Downregulation of miR-196b in regulating the growth and apoptosis of hepatoma cells by targeting nuclear apoptosis-inducing factor 1

Objective To investigate the mechanism of action of miR-196 b in regulating the growth and apoptosis of hepatoma cells by targeting nuclear apoptosis-inducing factor 1( NAIF1). Methods Real-time PCR was used to measure the expression of miR-196 b in hepatoma HuH-7,SNU-449,HepG2,and SMCC7721 cells versus normal human HL7702 hepatocytes. The hepatoma HepG2 cells were collected and divided into Control group( blank control),Anti-NC group( transfected with inhibitor control),Anti-miR-196 b group( transfected with miR-196 b inhibitor),si-NC group( transfected with siRNA control),si-NAIF1 group( transfected with NAIF1 siRNA),Anti-miR-196 b + si-NAIF1 group( co-transfected with miR-196 b inhibitor and NAIF1 siRNA),and Anti-miR-196 b + si-NC group( co-transfected with miR-196 b inhibitor and siRNA control). MTT assay was used to measure the change in proliferation,plate colony formation assay was used to measure colony formation ability,flow cytometry was used to measure cell apoptosis,and Western blot was used to measure the protein expression of Bax and C-caspase-3. Target gene prediction software predicted that NAIF1 might be a target gene of miR-196 b,and the luciferase reporting system was used to identify the targeting relationship. The t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups,and the SNK-q test was used for further comparison between two groups. Results There was a significant difference in the expression level of miR-196 b between hepatoma Hu H-7,SNU-449,Hep G2,and SMCC7721 cells and normal human HL7702 hepatocytes( 1. 85 ± 0. 16/1. 63 ±0. 12/2. 36 ± 0. 25/1. 92 ± 0. 13 vs 1. 00 ± 0. 09,F = 29. 05,P < 0. 001). Compared with the Anti-NC group,the Anti-miR-196 b group had significant reductions in the expression level of miR-196 b( 0. 42 ± 0. 03 vs 1. 02 ± 0. 10,P < 0. 05),cell proliferation( 0. 20 ±0. 02 vs 0. 30 ± 0. 05,P < 0. 05),and colony formation ability( 64. 35 ± 6. 97 vs 119. 54 ± 11. 82,P < 0. 05) and significant increases in apoptosis rate( 22. 30% ± 2. 09% vs 4. 26% ± 0. 35%,P < 0. 05) and relative protein expression of Bax( 0. 69 ± 0. 08 vs 0. 30 ± 0. 05,P < 0. 05) and C-caspase-3( 0. 63 ± 0. 05 vs 0. 21 ± 0. 04,P < 0. 05). Compared with the si-NC group,the si-NAIF1 group had significant increases in proliferation ability( 0. 46 ± 0. 05 vs 0. 31 ± 0. 04,P < 0. 05) and colony formation ability( 138. 92 ± 9. 66 vs 118. 47 ±8. 38,P < 0. 05) and significant reductions in apoptosis rate( 4. 12% ± 0. 40% vs 1. 23% ± 0. 12%,P < 0. 05),NAIF1( 0. 10 ± 0. 01 vs0. 17 ± 0. 02,P < 0. 05),and protein expression of Bax( 0. 18 ± 0. 02 vs 0. 29 ± 0. 03,P < 0. 05) and C-caspase-3( 0. 12 ± 0. 01 vs0. 20 ± 0. 03,P < 0. 05). Compared with the Anti-miR-196 b + si-NC group,the Anti-miR-196 b + si-NAIF1 group had significant increases in proliferation ability( 0. 28 ± 0. 02 vs 0. 21 ± 0. 03,P < 0. 05) and colony formation ability( 97. 12 ± 8. 23 vs 66. 35 ± 5. 20,P < 0. 05) and significant reductions in apoptosis rate( 9. 60% ± 1. 11% vs 21. 14% ± 1. 32%,P < 0. 05),NAIF1( 0. 30 ± 0. 04 vs 0. 52 ±0. 06,P < 0. 05),and protein expression of Bax( 0. 28 ± 0. 03 vs 0. 67 ± 0. 06,P < 0. 05) and C-caspase-3( 0. 22 ± 0. 05 vs 0. 60 ±0. 04,P < 0. 05). Conclusion Downregulation of miR-196 b can inhibit the growth and induce the apoptosis of hepatoma cells via negative regulation of NAIF1.