MiR-216b is involved in pathogenesis and progression of hepatocellular carcinoma through HBx-miR-216b-IGF2BP2 signaling pathway - PubMed (original) (raw)

MiR-216b is involved in pathogenesis and progression of hepatocellular carcinoma through HBx-miR-216b-IGF2BP2 signaling pathway

F-y Liu et al. Cell Death Dis. 2015.

Abstract

This study aims to investigate the expression status of miRNA-216b in familial hepatocellular carcinoma (HCC) and the correlation between miRNA-216b expression and pathogenesis, as well as the progression of HCC. The expression profile of miRNAs in plasma of peripheral blood between HCC patients with HCC family history and healthy volunteers without HCC family history was determined by microarray. Using real-time quantitative PCR to detect the expression in paired tissues from 150 patients with HCC, miR-216b was selected as its expression value in HCC patients was significantly lower compared with healthy volunteers. Next, miR-216b expression and the clinicopathological features of HCC were evaluated. The effect of miR-216b expression on tumor cells was investigated by regulating miR-216b expression in SMMC-7721 and HepG2 in vitro and in vivo. Finally, we explored mRNA targets of miR-216b. In 150 HCC, 37 (75%) tumors showed reduced miR-216b expression comparing with their adjacent liver tissues. The decreased expression of miR-216b was significantly correlated with tumor volume (P=0.044), HBV infection (P=0.026), HBV DNA quantitative (P=0.001) and vascular invasion (P=0.032). The 5-year disease-free survival and overall rates after liver resection in low expression and high expression groups of miR-216b are 62% and 54%, 25% and 20%, respectively. MiR-216b overexpression inhibited cell proliferation, migration and invasion, and miR-216b inhibition did the opposite. The expression of hepatitis B virus x protein (HBx) has tight correlation with downregulation of miR-216b. Furthermore, miR-216b downregulated the expression of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and exerted its tumor-suppressor function through inhibition of protein kinase B and extracellular signal-regulated kinase signaling downstream of IGF2. MiR-216b inhibits cell proliferation, migration and invasion of HCC by regulating IGF2BP2 and it is regulated by HBx.

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Figures

Figure 1

MiR-216b is downregulated in HCC tissues and plasma, direct correlation with prognosis of HCC patients. (a) miRNA array test of the expression levels of all kinds of miRNAs in plasma of HCC patients with HCC family history. (b) RT-PCR analysis of the expression levels of miR-216b between 10 pairs of HCC patients and healthy volunteer's plasma. The miR-216b expression levels in healthy volunteer plasma were much higher than in HCC patients' plasma. (c) RT-PCR analysis of the expression levels of miR-216b in 150 pairs of HCC patients' tissues and contrast the expression value between tumor and adjacent liver tissues. The expression of miR-216b in tumor tissues was significant higher than in adjacent liver. (d and e) After operation, the 5-year cumulative survival rate and disease-free survival rate of low miR-216b expression group is much lower than high expression group, and P<0.05

Figure 2

MiR-216b suppresses cell proliferation both in vitro and in vivo. (a) MiR-216b expression is lowest in HepG2 cells and highest in SMCC-7721 cells. (b) The mimics and inhibitors of miR-216b could significantly affect the expression of miR-216b and the negative control RNAs (NC) had no obvious effect on expression of miR-216b. (c) By CCK-8 assay, the miR-216b upregulated HepG2 cells have smaller value than control cells and Wild Type (WT) cells, and miR-216b downregulated SMCC-7721 cells have larger value than control cells and WT cells. (d) Soft agar colony formation assay shows the suppression role of miR-216b in HCC cells' proliferation. (e and f) MiR-216b attenuated HCC tumor growth in mouse xenograft models. The left panels show tumor formation upon subcutaneous injection of HepG2 cells that were continually transfected with the miR-216b mimics or control vector into nude mice. The right panels show tumor formation upon transplantation of SMCC-7721 cells was continually transfected with the miR-216b inhibitor or control vector into nude mice. (g) Flow cytometry study indicating the apoptotic rates in miR-216b overexpressing cells and control cells. MiR-216b induced apoptosis by regulating the important factors of the extrinsic apoptotic pathway

