Study Of PTEN Gene Expression In HCC And The Modulation Of HCV Core Protein On PTEN

PTEN (phosphatase and tensin homolog deleted in chromosome10), a tumor suppressor gene is the first tumor suppressor gene functioned with a dual-specificity lipid and protein phosphatase. PTEN negatively controls the phosphoinositide 3-kinase/AKT signaling pathway involved in the regulation of cell growth and survival in several cell systems. A reduced PTEN function determines a marked increase in PIP3 and activation of AKT survival signaling pathways, leading to inhibition of apoptosis and hyperplasia and contributing to tumor formation. Frequent genetic such as gene mutation and LOH, and epigenetic alterations have been found in a variety of human cancers. In the HCC, PTEN expression is downregulated. But up to now, the mechanism of it has not yet been clearly clarified.A total of 56 cases of HCC tissues were studied for the expression of PTEN by immunohistochemistry. The result demonstrated that the expression level of PTEN protein is much lower in HCC carcinoma tissues than paired surrounding non-carcinoma liver tissues. The reduced expression level of PTEN protein was significantly associated with histological type, tumor stage and intrahepatic metastasis, which provided further evidence that PTEN might serve as a tumor progression marker in HCC.To understand the mechanism explaining loss of PTEN protein expression in 32 of 56 HCC specimens, we first used PCR-SSCP and sequencing method to find any mutation of PTEN gene. But we found no mutations in exon5 and exon8. We revealed PTEN mutations in 5 of 56 HCC cases all in intron4. For the five mutations, three samples were found to be PTEN immunoreaction negative and two specimens were found to be immunoreaction positive. Mutation of PTEN seems to play a minor role in the pathogenesis of hepatocellular carcinomas. Then we identified the frequency of PTEN promoter hypermethylation in HCC samples by MSP assay. Of 56 HCCs, 9 cases for PTEN promoter methylation were detected (16.1%). PTEN methylation was present only in the area with loss of PTEN expression and PTEN methylation was related to histological grade. This suggests that loss of PTEN expression in HCC may be partly due to PTEN methylation and its methylation may play an important role in the development and invasion of HCC.Hepatitis C virus (HCV) is an important causative agent of the acute and chronic hepatitis, which frequently leads to liver cirrhosis and hepatocellular carcinoma. In the case of HCV infection, core protein plays an important role in progression of HCC through modulating apoptosis pathway or regulation of the cell cycle. Though there are many studies for PTEN, the studies of PTEN regulation are rare. So we want to explore how HCV core protein modulate PTEN gene.In this study, we first transfected cDNA encoding EGFP and EGFP-core in the mammalian expression vector pEGFP-C3 into HepG2 cells. By MTT and clone formation, we found cells expressing core protein grew faster than the other two group cells which did not express core protein. By FCM, it was shown that the S phase in core-cells was increased. These results demonstrated that HCV core protein can enhance cell growth and survival. By real time RT-PCR and Western blot, we also found that PTEN gene could be obviously down-regulated both at mRNA and protein level in a dose-dependant manner. In addition, a reduction in PTEN expression by HCV core protein results in the activation of AKT protein. The activation of AKT for cell growth and proliferation could be enhanced through the down-expression of PTEN by HCV core protein in HepG2 cells. Furthermore, we also noticed the activation of NF-κB in cells expressing core protein. After treating cells with PDTC, an inhibitor of NF-κB, we found the activation of NF-κB was decreased associated with the up-expression of PTEN. By Dual-Luciferase Reporter Assay System detection, we found that NF-κB may inhibit the transcriptional activity of PTEN in its promoter region.In conclusion, our results suggest that the level of PTEN protein is altered in part of hepatocellular carcinomas. The downregulation of PTEN expression may not be mainly responsible for the mutation of PTEN, but partly contribute to the promoter methylation and other epigenetic regulation. PTEN methylation may play an important role in the development and invasion of HCC. The growth and proliferation rate of HCV core protein-transfected cells is significantly higher than the others. Also we found PTEN gene could be obviously down-expressed both at mRNA and protein level in a dose-dependant manner by HCV core protein. And the reduction in PTEN expression by HCV core protein results in the activation of AKT protein. Furthermore, we found that HCV core protein can down-express PTEN by activating NF-κB which may inhibit the transcriptional activity of PTEN in its promoter region. Our studies would lay the foundation for completely understanding the tumorigenesis and development of HCC.