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Lin28B over-expression mediates the repression of let-7 by hepatitis B virus X protein in hepatoma cells.

ABSTRACT: The Let-7 microRNA (miRNA) family is frequently downregulated in multiple human tumors, including hepatocellular carcinoma (HCC). Our previous report demonstrated that hepatitis B virus X protein (HBx) suppressed the expression of let-7 in HepG2 hepatoma cells. However, the underlying mechanisms were not elucidated. Lin28B is known to negatively regulate the maturation of let-7, and this prompted us to determine whether HBx acts through Lin28B to suppress let-7. Real-time reverse-transcription polymerase chain reaction (qRT-PCR) was performed to examine let-7 expression before and after treatment with c-Myc-and Lin28B-specific siRNAs in HepG2 cells stably/transiently transfected with HBx. mRNA and protein analyses were employed to determine the correlation of HBx, c-Myc and Lin28B in HCC tissues and cells. Cell cycle and proliferation assays were performed to delineate the consequences of Lin28B repression in HepG2 cells expressing HBx. Lin28B was overexpressed in HBx-transfected cells and HBV-infected liver tissues. HBx-c-Myc-Lin28B axis mediated the repression of let-7 in HepG2 cells. Reduced expression of Lin28B inhibited the growth and cell cycle progression of HepG2 cells by derepressing let-7 and repressing c-Myc. There was not only a preliminary HBx-c-Myc-Lin28B-let-7 pathway but also another possible double-negative feedback loop between c-Myc/Lin28B and let-7 in HepG2 cells transfected with HBx, which together induced the deregulation of let-7. Lin28B has the potential to be a novel molecular target in the treatment of HBV(+) HCC.

SUBMITTER: Wu G

PROVIDER: S-EPMC4658883 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Lin28B over-expression mediates the repression of let-7 by hepatitis B virus X protein in hepatoma cells.

Wu Gang G Huang Pinbo P Ju Xueming X Li Zhixi Z Wang Youyu Y

International journal of clinical and experimental medicine 20150915 9

The Let-7 microRNA (miRNA) family is frequently downregulated in multiple human tumors, including hepatocellular carcinoma (HCC). Our previous report demonstrated that hepatitis B virus X protein (HBx) suppressed the expression of let-7 in HepG2 hepatoma cells. However, the underlying mechanisms were not elucidated. Lin28B is known to negatively regulate the maturation of let-7, and this prompted us to determine whether HBx acts through Lin28B to suppress let-7. Real-time reverse-transcription p ...[more]

PMID: 26628994

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Project description:Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small noncoding RNAs ( approximately 22 nt) that are important regulators of gene expression. Whether and how miRNAs regulate Bach1 or HCV are largely unknown. The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1, and/or HCV gene expression. HCV replicon cell lines (Con1 and 9-13) of the Con1 isolate and J6/JFH1-based HCV cell culture system were used in this study. The effects of miR-196 mimic on Bach1, HMOX1, and HCV RNA, and protein levels were measured by way of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The Dual Glo Luciferase Assay System was used to determine reporter activities. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression and inhibited HCV expression. Dual luciferase reporter assays demonstrated that transfection of miR-196 mimic resulted in a significant decrease in Bach1 3'-untranslated region (UTR)-dependent luciferase activity but not in mutant Bach1 3'-UTR-dependent luciferase activity. Moreover, there was no detectable effect of mutant miR-196 on Bach1 3'-UTR-dependent luciferase activity.miR-196 directly acts on the 3'-UTR of Bach1 messenger RNA and translationally represses the expression of this protein, and up-regulates HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype 1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a role in both HMOX1/Bach1 expression and the regulation of HCV expression in human hepatocytes. Overexpression of miR-196 holds promise as a potential novel strategy to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection.

Project description:BackgroundThe infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear.MethodsFifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting.Resultswe found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while L-02-X and HLE-X cells(stably transfected with pcDNA3.1-HBx) were silenced AFP expression by AFP-siRNA.ConclusionsHBx through stimulating expression of AFP to promote malignant behaviors of human normal liver cells and HCC cells; AFP maybe used as a novel biotarget for therapeutics of HCC patients.

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Project description:In addition to stellate cells and immune cells, inflamed hepatocytes and hepatoma cells express various kinds of chemokines that attract various kinds of immune cells. Previously, we reported that hepatitis B virus (HBV) replication can induce physiological stress. The aim of this study was to analyze the effect of chemokines produced by HBV-infected hepatocytes and hepatoma cells. A real-time PCR array targeting genes related to chemokines and enzyme-linked immunosorbent assay (ELISA) were carried out to detect the specific chemokines produced by Huh7 cells and HepG2 cells infected with various HBV genotypes. A migration assay, flow cytometry analysis, and immunohistochemistry were carried out to analyze the candidate immune cells that can affect the immunopathogenesis of HBV infection. The expressions of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and control cells (mock) (P < 0.05). CD56(+) NK cells and CD8(+) T cells migrated to the hepatoma cells with HBV replication. Moreover, the migration activity of both immune cells was partially cancelled after the treatment of CX3CL1 neutralizing antibody. The expression level of NKG2D on CX3CR1(+) NK cells in HCC with HBV infection was significantly lower than that in hepatocellular carcinoma (HCC) with HCV infection and chronic hepatitis B and C patients (P < 0.05). On the other hand, the frequency of PD-1(high) CX3CR1(+) CD8(+) T cells in HCC with HBV infection was significantly higher than that in HCC with HCV infection and chronic hepatitis B and C (P < 0.05). The expression of CX3CL1 in HBV-replicating hepatocytes and hepatoma cells could contribute to the immunopathogenesis of HBV infection.The progressions of the disease are significantly different among HBV genotypes. However, it has not been clear that how different HBV genotypes could induce different inflammatory responses. Here, we first report that the levels of expression of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and mock. Not only the differential expression of CX3CL1 among the genotypes but also the phenotype of CX3CR1(+) NK cells and T cells were gradually changed during the progression of the disease status. In addition to in vitro study, the analysis of immunohistochemistry with human samples and NOG mice with human lymphocytes and hepatoma cells supports this phenomenon. The quantification of CX3CL1 could contribute to better understanding of the disease status of HBV infection. Moreover, modifying CX3CL1 might induce an immune response appropriate to the disease status of HBV infection.

Project description:UnlabelledHepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis.ImportanceIt is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with the CHC background suggests that FBP1 has an important role in promoting HCV infection and HCC tumors by suppressing p53.

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Lin28B over-expression mediates the repression of let-7 by hepatitis B virus X protein in hepatoma cells.

Publications

Lin28B over-expression mediates the repression of let-7 by hepatitis B virus X protein in hepatoma cells.

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