Effect of up-regulation of miR-193b on HBV positive hepatocellular carcinoma cells

Abstract

Objective To investigate the effect of miRNA-193b (miR-193b) up-regulation on the apoptosis, invasion and metastasis of hepatitis B virus (HBV)-positive hepatocellular cells. Methods HepG2.2.15 cells were cultured until logarithmic growth phase and transfected with miR-193b mimic at a concentration of 25, 50, 100 pmol. The control group was transfected with blank mimic (0 pmol). After 48 hours, the expression of miR-193b in each group was detected by real-time quantitative polymerase chain reaction (qRT-PCR), the apoptosis rate in each group was detected by flow cytometry, the cell migration was detected by cell scratch assay, the cell invasion was detected by Transwell assay, and the expressions of Bax, Bcl-2, matrix metalloprotease (MMP)-9 and MMP-2 protein were detected by Western blotting. Results qRT-PCR results showed that the miR-193b expressions of miR-193b mimic (25, 50 and 100 pmol) groups and control group in HepG2.2.15 cells were 1.05±0.09, 1.53±0.12, 2.08±0.17 and 0.49±0.12, and the difference was statistically significant (F=261.35, P<0.001). Compared with the control group, miR-193b expression significantly increased in each transfection group (P=0.036; P=0.029; P=0.022). Flow cytometry results showed the apoptosis rates of miR-193b mimic (25, 50 and 100 pmol) groups and control group were (30.28±5.22)%, (53.41±6.18)%, (79.89±7.13)% and (1.02±0.13)%, and the difference was statistically significant (F=357.19, P<0.001). Compared with the control group, the apoptosis rate significantly increased in each transfection group (P=0.025; P=0.010; P=0.007). Cell scratch assay results showed that the migration distances of miR-193b mimic (25, 50 and 100 pmol) groups and control group were (27.53±1.54)mm, (19.24±2.12)mm, (13.42±1.53)mm and (34.95±1.92)mm, and the difference was statistically significant (F=408.62, P<0.001). Compared with the control group, the migration distance significantly decreased in each transfection group (P=0.032; P=0.007; P=0.006). Transwell experimental results showed that the absorbance values of miR-193b mimic (25, 50 and 100 pmol) groups and control group were 1.02±0.12, 0.59±0.13, 0.42±0.10 and 1.68±0.16, and the difference was statistically significant (F=511.68, P<0.001). Compared with the control group, the cell invasion ability significantly weakened in each transfection group (P=0.028; P=0.005; P=0.002). Western blotting results showed that the expressions of Bax, Bcl-2, MMP-9 and MMP-2 protein of miR-193b mimic (25, 50 and 100 pmol) groups and control group had statistically significant difference (F=264.38, P<0.001; F=437.19, P<0.001; F=377.46, P<0.001; F=208.79, P<0.001). Further paired comparison showed that the expressions of Bax, Bcl-2, MMP-9 and MMP-2 protein between mimics groups and control group were statistically significant (all P<0.05). Conclusion miR-193b can induce the apoptosis and inhibit the invasion, metastasis of HBV-positive hepatocellular cells, and the mechanism may be related to the regulation of Bax, Bcl-2, MMP-9 and MMP-2 protein expression. Key words: Hepatitis B virus; Liver neoplasms; MicroRNAs; Tumor infiltrating