Research On The Mechanism Of Hepatitis B Virus X Protein In Hepatocellular Carcinoma By Regulating Of MicroRNA

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Epidemiological studies have shown that millions of people are chronically infected with hepatitis B virus (HBV), and an abundance of evidence has proved that HBV infection is one of the important causes of HCC, given that the incidence of HCC in chronically HBV infected individuals is approximately 100-fold higher than in the uninfected population. The HBV X protein (HBx), a protein encoded by HBV, is thought to play a key role in the molecular pathogenesis of initiation, development and metastasis of HBV related HCC. Since there are few ideal animal models that can be used to demonstrate the role of HBx in HCC development, the molecular mechanisms underlying HBx protein-mediated tumorigenesis are not fully understood. Fortunately, a p21-HBx transgenic mouse model has been established by introducing the HBx gene into the p21 locus. In this model established by Wang Y et al., none of mice homozygously null for p21 over a 2-year period developed HCC, suggesting that p21 deficiency does not directly increase the susceptibility to HCC. Furthermore, in this model, HBx gene is driven by its own promoter (PGK promoter, strong promoter) which could express persistently for at least 24 months in the liver. HCCs often develop when the transgenic mice are 18 months of age. What's more, some of the transgenic mice can even develop to metastatic tumors. Thus, the HBx transgenic mouse model is an ideal model for screening for possible mechanisms by which HBx leads to the initiation/development/metastasis of HCC.MicroRNAs (miRNAs) are 21-23 nt long non-coding RNA sequences that are involved in various biological processes, including cell proliferation, cell death, stress resistance, and tumorigenesis. Recent studies indicate that miRNAs may play a role in several cancers. Altered miRNA levels can result in aberrant expression of gene products that may contribute to cancer biology, which suggests that expression profiling of miRNAs may significantly contribute to cancer development. Furthermore, it has also been shown that miRNA expression patterns have relevance to the biological and clinical behavior of human cancers. These and other data demonstrate that miRNAs play a substantial role in the pathogenesis of cancers, and that expression profiling of miRNAs can be used as potential diagnostic/prognostic markers. However, to date very limited information has been published about miRNAs that are specifically involved in the initiation/development/metastasis of HBx-related HCC. The HBx protein is a viral transcriptional transactivator. It can modulate many host cell functions through its integrations with a variety of host factors (such as AP-1, AP-2, NF-κB, ATF2, CREB). The presence of HBx gene fragments at many different locations within the host DNA can result in genetic instabilities. Genetic instabilities associated with integrations potentially alter the expression of oncogenes, tumor suppressor genes, and miRNAs that may contribute to tumorigenesis. This strongly indicates that although research on known genes and proteins has already yielded useful information, new discoveries about miRNA may also build insights into the initiation/development/metastasis of HCC in the context of HBx. Discovery of unique new target molecules is of paramount importance and will be essential if we are to understand the mechanisms and improve the prognoses associated with hepatic cancers as early as possible, therapeutic intervention and prevent from tumor invasion/metastasis and postsurgical recurrence. The result should be an increase in the likelihood of successful treatment.Based on these findings, we screened candidate specific miRNA molecules in the livers of p21-HBx transgenic mice compared with wide-type mice, using real-time PCR in order to elucidate the possible underlying mechanisms which lead from chronic HBV infection to HCC. The functional effects of these miRNAs were then examined to elucidate the possible mechanisms of HCC. Finally, liver samples of HCC patients were analyzed to validate the expression patterns of the unique miRNAs. The results demonstrated that five upregulated miRNAs including miR-212, miR-23a, miR-155, miR-17-5p and miR-20a and two downregulated miRNAs including miR-152 and miR-200a that were selectively expressed in HBx-expressing livers of p21-HBx transgenic mice. The expression levels of these unique miRNAs were corrected with the expression of HBx, and displayed the mode of dose-dependent. The five upregulated and two downregulated miRNAs were identified by in vitro analysis as being involved in regulating cell apoptosis, proliferation and migration. Overexpression of miR-212, miR-23a, miR-155, miR-17-5p and miR-20a can significantly reduced cell apoptosis, while overexpression of miR-152 and miR-200a can induced the cell apoptosis. Overexpression of miR-212, miR-17-5p and miR-20a can significantly increase cell proliferation, but overexpression of miR-152 and miR-200a can reduced the cell proliferation. Furthermore, overexpression of miR-212, miR-23a, miR-17-5p and miR-20a can significantly increase cell migration, but overexpression of miR-152 and miR-200a can reduced the cell migration. Our results were validated in ectopic HBx-expressing HCC patients. Our data strongly suggest that these unique miRNAs be involved in tumorigenesis in the context of HBx, and thus may be useful in the prognosis of HBV-related HCC of tumor metastasis in HBV-HCC.Meanwhile, we have found that miR-143 was significantly down-regulated in livers of ten-month old transgenic mice while dramatically up-regulated after the HCC developed in the transgenic mice especially when accompanied by tumor metastasis to the lung. Next, we use the HBV-HCC patients to validate the results in humans, and also find that miR-143 was dramatically up-regulated after the HCC developed in HBV-HCC patients, especially when accompanied by tumor metastasis. Functional study performed in vitro demonstrated that miR-143 has no role in regulating cell proliferation and apoptosis, but has signifigant affect on cell migration. Furthermore, we have found that miR-143 is directly regulated by the transcription factor NF-κB, and its direct target gene is FNDC3B (fibronectin type III domain containing 3B). The study in vitro and in vivo we performed has demonstrated that upregulated miRNA-143 induced by NF-κB enhances hepatocarcinoma metastasis by repression of fibronectin. And the novel HBx/NF-κB/ miR-143/ FNDC3B pathway is an important complement to the network.Altered expression profling of miRNA mediated by HBx may sheds new insight into elucidating the molecular mechanism of the development and progression of HBV-HCC and a target for early diagnose, therapy and inhibiting tumor metastasis and recurrence of HCC.