Down-regulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells while promoting their apoptosis via the PI3K/Akt signaling pathway

Fanggui Xu,Wenjie Gao,Feng Zhu,Xin Chen,Wulin Wang,Chunzhao Yu

doi:10.14715/cmb/2020.66.3.25

Abstract

The purpose of this study was to investigate the effects of microRNA-196b (miRNA-196b) on proliferation, migration, invasiveness and apoptosis of hepatocellular carcinoma cell line (HepG2), and the mechanism involved. MiRNA-196b inhibitor or negative control were transfected into HepG2 cells, while empty liposome vector was used as normal control. The results of transfection were assessed using real-time quantitative polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasiveness and apoptosis were determined using cell counting kit 8 (CCK-8), scratch test, Transwell invasion assay, and flow cytometric analysis, respectively. The expressions of PIK3, Akt and p-Akt proteins were determined using Western blotting. The HepG2 cells were also treated with PI3K/Akt signaling pathway inhibitor LY294002, and its effect on cell proliferation, migration, invasion, and apoptosis, and expressions of PIK3, Akt, and p-Akt proteins were determined. The results of RT-PCR showed that the relative expression of miRNA-196b in the inhibitor group (0.42 ± 0.13) was significantly lower than that in the blank control group (0.96 ± 0.10) and the negative control group (1.01 ± 0.32) (p < 0.05). The miRNA-196b inhibitor significantly and time-dependently reduced the invasiveness, proliferation migration abilities of HepG2 cells, while promoting their apoptosis (p < 0.05). The expressions of PIK3 and p-Akt proteins were significantly down-regulated in the inhibitor group, when compared with normal and negative control groups (p < 0.05). However, there were no significant differences in the expression of Akt protein among the groups (p > 0.05). After treatment of HepG2 cells with PI3K/Akt signaling pathway inhibitor LY294002, the proliferative, migratory and invasive abilities of cells in the treatment group were significantly enhanced, while cell apoptosis was significantly reduced (p < 0.05). Similarly, the protein expressions of PIK3 and p-Akt in the non-treatment group was significantly upregulated, relative to the treatment group (p < 0.05), but there was no significant difference in the expression of Akt protein between the two groups (p > 0.05). Downregulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells, while promoting their apoptosis via a mechanism involving the PI3K/Akt signaling pathway.

Highlights

Liver cancer is a common malignant tumor characterized by high incidence of mortality
The results showed that transfection of miRNA-196b inhibitor significantly and time-dependently reduced the proliferative and migratory abilities of HepG2 cells
These results suggest that the downregulation of miRNA-196b expression may inhibit the proliferation, migration and invasiveness of HepG2 cells, while enhancing their apoptosis

Summary

Introduction

Liver cancer is a common malignant tumor characterized by high incidence of mortality. MicroRNA (miRNA) is a small non-coding RNA molecule found in plants, animals and some viruses, and functions in RNA silencing and post-transcriptional regulation of gene expression. The expression and processing patterns of miRNA vary depending on the type of tumor. By targeting dozens-to-hundreds of genes, the basic biological functions and pathways involved in tumorigenesis can be unraveled [2]. MicroRNAs have been shown to play a role in the pathogenesis and progression of liver cancer. Studies on the role of miRNA-196b in liver cancer are scanty. It has been reported that miRNA-196b may promote the proliferation and invasion of hepatocellular carcinoma (HCC) cells by targeting forkhead box p2 gene (FOXP2) [7]

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