Functional analysis of miR-181a and Fas involved in hepatitis B virus-related hepatocellular carcinoma pathogenesis.

The hepatitis B virus (HBV) is responsible for most of hepatocellular carcinoma (HCC). However, whether HBV plays an important role during hepatocarcinogenesis through effecting miRNAs remains unknown. Here, we reported that HBV up-regulated microRNA-181a (miR-181a) by enhancing its promoter activity. Simultaneously, we found that miR-181a inhibited apoptosis in vitro and promoted tumor cell growth in vivo. TNF receptor superfamily member 6 (Fas) was further identified as a target of miR-181a. We also found that Fas could reverse the apoptosis-inhibition effect induced by miR-181a. Moreover, HBV could inhibit cell apoptosis by down-regulating Fas expression, which could be reversed by miR-181a inhibitor. Our data demonstrated that HBV suppressed apoptosis of hepatoma cells by up-regulating miR-181a expression and down-regulating Fas expression, which may provide a new understanding of the mechanism in HBV-related HCC pathogenesis.

Functional analysis of miR-101-3p and Rap1b involved in hepatitis B virus-related hepatocellular carcinoma pathogenesis.

MicroRNA-101(miR-101) has been shown to be down-regulated in hepatocellular carcinoma (HCC). The hepatitis B virus (HBV) is a major risk factor in the development and progression of HCC. However, the correlation between HBV and miR-101 has not yet been fully elucidated. In this study, we reported that HBV could repress miR-101-3p by inhibiting its promoter activity and identified the potential effects of miR-101-3p on some important biological properties of HCC cells by targeting Rap1b. Dual-luciferase reporter assays showed that HBV down-regulated miR-101-3p by inhibiting its promoter activity. Down-regulation of miR-101-3p promoted cell proliferation, migration, and reduced apoptosis, and resulted in up-regulation of Rap1b, while overexpression of miR-101-3p inhibited these processes. Moreover, overexpression of Rap1b was able to reverse the suppressed cell proliferation and migration mediated by miR-101-3p. Our data showed that HBV down-regulated miR-101-3p expression by inhibiting its promoter activity, which resulted in up-regulation of Rap1b, and down-regulation of miR-101-3p or up-regulation of Rap1b promoted proliferation and migration of HCC cells. This provides a new understanding of the mechanism of HBV-related HCC pathogenesis and the potential application of miR-101-3p in cancer therapy.

Up-regulated MicroRNA-181a induces carcinogenesis in hepatitis B virus-related hepatocellular carcinoma by targeting E2F5.

BACKGROUND: Accumulating evidence showed that microRNAs are involved in development and progression of multiple tumors. Recent studies have found that miR-181a were dysregulated in several types of cancers, however, the function of miR-181a in hepatocellular carcinoma (HCC) remains unclear. In this study we assessed the potential association between miR-181a, HBV and HCC. METHODS: The expression of miR-181a in HBV-expressing cells was determined by using qRT-PCR. Dual-Luciferase reporter Assay, qRT-PCR and western blot were performed to investigate the target genes of miR-181a. The effects of miR-181a on HCC proliferation were analyzed by MTS and colony formation assay. Tumor growth assay was used to analyze the effect of miR-181a on tumor formation. RESULTS: HBV up-regulated miR-181a expression by enhancing its promoter activity. Overexpression of miR-181a in hepatoma cells promoted cell growth in vitro and tumor formation in vivo. Conversely, inhibition of miR-181a suppressed the proliferation of HBV-expressing cells. Mechanism investigation revealed that miR-181a inhibited the expression of transcription factor E2F5 by specifically targeting its mRNA 3'UTR. Moreover, E2F5 inhibition induced cell growth and rescued the suppressive effect of miR-181a inhibitor on the proliferation of SMMC-7721 cells. Interestingly, we also discovered that HBV could down-regulate E2F5 expression. CONCLUSIONS: Those results strongly suggested that HBV down-regulated E2F5 expression, in part, by up-regulating the expression of miR-181a. Up-regulation of miR-181a by HBV in hepatoma cells may contribute to the progression of HCC possibly by targeting E2F5, suggesting miR-181a plays important role in HCC development.

