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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参考文献(22)

[1] LIU H,XU Y,XIANG J,et al. Targeting alpha-fetoprotein(AFP)-MHC complex with CAR T-cell therapy for liver cancer[J]. Clin Cancer Res,2017,23(2):478-488.

[2] ZHAO J,YANG XW,LI JT,et al. Construction and functional enrichment analysis of circRNA-miRNA-mRNA regulatory network for liver cancer based on high-throughput sequencing[J]. J Clin Hepatol,2019,35(8):1740-1744.(in Chinese)赵静，杨兴武，李京涛，等．基于高通量测序的肝癌circRNAmiRNA-mRNA调控网络构建及功能富集分析[J]．临床肝胆病杂志，2019,35(8):1740-1744.

[3] REN D,LIN B,ZHANG X,et al. Maintenance of cancer stemness by miR-196b-5p contributes to chemoresistance of colorectal cancer cells via activating STAT3 signaling pathway[J]. Oncotarget,2017,8(30):49807-49823.

[4] XU L,GUO Y,YAN W,et al. High level of miR-196b at newly diagnosed pediatric acute myeloid leukemia predicts a poor outcome[J]. Excli J,2017,16:197-209.

[5] WEN P,CHEN SD. Changes of miR-196b expression in liver cancer and its relationship with clinicopathological parameters and prognosis[J]. Shandong Med J,2018,58(12):75-77.(in Chinese)温萍，陈盛铎．肝癌组织miR-196b表达变化及其与患者临床病理参数和预后的关系[J]．山东医药，2018,58(12):75-77.

[6] LI JH,SUN SS,FU CJ,et al. Diagnostic and prognostic value of microRNA-628 for cancers[J]. J Cancer,2018,9(9):1623-1634.

[7] XIN R,LI D,XU HY,et al. Influence of miRNA-21 in radiosensitivity of breast cancer cells and its mechanism[J]. J Jilin Univ(Med Edit),2019,45(4):772-778.(in Chinese)信瑞，李冬，徐慧英，等．miRNA-21对乳腺癌细胞放射敏感性的影响及其机制[J]．吉林大学学报(医学版)，2019,45(4):772-778.

[8] XU M,CUI P,YE M,et al. Insights into tumor suppression mechanism of miRNA-101 in medullary thyroid carcinoma[J]. Chin J Clin Pharmacol Ther,2018,23(5):524-530.(in Chinese)徐明，崔鹏，叶敏，等．miRNA-101在甲状腺髓样癌中抑癌机制的研究[J]．中国临床药理学与治疗学，2018,23(5):524-530.

[9] WANG CL. Regulation of miR-196b-5p on adipocyte differentiation[D]. Tianjin:Tianjin Medical University,2016.(in Chinese)王昌兰．miR-196b-5p对脂肪细胞分化的调控[D]．天津:天津医科大学，2016.

[10] VELU CS,BAKTULA AM,GRIMES HL. Gfi1 regulates miR-21 and miR-196b to control myelopoiesis[J]. Blood,2009,113(19):4720-4728.

[11] LI H,FENG C,SHI S. miR-196b promotes lung cancer cell migration and invasion through the targeting of GATA6[J].Oncol Lett,2018,16(1):247-252.

[12] LIU Y,GONG W,YANG ZY,et al. Quercetin induces protective autophagy and apoptosis through ER stress via the pSTAT3/Bcl-2 axis in ovarian cancer[J]. Apoptosis,2017,22(4):544-557.

[13] TOKUNAGA T,ANDO T,SUZUKI-KARASAKI M,et al. Plasma-stimulated medium kills TRAIL-resistant human malignant cells by promoting caspase-independent cell death via membrane potential and calcium dynamics modulation[J]. Int J Oncol,2018,52(3):697-708.

[14] REN L,LI Z,DAI C,et al. Chrysophanol inhibits proliferation and induces apoptosis through NF-κB/cyclin D1 and NF-κB/Bcl-2 signaling cascade in breast cancer cell lines[J].Mol Med Rep,2018,17(3):4376-4382.

[15] YANG Z,GENG Y,YAO Z,et al. Spatiotemporal expression of Bcl-2/Bax and neural cell apoptosis in the developing lumbosacral spinal cord of rat fetuses with anorectal malformations[J]. Neurochem Res,2017,42(11):3160-3169.

[16] FENG X,YU Y,HE S,et al. Dying glioma cells establish a proangiogenic microenvironment through a caspase 3 dependent mechanism[J]. Cancer Lett,2017,385:12-20.

[17] CHEN F,LIU X,CHENG Q,et al. RUNX3 regulates renal cell carcinoma metastasis via targeting miR-6780a-5p/E-cadherin/EMT signaling axis[J]. Oncotarget,2017,8(60):101042-101056.

[18] JIA D,NIU Y,LI D,et al. lncRNA C2dat1 promotes cell proliferation,migration,and invasion by targeting miR-34a-5p in osteosarcoma cells[J]. Oncol Res,2018,26(5):753-764.

[19] ZHAO G,LIU L,ZHAO T,et al. Upregulation of miR-24 promotes cell proliferation by targeting NAIF1 in non-small cell lung cancer[J]. Tumour Biol,2015,36(5):3693-3701.

[20] LUO Q,ZHAO M,ZHONG J,et al. NAIF1 is down-regulated in gastric cancer and promotes apoptosis through the caspase-9pathway in human MKN45 cells[J]. Oncol Rep,2011,25(4):1117-1123.

[21] GU YY,ZHAO M,YANG M,et al. Effects of NAIF1 on cell proliferation and migration in hepatoma cell HepG2[J]. Prog Mod Biomed,2015,15(6):1001-1004.(in Chinese)谷愉愉，赵玫，杨梅，等．NAIF1对肝癌细胞HepG2增殖与迁移能力的影响[J]．现代生物医学进展，2015,15(6):1001-1004.

[22] LUO Q. Study on the differential expression and function of human nuclear inducing apoptosis factor 1(NAIF1)gene in gastric cancer and breast cancer[D]. Beijing:Peking Union Medical College,2011.(in Chinese)罗清．人核诱导凋亡因子1(NAIF1)基因在胃癌及乳腺癌中的差异表达及功能[D]．北京:北京协和医学院，2011.

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