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MicroRNA-212 inhibits hepatocellular carcinoma cell proliferation and induces apoptosis by targeting FOXA1.

Tu H, Wei G, Cai Q, Chen X, Sun Z, Cheng C, Zhang L, Feng Y, Zhou H, Zhou B, Zeng T - Onco Targets Ther (2015)

Bottom Line: In this study, the miR-212 expression was found to be obviously downregulated in hepatocellular carcinoma (HCC) tissues as compared with adjacent nontumor tissues.Interestingly, we found that upregulation of miR-212 decreased FOXA1 expression in HepG2 cells.In conclusion, miR-212 is a potent prognostic marker and may suppress HCC tumor growth by inhibiting FOXA1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Hubei University of Medicine, Shiyan, People's Republic of China.

ABSTRACT
MircroRNA-212 (miR-212) is proposed as a novel tumor-related miRNA and has been found to be significantly deregulated in human cancers. In this study, the miR-212 expression was found to be obviously downregulated in hepatocellular carcinoma (HCC) tissues as compared with adjacent nontumor tissues. Clinical association analysis indicated that low expression of miR-212 was prominently correlated with poor prognostic features of HCC, including high AFP level, large tumor size, high Edmondson-Steiner grading, and advanced tumor-node-metastasis tumor stage. Furthermore, the miR-212 expression was an independent prognostic marker for predicting both 5-year overall survival and disease-free survival of HCC patients. Our in vitro studies showed that upregulation of miR-212 inhibited cell proliferation and induced apoptosis in HepG2 cells. On the contrary, downregulation of miR-212 promoted cell proliferation and suppressed apoptosis in Huh7 cells. Interestingly, we found that upregulation of miR-212 decreased FOXA1 expression in HepG2 cells. Significantly, FOXA1 was identified as a direct target of miR-212 in HCC. FOXA1 was downregulated in HCC tissues as compared with noncancerous tissues. An inverse correlation between FOXA1 and miR-212 expression was observed in HCC tissues. Notably, FOXA1 knockdown inhibited cell proliferation and induced apoptosis in HepG2 cells. In conclusion, miR-212 is a potent prognostic marker and may suppress HCC tumor growth by inhibiting FOXA1 expression.

No MeSH data available.


Related in: MedlinePlus

FOXA1 is identified as a functional target of miR-212 in HCC.Notes: (A) qRT-PCR and (B) Western blot analysis of FOXA1 expression in HepG2 cells with miR-212 or control vectors transfection. n=3 independent experiments, *P<0.05. (C) miR-212 and its putative binding sequence in the 3′-UTR of FOXA1. The mutant miR-212 binding site was generated in the complementary site for the seed region of miR-212 (wt, wild type; mt, mutant type). (D) miR-212 significantly suppressed the luciferase activity that carried wt but not mt 3′-UTR of FOXA1. Anti-miR-212 led to a noticeable increase in luciferase activity of wt 3′-UTR of FOXA1. n=3 repeats with similar results, *P<0.05.Abbreviations: FOXA1, forkhead box protein A1; HCC, hepatocellular carcinoma; miR-212, microRNA 212; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; UTR, untranslated region; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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f3-ott-8-2227: FOXA1 is identified as a functional target of miR-212 in HCC.Notes: (A) qRT-PCR and (B) Western blot analysis of FOXA1 expression in HepG2 cells with miR-212 or control vectors transfection. n=3 independent experiments, *P<0.05. (C) miR-212 and its putative binding sequence in the 3′-UTR of FOXA1. The mutant miR-212 binding site was generated in the complementary site for the seed region of miR-212 (wt, wild type; mt, mutant type). (D) miR-212 significantly suppressed the luciferase activity that carried wt but not mt 3′-UTR of FOXA1. Anti-miR-212 led to a noticeable increase in luciferase activity of wt 3′-UTR of FOXA1. n=3 repeats with similar results, *P<0.05.Abbreviations: FOXA1, forkhead box protein A1; HCC, hepatocellular carcinoma; miR-212, microRNA 212; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; UTR, untranslated region; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Mentions: To disclose the molecular mechanisms by which miR-212 inhibits HCC tumor growth, predicted target genes of miR-212 were retrieved and analyzed using publicly available databases (TargetScan 6.2 and MiRanda). FOXA1, an important transcription factor in liver development and cancer progression,16 was predicted as one of the targets of miR-212. As measured by qRT-PCR and Western blot, both the levels of FOXA1 mRNA and protein were significantly reduced by upregulation of miR-212 in HepG2 cells (P<0.05, respectively, Figure 3A and B). Next, we investigated whether the miR-212 directly interacted with the 3′-UTR of FOXA1 mRNA using a Dual-Luciferase® Reporter Assay System. As expected, miR-212 significantly inhibited the luciferase activity of FOXA1 containing a wild-type (wt) 3′-UTR but did not suppress the activity of FOXA1 with a mutant (mt) 3′-UTR (P<0.05, Figure 3C and D). When anti-miR-212 was transfected, an increase in luciferase activity of wt FOXA1 3′-UTR was observed. However, with the mt FOXA1 3′-UTR constructs, there was no relative increase in activity (P<0.05, Figure 3C and D). Thus, our data strongly suggest that FOXA1 is a target of miR-212 in HCC.


