Effect of up-regulation of miR-193b on HBV positive hepatocellular carcinoma cells / 国际肿瘤学杂志

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 HepG2.2.15cells 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 hepatoeellular cells,and the mechanism may be related to the regulation of Bax,Bcl-2,MMP-9 and MMP-2 protein expression.