Figure 3

IGF2BP2 is a direct target of miR-216b. (a) Sequence alignment of miRNAs of the miR-216b with the IGF2BP2 3'-UTR. The seed-recognizing sites in the IGF2BP2 15 matched bases with the seed regions of miR-216b. (b) Luciferase assay on HepG2 cells and SMCC-7721 cells show that miR-216b mimics markedly suppressed luciferase activity in wild-type reporter constructs. MiR-216b inhibitor markedly promoted luciferase activity in wild-type constructs. The data are means ±S.D. (c) By RT-PCR, the level of IGF2BP2 mRNA was not affected by miR-216b or anti-miR-216b transfection. All data are shown as means ±S.D. (d) MiR-216b or anti-miR-216b transfection affects IGF2BP2 protein levels and the expression of IGF2BP2 was negative correlated with the expression of miR-216b. HepG2 cells were transfected with miR-216b mimics or negative control (NC), and SMCC-7721 cells were transfected with miR-216b inhibitor or NC. (e) The CCK-8 assay further showed that the knockdown of IGF2BP2 by si-IGF2BP2 overcame the growth promotion induced by miR-216b inhibitor in SMCC-7721 cells after days 5 or 4 of culture. (f) After 48 h, overexpression of miR-216b suppressed HepG2 cell motility by wound-healing assay, but the overexpression of IGF2BP2 could offset this effect. (g) Overexpression of miR-216b suppressed HepG2 cell invasion by transwell assay, but the overexpression of IGF2BP2 could offset this effect. (h) miR-216b knockdown increased cell invasion in SMCC-7721 cells. All cells were subjected to a Matrigel invasion assay. (i) MiR-216b overexpression reduced the activity of AKT/mTOR and MAPK/ERK pathways in HepG2 cells. Knockdown of miR-216b by inhibitor increased the activity of AKT/mTOR and MAPK/ERK signaling in SMCC-7721 cells. All data are shown as means ±S.D. *P<0.05; **P<0.01

Figure 4

HBx expression correlates with IGF2BP2 expression. (a) With the downregulation of miR-216b expression in 50 tumor tissues, the HBx expression and IGF2BP2 expression are increased. (Protein expression was evaluated by IPP6.0). (b) The HBx and IGF2BP2 expression are upregulated the same time in HCC tumor tissues than in adjacent liver tissues (A, adjacent liver; T, tumor tissue). (c) IGF2BP2 expression is significant higher in HCC tumor tissues with high HBx expression (A1, adjacent liver 1; T1, tumor tissue 1) than in adjacent liver in IHC assay. (d) The overexpression of HBx could obviously downregulate the expression of miR-216b in HepG2 cells (P<0.01). (e) When miR-216b expression is upregulated, the HBx upregulating effects on IGF2BP2 expression and the following pathways are offset. (f) When miR-216b expression is downregulated, the siHBx downregulating effects on IGF2BP2 expression and the following pathways are offset

Figure 5

HBx inhibits p53-mediated activation of miR-216b and upregulates IGF2BP2. (a) RT-PCR assay for miR-216b and western blotting analysis for IGF2BP2 in vector-, HBs-, HBc-, HBp- or HBx-expressing cells. (b) RT-PCR analysis for miR-216b and pre-miR-216b and western blotting analysis for IGF2BP2 in two HCC cell lines, HepG2 and SMMC-7721, transfected with HBx at different doses and controls. (c) RT-PCR analysis for miR-216b and pre-miR-216b and western blotting analysis for IGF2BP2 in HepG2 and HepG2.2.15 cells with or without HBx inhibition. (d) Dual-luciferase assay of the putative miR-216b promoter in HepG2 and SMMC-7721 cells transfected with HBx and controls. (d) HepG2 cells were transfected with p53 or p53 mutant (R249S) and analyzed for miR-216b expression by real-time RT-PCR and for IGF2BP2 expression by western blotting. (e and f) HepG2 cells were transfected with HBx and p53 siRNAs or control siRNA and analyzed as in a. (g) ChIP analysis showed that HBx inhibited p53 occupying on the putative miR-216b promoter in HepG2 cells. The data represent the mean±S.D. (*P<0.05)

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MiR-216b is involved in pathogenesis and progression of hepatocellular carcinoma through HBx-miR-216b-IGF2BP2 signaling pathway - PubMed (original) (raw)