| The most proteoglycans on the cell surface are heparan sulfate proteoglycans(HSPG). These HSPGs are diverse and can be transmembrane (syndecans), bound by a glycosyl phosphatidylinositol (GPI) linkage to plasma membrane lipid (glypicans), or "part-time" proteoglycans,such as (3-glycan and CD44E. HSPG are abundant cell-surface molecules that consist of a protein core to which heparan sulphate glycosaminoglycan chains are attached. Biochemical studies and cell-culture assays have implicated HSPGs as co-receptors in processes ranging from mechanical support to functions in adhesion, motility, proliferation, differentiation, morphogenesis and carcinogenesis and development. Glypican are a family of HSPGs linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3),may act as an important member.First part: gpc3/mxr7 gene expression in human hepatocellular carcinoma and some other tumorsTo investigated the gpc3/mxr7 gene expression in human HCC, renal cell carcinoma, primary colorectal cancer and normal tissues. Using 32P labeled gpc3/mxr7cDNA as probe, gene expression was detected by Northern blot analysis. gpc3/mxr7 mRNA was expressed in high levels in the placenta, fetal liver, lung, and kidney, but it was undetectable in adult liver and was expressed in low levels in adult lung and kidney. Gpc3/mxr7 mRNA was expressed in high levels in the human hepatocellular carcinoma, but it was undetectable in renal cell carcinoma, primary colorectal cancers.In situ hybridisation analysis demonstrated intense expression of gpc3/mxr7 in hepatic cancer cells with even higher levels in cancer nest.The gpc3/mxr7 mRNA was detected in 58 of 79 (73.41%) primary HCCs but trace levels in the paracarcinomous tissues. Our observations suggest that gpc3/mxr7 gene is regulated developmentally and expressed preferentially in HCC GPC3 mRNA wasdetectable in 42 of 55 AFP-negative HCCs (76.36%,42/55). The expression rate of gpc3/mxr7 mRNA in large tumor (>5 cm) was significantly higher than that in small tumor (<5 cm, 81.36% vs 55.0%, P<0.01).The expression rate of gpc3/mxr7 mRNA in poorly differentiated HCC was significantly higher than that in well differentiated HCC (77.14% vs 44.44%,PO.05).The expression of gpc3/mxr7 mRNA was not correlated with age, sex, HbsAg positivity, fibrocapsule, portal venous embolus and intrahepatic metastasis. No gpc3/mxr7 mRNA was detected in non-hepatocellular carcinoma. gpc3/mxr7 gene was specifically overexpressed in HCC, indicating that gpc3/mxr7 gene might be a new genetic marker of HCC and play an important role in hepatocarcinogenesis.Thus, gpc3/mxr7 may serve as a sensitive tumor marker for early diagnosis of HCC and warrants further study to better understand its biological function.Second part: transcriptional regulation of gpc3/mxr7\n HCC Gpc3/mxr7 was tissue-specific expression, which is limited predominantly to mesodermally derived tissues in early development.To investigate the relationship between methylation patterns of the CpG dinucleotide sites within gpc3/mxr7 promoter in HCC. Methylation status of gpc3/mxr7 promoter were analyzed in HCC tissue by using the southern analysis. Among the analyzed samples, 7 tumors were demethylated, 1 was partially methylated,and the normal liver tissues were hypermethylated.Demethylation of the promoter may be one of the mechanisms leading to the activation ofgpc3/mxr7 gene in HCC. To determine whether gpc3/mxr7 gene is mutated in HCC, PCR-SSCP analysis was performed on genomic in 8 pairs of HCC, we didn't detect mutations in hepatocellular carcinoma. These datas indicated that methylation status of gpc3/mxr7 promoter were important to gene expression and the gene mutation is unclear in hepatocellular carcinoma. Third part: Protein expression and preparation of antibody against GPC3 The study was aimed at obtaining gpc3/mxr7 protein and preparing the anti-GPC3 polyclonal antibody. Firstly,We constructed eukaryotic expressed vector pcDNA3, and gpc3/mxr7 cDNA fused to the 3'end of the gene enc... |
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