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

Liu FY, Zhou SJ, Deng YL, Zhang ZY, Zhang EL, Wu ZB, Huang ZY, Chen XP - Cell Death Dis (2015)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Surgery, Wuhan Center Hospital, Wuhan, Hubei, China [2] Research Laboratory and Hepatic Surgical Center, Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, HuBei, China.

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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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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fig5: 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)

Mentions: Next, we examined the effects of four HBV proteins on miR-216b by transfecting HBs, HBc, HBp and HBx in HepG2 and SMMC-7721 cells. At 48 h after transfection, only HBx reduced miR-216b and activated IGF2BP2 expression levels (Figure 5a). Furthermore, transfection of different doses of HBx in HepG2 and SMMC-7721 cells for 48 h reduced the levels of miR-216b and pre-miR-216b in a dose-dependent manner (Figure 5b). Taken together, these results indicated that HBx, but not other HBV proteins, reduced miR-216b in HCC cells. Then siHBx was transfected into HepG2.2.15 cells to silence HBx, which increased the levels of miR-216b and pre-miR-216b (P<0.05; Figure 5c). To further investigate the mechanism of miR-216b downregulation by HBx, we examined the effect of HBx on the activity of the putative miR-216b promoter. We found that the activity of the putative miR-216b promoter was reduced in HBx-expressing cells compared with that in control cells (Figure 5d). These results suggested that HBx downregulated miR-216b by regulating its transcription.


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

Liu FY, Zhou SJ, Deng YL, Zhang ZY, Zhang EL, Wu ZB, Huang ZY, Chen XP - Cell Death Dis (2015)

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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385924&req=5

fig5: 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)
Mentions: Next, we examined the effects of four HBV proteins on miR-216b by transfecting HBs, HBc, HBp and HBx in HepG2 and SMMC-7721 cells. At 48 h after transfection, only HBx reduced miR-216b and activated IGF2BP2 expression levels (Figure 5a). Furthermore, transfection of different doses of HBx in HepG2 and SMMC-7721 cells for 48 h reduced the levels of miR-216b and pre-miR-216b in a dose-dependent manner (Figure 5b). Taken together, these results indicated that HBx, but not other HBV proteins, reduced miR-216b in HCC cells. Then siHBx was transfected into HepG2.2.15 cells to silence HBx, which increased the levels of miR-216b and pre-miR-216b (P<0.05; Figure 5c). To further investigate the mechanism of miR-216b downregulation by HBx, we examined the effect of HBx on the activity of the putative miR-216b promoter. We found that the activity of the putative miR-216b promoter was reduced in HBx-expressing cells compared with that in control cells (Figure 5d). These results suggested that HBx downregulated miR-216b by regulating its transcription.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Surgery, Wuhan Center Hospital, Wuhan, Hubei, China [2] Research Laboratory and Hepatic Surgical Center, Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, HuBei, China.

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.

Show MeSH
Related in: MedlinePlus