Study Of HBx Regulate The Expression Of Caveolin-1 And The Molecular Mechanisms Of It In Hepatocellular Carcinoma

Background and objectiveHepatitis B virus (HBV) infection is the major etiological factor of hepatocellular carcinoma (HCC) in China. Most of the HCC patients complicated with HBV infection. HBV can promote the invasion and metastasis of HCC, but the mechanisms are not clear. The protein product encoded by HBV genome, Hepatitis B virus X (HBx) closely related to the development of HCC. One of the mechanisms is that HBx can lead to inactivation of tumor suppressor genes, so that they lose the inhibition of invasion and metastasis of tumor cells.Previous studies of our institute showed that, as well as reported, HBx could activate the oncogene c-Src, but the mechanisms were not clear. The candidate tumor suppressor Caveolin-1 is the major inhibitor of c-Src. We supposed Caveolin-1 played an important role in the mechanisms of HBx activate c-Src. Previous reports showed, DNA methylation was the important role of tumor suppressors down-regulation in carcinoma. methylation of Caveolin-1 was detected in several kinds of tumors. And some viruses including HBV can induce DNA methylation of tumor suppressors and down-regulate the expression of the genes. So, we made a hypothesis: HBx can induce methylation of the promoter of Caveolin-1, sequential reduce the transcriptional activity and gene expression of it. This study focused on this hypothesis.Methods1. We surveyed the expression of Caveolin-1 in 123 cases of HCC tissue with Real-Time PCR and immunohistochemical staining, and correlated these data with the clinical pathological data of the patients. The expression of Caveolin-1 was measured in MHCC-97L and MHCC-97H cell lines that with disparate invasion and metastasis.2. We inserted Caveolin-1 coding domain into adenovirus system. The Caveolin-1 adenovirus then transferred into HepG-2 cells to study the regulation of c-Src activity and tumor invasion and metastasis by Caveolin-1.3. Adenovirus which combined with HBx gene was used to transfer SMMC-7721 cells and to study the regulation of expression of Caveolin-1 by HBx. Then we investigated whether HBx could induce Caveolin-1 methylation by nest-methylation Special PCR (n-MSP) and sequencing. We also investigated the methylation status in HCC tissues that complicated HBV infection.4. There are two SP1 binding sites in Caveolin-1 promoter. As we know, DNA methylation can inhibit the activation of transcription that by SP1. Then we designed series promoters, each contains complete sequence of Caveolin-1 promoter, missing one SP1 binding site from the 5'- flanking region and missing both of the two SP1 binding site. Then we subcloned these DNA fragments into pGL3-basic vectors respectively as promoter-reporters. SMMC-7721 cells were transferred with HBx and the luciferase assay was performed to study the effect of HBx on transcriptional activities of Caveolin-1 promoter, and to make sure the role of SP1 binding site in this procedure.5. DNMT is the main kinase for DNA methylation. We Transfected SMMC-7721 cells with HBx, assayed total DNMT activity and the mRNA expression of DNMT1, DNMT3a and DNMT3b to investigate the effect of HBx on DNMTs . We designed and inserted the promoters of DNMT1, DNMT3a and DNMT3b into pGL3-basic vectors respectively. Luciferase assay was performed to study the effect of HBx on the activities of DNMTs promoters.Results1. The expression of caveolin-1 decreased in HCC tissues significantly compared with adjacent noncancerous tissues. In addition, it was inverse correlate with five clinical factors including tumor size, expression of AFP, major venous-invasion, mono/multi-tumor and pTNM staging which are close to the Prognosis of HCC. Caveolin-1 was hypo-expressed in MHCC-97H significantly compared with MHCC-97L cells. These results showed that the expression of Caveolin-1 was associated with invasion and metastasis of HCC, and low expression of caveolin-1 means poor prognosis. More additionally, we found that the expression of caveolin-1 was inverse correlated with that of HBx. So we supposed caveolin-1 can be down-regulated by HBx protein. 2. To subcloned the adenoviral vector that combined with Caveolin-1 coding domain successfully. Then we transferred it into HepG-2 cell, the target gene over-expressed. The activated c-Src kinase was down-regulated and the invasion & migratory potential decreased with Caveolin-1 transfection.3. Aberrant methylation of Caveolin-1 was observed in 84.8%(28/33) of 33 cases which complicated with HBV infection. The promoter of Cveolin-1 was partially methylated and the expression of the gene was down-regulated with HBx transfection. It was demethylated and the gene expression was recovered after 5-Aza-2'-DC treatment. It showed that DNA methylation is one of the causes of down-regulation of Caveolin-1 by HBx.4. We subcloned a series of DNA fragments, which sequentially knocked out SP1 binding sites from the 5'- flanking region of the promoter of Caveolin-1 into pGL3-basic vector as promoter-reporters successfully. The data of luciferase assay showed, HBx could down-regulate the transcriptional activity of the promoter that with whole sequence. But it did not affect notable on the promoters that without SP1 binding sites. That showed maybe SP1 is the key point in transcriptional regulation of Caveolin-1 and it played an important role in the mechanism of HBx down-regulate Caveolin-1.5. It showed that HBx up-regulated the activity of total DNMT. We subcloned the promoter-reporters of DNMT1, DNMT3a and DNMT3b successfully. The transcriptional activities of DNMT1 and DNMT3a were increased with HBx transfection and so were the expression of mRNA of these two genes, but DNMT3b did not affected by HBx significantly. Therefore, we supposed DNMT1 and DNMT3a were the major catalysis in Caveolin-1 methylation that induced by HBx.ConclusionsCaveolin-1 plays the role of tumor suppressor in HCC. Low expression of caveolin-1 means poor prognosis. HBx could down-regulated the expression of Caveolin-1. The mechanisms were: HBx up-regulated the transcriptional activity of DNMT1 and DNMT3a, and then the CpG island was methylated in the promoter of Caveolin-1. So, the transcriptional activity decreased caused by failure of SP1 protein binding to DNA.