. Overexpression of TF was found in 42 brain glioma samples, mean value is 2.9 x 10(6) copy/microg RNA.
TF mRNA transcript is expressed in glioma and the level of expression correlates with histologic grade of malignancy. This new simple, rapid, semiautomated assay is a major alternative to Northern blot and competitive quantitative PCR for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and support future TF-based clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Lipocalin-type prostaglandin D synthase (L-PGDS) has recently been shown to be expressed in human brain tumors and breast tumors. However, L-PGDS expression has not been investigated in ovarian cancer. The objective of this study was to determine whether L-PGDS is expressed in human ovarian cancer. Lipocalin prostaglandin D synthase mRNA was cloned and sequenced by RT-PCR. Using in situ hybridization (ISH) technique, the expression of L-PGDS mRNA in 54 ovarian cancer was investigated. Expression of L-PGDS mRNA was found in tumor cells of all various types of ovarian cancers. Patterns of staining of tumor cells varied among different histological types of ovarian cancer. Significant discrepancy between the intensity of the staining and histological types of ovarian cancer could be established (p<0.01). It is reported for the first time that the expression of mRNA of L-PGDS exists in the ovarian cancer, and is related to the cancer type. This may have significance for the progress of ovarian cancer.
Clinical Chemistry and Laboratory Medicine 12/2001; 39(12):1198-203. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The sugar-chain structures of many glycoproteins are altered in the hepatoma tissues. The molecular mechanism by which these alterations occur remains largely unknown. Methods: Messeger RNA expression of N-acetylglucosaminyltransferase (GlcNAcT-V), N-acetylglucosaminyltransferase III (GlcNAcT-III) and α1–6 fucosyltransferase (α1–6 FucT), were investigated in normal liver tissues samples (n=7), primary hepatic cancer (PHC) tissues (n=15) and noncancerous tissues surrounding PHC (n=15) by reverse transcriptase-polymerase chain reaction (RT-PCR). Results: The mRNA expression of the three glycosyltransferases in PHC tissues were enhanced by 3- to 10-fold in comparison with that in normal liver tissues. There were significantly higher mRNA expressions of GlcNAcT-V in invasive PHC than non-invasive. Conclusions: The change of glycoprotein sugar-chain structures in PHC may be related to the abnormal mRNA expression of some glycosyltransferases and the levels of mRNA expression of GlcNAcT-V associated with PHC invasiveness.