Upregulated in Hepatitis B virus-associated hepatocellular carcinoma cells, miR-331-3p promotes proliferation of hepatocellular carcinoma cells by targeting ING5

Abstract

Hepatitis B virus (HBV) is a major risk factor for development and progression of hepatocellular carcinoma (HCC). It has been reported that viral infection can interfere with cellular microRNA (miRNA) expression and participate in the pathogenesis of oncogenicity. Our miRNAs array data indicated that miR-331-3p expression in HCC cell lines increased, but the relationship between miR-331-3p expression and HBV activity is unclear. Here, we observed elevated expression of miR-331-3p in different HCC cell lines expressing HBV. HBV, especially HBx, promotes miR-331-3p expression by enhancing its promoter activity. Using a miRNA target prediction database miRBase, we identified ING5 to be a novel target gene of miR-331-3p. miR-331-3p could inhibit ING5 expression by directly targeting its 3'-untranslated region (3'-UTR). As predicted, HBV was confirmed to repress ING5 at both mRNA and protein levels by promoting miR-331-3p expression. Our result indicated that miR-331-3p expression promotes proliferation of SMMC7721 cells by inhibiting ING5. ING5 overexpression promoted cell apoptosis in HCC cell lines. We also found ING5 expression was decreased in tumor tissue of HCC patient with HBV infection compared to its expression in para-carcinoma tissues.

Conclusion: These results showed that miR-331-3p is upregulated by HBV and promotes proliferation of HCC cells though repression of ING5 expression. These data provide new insights for understanding the mechanisms of HBV-related HCC pathogenesis.

Keywords: HBV; HCC; ING5; miR-331-3p; miRNA.

Figure 1. HBV upregulates miR-331-3p expression by enhancing its promoter activity

A–C. Relative miR-331-3p expression in HepG2 and HepG2.2.15 cells (A), SMMC7721 cells transfected with pCH9 or pCH9/3091 plasmids (B) and HepAD38 cells with/without Tet treatment (C) miRNA was normalized to U6 RNA. D. HBsAg and HBeAg expression in HCC cell lines. E. miR-331-3p promoter activity in SMMC7721 cells transfected with pCH9 or pCH9/3091 measured by Dual luciferase reporter analysis. F. miR-331-3p promoter activity in SMMC7721 cells co-transfected with DNAs or RNAs as indicated and measured by Dual luciferase reporter analysis. *P < 0.05, **P < 0.01.

Figure 2. miR-331-3p promotes proliferation of HCC cells

A. Relative miR-331-3p expression in pTARGET-miR-331-3p and pTARGET transfected SMMC7721 cells and effect of miR-331-3p over-expression on cell proliferation measured by MTS assay. B. Relative miR-331-3p expression in miR-331-3p inhibitor or NC transfected SMMC7721 cells and effect of miR-331-3p inhibitor on cell proliferation measured by MTS assay. C. Relative miR-331-3p expression in p-miR-331-3p compares to p-pTARGET stable cell lines, p-miR-331-3p and p-pTARGET cell proliferation were measured by MTS assay. miRNA was normalized to U6 RNA. D. Representative pictures of colony formation assay with p-pTARGET and p-miR-331-3p. Colonies were counted and values are reported as ratios. *P < 0.05, **P < 0.01.

Figure 3. ING5 is a target gene of miR-331-3p

A. Relative gene expression in pCH9 and pCH9/3091 transfected SMMC7721 cells (upper panel) and in HepG2 and HepG2.2.15 cells (lower panel). B. Schematic diagram of predicted miR-331-3p binding site WT or MUT in the ING5 3′-UTR. C. Luciferase reporter assays in SMMC7721 (left panel) or HepG2.2.15 cells (right panel), co-transfected with DNAs or RNAs as indicated. D. ING5 mRNA and protein expression in SMMC7721 cells transfected with pTARGET-miR-331-3p or its control. E. ING5 mRNA and protein expression in SMMC7721 cells transfected with miR-331-3p inhibitor or NC. F. ING5 mRNA and protein expression in p-pTARGET and p-miR-331-3p. β-actin was used as an internal quantitative control. *P < 0.05, **P < 0.01.

Figure 4. HBV represses ING5 expression by upregulating miR-331-3p

A–C. ING5 mRNA and protein expression in HepG2 and HepG2.2.15 cells (A), pCH9 and pCH9/3091 transfected SMMC7721 cells (B) and HepAD38 cells treated with Tet or not (C) β-actin was used as an internal quantitative control. *P < 0.05, **P < 0.01. D. ING5 protein expression in SMMC7721 cells cotransfected with DNAs or RNAs as indicated.

Figure 5. ING5 inhibited HCC cell proliferation

A. ING5 mRNA and protein expression in SMMC7721 cells transfected with pcDNA3.1 or pcDNA3.1-ING5, effect of ING5 overexpression on cell proliferation was measured by MTS assay. B. ING5 mRNA and protein expression in siING5 transfected SMMC7721 cells compared to NC; effect of ING5 silencing on cell proliferation was measured by MTS assay. C. ING5 mRNA and protein expression in p-pcDNA3.1 and p-ING5 stable cell lines. (A-C) β-actin was used as an internal quantitative control. D. p-ING5 and p-pcDNA3.1 cell proliferation measured by MTS and colony formation assays. Colonies were counted and values are reported as ratios. E. ING5 protein expression in SMMC7721 cells co-transfected with DNAs as indicated. MTS assay was used to measure cell proliferation of SMMC7721 cells co-transfected with DNAs as indicated. *P < 0.05, **P < 0.01.

Figure 6. ING5 induces HCC cell apoptosis

A. ING5 stable overexptession HCC cells (p-ING5) apoptosis were analyzed. p-pcDNA3.1 stable HCC cells were used as control. B. p-miR-331-3p, p-pTARGET (control) plasmids were transfected into ING5 stable overexptession HCC cells, apoptosis were analyzed by flow cytometry.

Figure 7. miR-331-3p or ING5 overexpression influences tumor growth in nude mice

A. Representative pictures of tumors formed in p-pTARGET and p-pTARGET-miR-331-3p injected nude mice. B. Tumor growth curve of each group in A. C. Representative pictures of tumors formed in p-pcDNA3.1 and p-ING5 injected nude mice. D. Tumor growth curve of each group in C. *P < 0.05, **P < 0.01. E. Ki-67 and ING5 stained sections of transplanted tumors. Original magnification: 400×.

Figure 8. ING5 is downregulated in HBV-infected HCC tissue

Human HBV-infected HCC tumor and pericarcinous tissue sections were immunohistochemistry staining with Ki-67 and ING5 (magnification 400×).