miR-199a-3p inhibits cell proliferation and induces apoptosis by targeting YAP1, suppressing Jagged1-Notch signaling in human hepatocellular carcinoma

Abstract

Background: miR-199a-3p was significantly downregulated in the majority of human hepatocellular carcinoma (HCC) tissues and HCC cell lines. Yes associated protein 1 (YAP1) was overexpressed in human HCC, which promoted HCC development and progression by upregulating Jagged1 and activating the Notch pathway. We searched potential targets of miR-199a-3p with DIANA, TargetScan and PicTar tools, and found that YAP1 is one of the potential targets. Based on these findings, we speculated that miR-199a-3p might suppress HCC growth by targeting YAP1, downregulating Jagged1 and suppressing the Notch pathway.

Results: We determined the expression of miR-199a-3p and YAP1 by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found downregulation of miR-199a-3p and upregulation of YAP1 in HCC cell lines. Cell proliferation and apoptosis assays showed that miR-199a-3p suppresses HCC cell proliferation and promotes apoptosis, and knockdown of YAP1 has similar role. Furthermore, we verified that miR-199a-3p can directly target YAP1. We further investigated and confirmed that miR-199a-3p and YAP1 regulate HCC cell proliferation and apoptosis through Jagged1-Notch signaling.

Conclusion: miR-199a-3p targets YAP1, downregulates Jagged1 and suppresses the Notch signaling to inhibit HCC cell proliferation and promote apoptosis. These findings provide new insights into the mechanism by which miR-199a-3p suppresses HCC cell proliferation and induces apoptosis.

Keywords: Hepatocellular carcinoma; Jagged1; Notch signaling; Yes associated protein 1; miR-199a-3p.

Fig. 1

The expression levels of miR-199a-3p and YAP1 in HCC cell lines. a Comparing differences in the expression levels of miR-199a-3p between HCC cell lines and normal liver cell line HL-7702. b and c The expression levels of YAP1 mRNA and protein are determined by qRT-PCR and western blot. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 2

Overexpression of miR-199a-3p and knockdown of YAP1 inhibit proliferation and induce apoptosis in HCC cells. a and d The effects of overexpression of miR-199a-3p and knockdown of YAP1 on Huh7 cell proliferation are detected by MTT assay. b and e The effects of overexpression of miR-199a-3p and knockdown of YAP1 on Huh7 cell apoptosis are assessed by flow cytometry assay. c and f Caspase 3/7 activity is measured in Huh7 cells transfected with miR-control, miR-199a-3p mimic, si-control or si-YAP1. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 3

YAP1 is a functional target of miR-199a-3p in HCC cells. a Bioinformatics-based target prediction analysis represents that YAP1 is a potential target of miR-199a-3p and the putative binding sequence is in the 3’-UTR of YAP1 mRNA. b Luciferase reporter assay shows that the luciferase activity in cells containing miR-199a-3p and 3’UTR-WT of YAP1 is markedly decreased compared with the negative control and 3’UTR-MUT groups. c and d qRT-PCR and western blot analyses shows that miR-199a-3p overexpression signigficantly downregulates mRNA and protein levels of YAP1.e–g MTT, flow cytometry and Caspase 3/7 activation assays show that the upregulation of YAP1 partially reverses miR-199a-3p’s effect on the proliferation and apoptosis of Huh7 cells. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 4

The Notch ligand Jagged1 is a functional YAP1 target. a and b High expression of miR-199a-3p and YAP1 silencing significantly inhibit protein level of Jagged1. c and g Wetern blot assays confirm that transfection is successful for all combinations. d–f Overexpression of miR-199a-3p reduces cell proliferation and induces apoptosis, however, Jagged1 relieves these effects in Huh7 cells. h–j si-YAP1 significantly inhibits cell proliferation and induces apoptosis in Huh7 cells, while Jagged1 reverses these effects. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 5

miR-199a-3p and YAP1 regulated proliferation and apoptosis of HCC cells through Jagged1-Notch signaling. a and b Huh7 cells transfected with miR-199a-3p mimics and si-YAP1 show signigficantly lower protein levels of NICD and Hes-1 than control cells. c–e Huh7 cells treated by miR-199a-3p mimic and DAPT have an obviously lower cell proliferation rate and a significantly higher apoptosis rate than the cells treated by miR-control and DAPT. *P < 0.05 and **P < 0.01