MicroRNA-212 inhibits hepatocellular carcinoma cell proliferation and induces apoptosis by targeting FOXA1.

Tu H, Wei G, Cai Q, Chen X, Sun Z, Cheng C, Zhang L, Feng Y, Zhou H, Zhou B, Zeng T - Onco Targets Ther (2015)

FOXA1 is identified as a functional target of miR-212 in HCC.Notes: (A) qRT-PCR and (B) Western blot analysis of FOXA1 expression in HepG2 cells with miR-212 or control vectors transfection. n=3 independent experiments, *P<0.05. (C) miR-212 and its putative binding sequence in the 3′-UTR of FOXA1. The mutant miR-212 binding site was generated in the complementary site for the seed region of miR-212 (wt, wild type; mt, mutant type). (D) miR-212 significantly suppressed the luciferase activity that carried wt but not mt 3′-UTR of FOXA1. Anti-miR-212 led to a noticeable increase in luciferase activity of wt 3′-UTR of FOXA1. n=3 repeats with similar results, *P<0.05.Abbreviations: FOXA1, forkhead box protein A1; HCC, hepatocellular carcinoma; miR-212, microRNA 212; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; UTR, untranslated region; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556035&req=5

f3-ott-8-2227: FOXA1 is identified as a functional target of miR-212 in HCC.Notes: (A) qRT-PCR and (B) Western blot analysis of FOXA1 expression in HepG2 cells with miR-212 or control vectors transfection. n=3 independent experiments, *P<0.05. (C) miR-212 and its putative binding sequence in the 3′-UTR of FOXA1. The mutant miR-212 binding site was generated in the complementary site for the seed region of miR-212 (wt, wild type; mt, mutant type). (D) miR-212 significantly suppressed the luciferase activity that carried wt but not mt 3′-UTR of FOXA1. Anti-miR-212 led to a noticeable increase in luciferase activity of wt 3′-UTR of FOXA1. n=3 repeats with similar results, *P<0.05.Abbreviations: FOXA1, forkhead box protein A1; HCC, hepatocellular carcinoma; miR-212, microRNA 212; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; UTR, untranslated region; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Mentions: To disclose the molecular mechanisms by which miR-212 inhibits HCC tumor growth, predicted target genes of miR-212 were retrieved and analyzed using publicly available databases (TargetScan 6.2 and MiRanda). FOXA1, an important transcription factor in liver development and cancer progression,16 was predicted as one of the targets of miR-212. As measured by qRT-PCR and Western blot, both the levels of FOXA1 mRNA and protein were significantly reduced by upregulation of miR-212 in HepG2 cells (P<0.05, respectively, Figure 3A and B). Next, we investigated whether the miR-212 directly interacted with the 3′-UTR of FOXA1 mRNA using a Dual-Luciferase® Reporter Assay System. As expected, miR-212 significantly inhibited the luciferase activity of FOXA1 containing a wild-type (wt) 3′-UTR but did not suppress the activity of FOXA1 with a mutant (mt) 3′-UTR (P<0.05, Figure 3C and D). When anti-miR-212 was transfected, an increase in luciferase activity of wt FOXA1 3′-UTR was observed. However, with the mt FOXA1 3′-UTR constructs, there was no relative increase in activity (P<0.05, Figure 3C and D). Thus, our data strongly suggest that FOXA1 is a target of miR-212 in HCC.

Bottom Line: In this study, the miR-212 expression was found to be obviously downregulated in hepatocellular carcinoma (HCC) tissues as compared with adjacent nontumor tissues.Interestingly, we found that upregulation of miR-212 decreased FOXA1 expression in HepG2 cells.In conclusion, miR-212 is a potent prognostic marker and may suppress HCC tumor growth by inhibiting FOXA1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Hubei University of Medicine, Shiyan, People's Republic of China.

ABSTRACT
MircroRNA-212 (miR-212) is proposed as a novel tumor-related miRNA and has been found to be significantly deregulated in human cancers. In this study, the miR-212 expression was found to be obviously downregulated in hepatocellular carcinoma (HCC) tissues as compared with adjacent nontumor tissues. Clinical association analysis indicated that low expression of miR-212 was prominently correlated with poor prognostic features of HCC, including high AFP level, large tumor size, high Edmondson-Steiner grading, and advanced tumor-node-metastasis tumor stage. Furthermore, the miR-212 expression was an independent prognostic marker for predicting both 5-year overall survival and disease-free survival of HCC patients. Our in vitro studies showed that upregulation of miR-212 inhibited cell proliferation and induced apoptosis in HepG2 cells. On the contrary, downregulation of miR-212 promoted cell proliferation and suppressed apoptosis in Huh7 cells. Interestingly, we found that upregulation of miR-212 decreased FOXA1 expression in HepG2 cells. Significantly, FOXA1 was identified as a direct target of miR-212 in HCC. FOXA1 was downregulated in HCC tissues as compared with noncancerous tissues. An inverse correlation between FOXA1 and miR-212 expression was observed in HCC tissues. Notably, FOXA1 knockdown inhibited cell proliferation and induced apoptosis in HepG2 cells. In conclusion, miR-212 is a potent prognostic marker and may suppress HCC tumor growth by inhibiting FOXA1 expression.

No MeSH data available.


Related in: MedlinePlus