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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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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
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fig3: 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

Mentions: To study the mechanism by which miR-216b inhibited tumor cell growth, four computational algorithms, namely, DIANAmT, TargetScan, miRWalk and miRanda, were used to determine the potential target genes of miR-216b. Ideal base pairing was observed between the seed sequence of mature miR-216b and 3′-UTR of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) mRNA; moreover, this seed sequence was highly conserved across species (Figure 3a).


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)

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
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385924&req=5

fig3: 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
Mentions: To study the mechanism by which miR-216b inhibited tumor cell growth, four computational algorithms, namely, DIANAmT, TargetScan, miRWalk and miRanda, were used to determine the potential target genes of miR-216b. Ideal base pairing was observed between the seed sequence of mature miR-216b and 3′-UTR of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) mRNA; moreover, this seed sequence was highly conserved across species (Figure 3a).

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