Downregulation of miR-101-3p by hepatitis B virus promotes proliferation and migration of hepatocellular carcinoma cells by targeting Rab5a.

RAB GTPase 5A (RAB5A), a member of the Rab subfamily of small GTPases, acts as an oncogene and has been associated with various key cellular functions, including cell growth, differentiation, apoptosis and angiogenesis. Recently, it has been reported that the Rab5a gene is involved in the progression of cancer. Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers, and it is usually associated with persistent hepatitis B virus (HBV) infections. Emerging evidence suggests that HBV alters microRNA (miRNA) expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. Here, we examine how HBV affects the production of miR-101-1, which has been shown to be downregulated in HCC. We found that HBV could repress miR-101-3p by inhibiting its promoter activity. Downregulation of miR-101-3p promoted cancer cell growth and migration, and a specific miR-101-3p inhibitor was able to enhance proliferation and migration. Moreover, we identified Rab5a was one of the target genes of miR-101-3p in HBV-related HCC. Forced expression of miR-101-3p in liver cell lines resulted in a marked reduction of the expression of Rab5a at both the mRNA and protein level by directly targeting the 3'untranslated region of Rab5a. Overexpression of Rab5a resulted in a reversal of the suppression of proliferation and migration of SMMC-7721 cells mediated by miR-101-3p. Taken together, our data show that HBV can downregulate miR-101-3p expression by inhibiting its promoter activity and that downregulation of miR-101-3p promotes HCC cell proliferation and migration by targeting Rab5a. This provides new insights into the mechanisms of HBV-related HCC pathogenesis.

HBx and HBs regulate RhoC expression by upregulating transcription factor Ets-1.

The available evidence suggests that HBV proteins play an important role in the development of hepatocellular carcinoma (HCC). RhoC, a member of the Rho subfamily of the Ras superfamily of homologous genes, had been implicated in tumorigenesis and tumor progression. In a previous study, we demonstrated that HBx and HBs could up-regulate RhoC expression by enhancing its promoter activity. However, the specific mechanisms remain unclear. Here, we demonstrate that overexpression of Ets-1 results in upregulation of RhoC promoter activity and mRNA and protein levels. Expression of transcription factor Ets-1 was significantly higher in HepG2.2.15 cells than that in HepG2 cells. Meanwhile, infection of HepG2 cells with an HBV-adenovirus recombinant virus led to up-regulation of Ets-1. Of the four HBV proteins, HBx and HBs, could increase expression of Ets-1, which consequently contributed to the upregulation of RhoC. These findings might provide a novel insight into HBV-induced HCC metastasis.

Hepatitis B virus regulation of Raf1 promoter activity through activation of transcription factor AP-2α.

The X protein of hepatitis B virus (HBx) is one of the important factors in the development of hepatocellular carcinoma. Raf1 kinase is a central component of many signaling pathways that are involved in normal cell growth and oncogenic transformation. We previously demonstrated that hepatitis B virus regulates Raf1 expression in HepG2.2.15 cells by enhancing its promoter activity and that HBx and HBs might play an important role in this process. However, the underlying molecular mechanisms remain unclear. In this study, we show that nucleotides -209 to -133 of the Raf1 promoter sequence constitute the core region where hepatitis B virus is regulated. This regulation was found to require the involvement of cis-regulatory element AP-2α. We further demonstrated that AP-2α expression was higher in HepG2.2.15 cells (HBV-expressing cells) than in HepG2 cells in vitro. Silencing AP-2α expression by siRNA significantly inhibited the Raf1 promoter activity in HepG2.2.15 cells. These findings indicated that HBV regulates Raf1 promoter activity, possibly through AP-2α.

Hypoxia-inducible lipid droplet-associated (HILPDA) facilitates the malignant phenotype of lung adenocarcinoma cells in vitro through modulating cell cycle pathways.

