下调miR-196b靶向核凋亡诱导因子1调控肝癌细胞生长和凋亡的机制

肖二辉; 宁会彬; 康月花; 殷辉; 马力; 毛重山; 赵岩; 尚佳

doi:10.3969/j.issn.1001-5256.2020.10.014

下调miR-196b靶向核凋亡诱导因子1调控肝癌细胞生长和凋亡的机制

DOI: 10.3969/j.issn.1001-5256.2020.10.014

1. 河南省人民医院感染科郑州大学人民医院河南大学人民医院
2. 阜外华中心血管病医院急诊科河南省人民医院心脏中心

基金项目:

河南省医学科技攻关计划项目(201702204)；河南省科技攻关项目(162102310156)；

详细信息

中图分类号: R735.7
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出版历程
- 收稿日期: 2020-04-29
- 出版日期: 2020-10-20

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

Department of Infectious Diseases,Henan Province People's Hospital,Zhengzhou University People's Hospital & Henan University People's Hospital
Emergency Department of Fuwai Central China Cardiovascular Hospital & Heart Center of Henan Provincial People's Hospital

Research funding:

摘要

摘要: 目的探讨miR-196b靶向核凋亡诱导因子1（NAIF1）调控肝癌细胞生长和凋亡的机制。方法 Real-time PCR检测miR-196b在肝癌细胞HuH-7、SNU-449、HepG2、SMCC7721和人正常肝细胞HL7702中的表达差异。取肝癌细胞HepG2,分成Control（空白对照）、Anti-NC（转染inhibitor control）、Anti-miR-196b（转染miR-196b inhibitor）、si-NC（转染siRNA control）、siNAIF1（转染NAIF1 siRNA）、Anti-miR-196b+si-NAIF1（共转染miR-196b inhibitor、NAIF1 siRNA）和Anti-miR-196b+siNC（共转染miR-196b inhibitor、siRNA control）组,MTT测定增殖变化,平板克隆实验检测克隆形成能力,流式细胞术检测细胞凋亡,Western Blot检测Bax、C-caspase-3蛋白表达变化。靶基因预测软件预测NAIF1可能为miR-196b的靶基因,荧光素酶报告系统鉴定靶向关系。计量资料两组...
- 肝肿瘤 /
- 微RNAs /
- 细胞增殖 /
- 细胞凋亡
Abstract: 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.
- liver neoplasms /
- microRNAs /
- cell proliferation /
- apoptosis

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