MiR-19a, miR-122 and miR-223 are differentially regulated by hepatitis B virus X protein and involve in cell proliferation in hepatoma cells - PubMed (original) (raw)

MiR-19a, miR-122 and miR-223 are differentially regulated by hepatitis B virus X protein and involve in cell proliferation in hepatoma cells

Guifang Yu et al. J Transl Med. 2016.

Abstract

Background: Hepatitis B virus (HBV) X protein (HBx) is a type of oncogenic protein involved in the progression of hepatocellular carcinoma (HCC) via interacting with host genes. Dysregulation of microRNAs (miRNAs) has been observed in HCC. This study aimed to investigate the role of HBx protein in the regulation of miR-19a, miR-122 and miR-223, and examine if these miRNAs involve in progression of malignant hepatocytes.

Methods: Quantitative real time PCR (qRT-PCR) was used to measure the expression of miR-19a, miR-122 and miR-223 in patient samples and in HepG2 cells transfected with HBx or 1.3 fold HBV genome and also in HepG2.2.15 cells, which stably produces HBV. Their target mRNAs and proteins-PTEN, cyclin G1 and c-myc were measured by qRT-PCR and western blot, respectively. The effect of miR-19a, miR-122 and miR-223, and their respective target genes, on cell proliferation was analyzed using 5-ethynyl-2-deoxyuridine incorporation and MTT assay.

Results: MiR-19a showed an up-regulation in HBV-positive HCC patients compared to healthy controls and HBV-negative HCC patients, while miR-122 and miR-223 showed a down-regulation compared to healthy controls, and miR-122 in HBV-positive HCC patients was also down-regulated when compared to HBV-negative HCC patients. MiR-19a was found to be up-regulated in HepG2 cells transfected with HBx or 1.3 fold HBV genome, but down-regulated in HepG2.2.15 cells. MiR-122 and miR-223 were down-regulated in HBx or 1.3 fold HBV transfected HepG2 cells as well as in HepG2.2.15 cell. Their target mRNAs and corresponding proteins-PTEN was down-regulated, while cyclin G1 and c-myc were found to be up-regulated. Modulated expression of miR-19a, miR-122 and miR-223 enhanced cell proliferation of HBx-transfected HepG2 cells, and rescue experiment further showed that their target genes-PTEN, cyclin G1and c-myc involved in cell proliferation of HBx-transfected HepG2 cells.

Conclusions: The expression of miR-19a, miR-122 and miR-223 were differentially regulated by HBx protein, the differential expression of miR-19a, miR-122 and miR-223 plays an important role in cell proliferation of HCC. This study provides new insight into understanding how HBx protein interacts with miRNAs and subsequently regulates host function.

Keywords: Cyclin G1; HepG2; Hepatitis B virus X protein; Hepatocellular carcinoma; PTEN; c-myc; microRNAs.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Expression levels of miR-19a, miR-122, and miR-223 in healthy subjects and patients samples. qRT-PCR analysis of a miR-19a, b miR-122, and c miR-223 from blood serum samples in healthy control, HBV-negative HCC patients and HCC patients with HBV infection. HCC hepatocellular carcinoma, HBV hepatitis B virus. Data represents the mean ± SEM, n = 3, *P < 0.05; **P < 0.01 (One-way ANOVA followed by Bonferroni test)

Fig. 2

Fig. 2

Expression levels of miR-19a, miR-122, and miR-223 in HepG2 cells transfected with HBx or HBV and in HepG2.2.15 cells. a qRT-PCR analysis of miR-19a, miR-122, and miR-223 in HepG2 cells transfected with HBx expressing plasmid pcDNA3.1-HBx or its control vector, pcDNA3.1; b qRT-PCR analysis of miR-19a, miR-122, and miR-223 in HepG2 cells transfected with 1.3 fold HBV expressing plasmid pcDNA3.1-HBV or its control vector, pcDNA3.1; c qRT-PCR analysis of miR-19a, miR-122, and miR-223 in HepG2.2.15 cells and its control, HepG2 cell lines. HBV hepatitis B virus, HBx HBV X protein. Data represents the mean ± SEM, n = 3, *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired t-test)

Fig. 3

Fig. 3

mRNA expression levels of PTEN, cyclin G1 and c-myc in HepG2 cell lines transfected with HBx or HBV and in HepG2.2.15 cells. a mRNA expression levels of PTEN, cyclin G1 and c-myc in HepG2 cells transfected with HBx expression plasmid pcDNA3.1-HBx or its control vector, pcDNA3.1; b mRNA expression levels of PTEN, cyclin G1 and c-myc in HepG2 cells transfected with 1.3 fold HBV expression plasmid pcDNA3.1-HBV or its control vector, pcDNA3.1; c mRNA expression levels of PTEN, cyclin G1 and c-myc in HepG2.2.15 cells and its control, HepG2 cells. HBV hepatitis B virus, HBx HBV X protein. Data represents the mean ± SEM, n = 3, *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired t-test)