BACKGROUND: Hypoxia-inducible lipid droplet-associated (HILPDA) is considered to have tumorigenic activity, but its function in lung adenocarcinoma (LUAD) is rarely known. This work aimed to assess the regulatory functions as well as the in-depth mechanism of HILPDA in LUAD. METHODS: The expression of HILPDA in LUAD tissues was analyzed based on TCGA database, and then qRT-PCR was performed to confirm the HILPDA expression in LUAD cell lines. Kaplan-Meier analysis was used to measure the correlation of HILPDA expression and overall survival in patients with LUAD. Then, Cell-Counting Kit-8 (CCK-8), colony formation and transwell assays were performed to detect cell proliferation, invasion and migration. Moreover, the pathways closely related to the high HILPDA expression was analyzed by Kyoto Encyclopedia of genes and Genomes (KEGG) analysis. The levels of Cell cycle pathway-related proteins were assessed using western blotting. RESULTS: Herein, we revealed that HILPDA was expressed at high levels in LUAD tissues and cell lines, and LUAD patients with the higher HILPDA expression presented the shorter survival time. Down-regulation of HILPDA in Calu-3 cells can retard cell proliferation, migration and invasion as well as arrest cells in the G1 phase, whereas overexpression of HILPDA in A549 cells presented a marked promotion on these phenotypes. Moreover, we surveyed that knockdown of HILPDA restrained the activation of cell cycle pathway, while up-regulation of HILPDA led to opponent outcomes. CONCLUSIONS: In summing, HILPDA may act as an oncogenic factor in LUAD cells via modulating cell cycle pathway, which represent a novel biomarker of tumorigenesis in LUAD patients.

LINC00052 regulates the expression of NTRK3 by miR-128 and miR-485-3p to strengthen HCC cells invasion and migration.

Long non-coding RNAs (LncRNAs) are a group of RNAs that are more than 200 nt in length but cannot encode proteins. Accumulating evidences showed that abnormal LncRNA expressions are highly involved in many kinds of tumor. By using gene trap methods which could knockdown gene expression to find important genes, we found one LncRNA which called intergenic non-protein coding RNA 52 (LINC00052) has the ability to inhibit invasion and migration of hepatocarcinoma cells. We found that invasion, migration and proliferation abilities in SMMC7721 cell were inhibited after up-expressing LINC00052. We identified that NTRK3 was the target gene of LINC00052. Down-expression of NTRK3 could increase SMMC7721 cell invasion, migration and proliferation. Meanwhile, we discovered that LINC00052 could regulate NTRK3 expression by forming complementary base pairing with miR-128 and miR-485-3p to reduce the luciferase activity of NTRK3 3'UTR. These results reveal a new mechanism for understanding hepatocarcinoma cells invasion and migration.

Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells.

BACKGROUND: The hepatitis B virus X (HBx) protein has long been recognized as an important transcriptional transactivator of several genes. Human aldo-keto reductase family 1, member C1 (AKR1C1), a member of the family of AKR1CS, is significantly increased in HBx-expressed cells. OBJECTIVES: This study aimed to investigate the possible mechanism of HBx in regulating AKR1C1 expression in HepG2.2.15 cells and the role of AKR1C1 for HBV-induced HCC. MATERIALS AND METHODS: RT-PCR was performed to detect AKR1C1 expression on mRNA level in HepG2 and HepG2.2.15 cell. The promoter activity of AKR1C1 was assayed by transient transfection and Dual-luciferase reporter assay system. The AKR1C1 promoter sequence was screened using the TFSEARCH database and the ALIBABA 2.0 software. The potential transcription factors binding sites were identified using 5' functional deletion analysis and site-directed mutagenesis. RESULTS: In this study, we found that HBx promoted AKR1C1 expression in HepG2.2.15 cells. Knockdown of HBx inhibited AKR1C1 activation. The role of HBx expression in regulating the promoter activity of human AKR1C1 gene was analyzed. The 5'functional deletion analysis identified that the region between -128 and -88 was the minimal promoter region of HBx to activate AKR1C1 gene expression. Site-directed mutagenesis studies suggested that nuclear factor-Y (NF-Y) plays an important role in this HBx-induced AKR1C1 activation. CONCLUSIONS: In HepG2.2.1.5 cell, HBx can promote AKR1C1 promoter activity and thus activates the basal transcription of AKR1C1 gene. This process is mediated by the transcription factor NF-Y. This study explored the mechanism for the regulation of HBV on AKR1C1 expression and has provided a new understanding of HBV-induced HCC.