Fig. 4

Fig. 4

Protein expression levels of PTEN, cyclin G1 and c-myc in HepG2 cell lines transfected with HBx or 1.3 fold HBV and in HepG2.2.15 cells. a Western blot analysis of PTEN, cyclin G1 and c-myc in HepG2 cells transfected with HBx expression plasmid pcDNA3.1-HBx or its control vector, pcDNA3.1; b Western blot analysis of PTEN, cyclin G1 and c-myc in HepG2 cells transfected with 1.3 fold HBV expression plasmid pcDNA3.1-HBV or its control vector, pcDNA3.1; c Western blot analysis of PTEN, cycling G1 and c-myc in HepG2.2.15 cells and its control, HepG2 cells. HBV hepatitis B virus, HBx HBV X protein. Data represents the mean ± SEM, n = 3, *P < 0.05 (unpaired t-test)

Fig. 5

Fig. 5

Expression levels of miR-19a, miR-122, and miR-223 after silence of HBx in HepG2 cell lines transfected with HBx or HBV and in HepG2.2.15 cells. a qRT-PCR analysis of miR-19a, miR-122, and miR-223 in HepG2 cells co-transfected with pcDNA3.1-HBx and HBx-siRNA or its negative control (NC); b qRT-PCR analysis of miR-19a, miR-122, and miR-223 in HepG2 cells co-transfected with pcDNA3.1-HBV and HBx-siRNA or its negative control (NC); c qRT-PCR analysis of miR-19a, miR-122, and miR-223 in HepG2.2.15 cells and its negative control, HepG2 cells. HBV hepatitis B virus, HBx HBV X protein. Data represents the mean ± SEM, n = 3, *P < 0.05; **P < 0.01 (unpaired t-test)

Fig. 6

Fig. 6

Effect of HBx on the cell proliferation in HepG2 cells. a The cell proliferation ability of hepatoma cells was analyzed using the EdU incorporation assay; b the cell proliferation ability of hetatoma cells was analyzed using MTT assay. HBx HBV X protein. Data represents the mean ± SEM, n = 3, *P < 0.05 (One-way ANOVA followed by Bonferroni test)

Fig. 7

Fig. 7

The role of miR-19a, miR-122, and miR-223 in HBx-mediated growth of HepG2 cells. a The proliferation ability of HepG2-pcDNA3.1-HBx cells was analyzed using the EdU incorporation and MTT assays after miR-19a inhibitor treatment; b the proliferation ability of HepG-pcDNA3.1 cells was analyzed using the EdU incorporation and MTT assays after miR-122 mimics treatment; c the proliferation ability of HepG-pcDNA3.1 cells was analyzed using the EdU incorporation and MTT assays after miR-223 mimics treatment. Data represents the mean ± SEM, n = 3, *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired t-test)

Fig. 8

Fig. 8

The role of PTEN, cyclin G1, and c-myc in HBx-mediated growth of HepG2 cells. The proliferation ability of HepG2-pcDNA3.1-HBx was analyzed using the EdU incorporation assays a after transfection with pcDNA3.1-PTEN or co-transfection with pcDNA3.1-PTEN and miR-19a inhibitor; b after transfection with cyclin G1 siRNA (si-cyclin G1) or co-transfection with pcDNA3.1-cyclin G1 and miR-223 mimics; c after transfection with c-myc siRNA (si-c-myc) or co-transfection with pcDNA3.1-c-myc and miR-122 mimics. Data represents the mean ± SEM, n = 3, *P < 0.05; **P < 0.01 (One-way ANOVA followed by Bonferroni test)

References

    1. McGlynn KA, Petrick JL, London WT. Global epidemiology of hepatocellular carcinoma: an emphasis on demographic and regional variability. Clin Liver Dis. 2015;19:223–238. doi: 10.1016/j.cld.2015.01.001. - DOI - PMC - PubMed
    1. Li Y, Zhang Z, Shi J, Jin L, Wang L, Xu D, Wang FS. Risk factors for naturally-occurring early-onset hepatocellular carcinoma in patients with HBV-associated liver cirrhosis in China. Int J Clin Exp Med. 2015;8:1205–1212. - PMC - PubMed
    1. Zhu M, Guo J, Li W, Xia H, Lu Y, Dong X, Chen Y, Xie X, Fu S, Li M. HBx induced AFP receptor expressed to activate PI3 K/AKT signal to promote expression of Src in liver cells and hepatoma cells. BMC Cancer. 2015;15:362. doi: 10.1186/s12885-015-1384-9. - DOI - PMC - PubMed
    1. Lee YH, Yun Y. HBx protein of hepatitis B virus activates Jak1-STAT signaling. J Biol Chem. 1998;273:25510–25515. doi: 10.1074/jbc.273.39.25510. - DOI - PubMed
    1. Lee YI, Kang-Park S, Do SI, Lee YI. The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J Biol Chem. 2001;276:16969–16977. doi: 10.1074/jbc.M011263200. - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources