miR-150-5p与NCAPG的靶向关系及其对肝细胞肝癌Huh7细胞的抑制作用  

Targeting relationship between miR-150-5p and NCAPG and its inhibitory effect on hepatocellular carcinoma Huh7 cells

作　　者：金俊伊 李木 胡耀元 王熠辉 JIN Junyi;LI Mu;HU Yaoyuan;WANG Yihui(Department of General Surgery,Shenbei Hospital,Shengjing Hospital,China Medical University,Shenyang 110100,China)

机构地区：[1]中国医科大学附属盛京医院沈北院区普外科,辽宁沈阳110100

出　　处：《吉林大学学报（医学版）》2023年第1期122-130,共9页Journal of Jilin University：Medicine Edition

基　　金：辽宁省科技厅自然科学基金面上项目(2021-BS-121)。

摘　　要：目的:探究miR-150-5p与非SMC凝聚体Ⅰ复合物亚基G(NCAPG)的靶向关系及其对肝细胞肝癌(HCC)细胞生物学功能的影响,为HCC的临床诊断和治疗提供潜在靶点。方法:通过双荧光素酶报告基因实验验证miR-150-5p与NCAPG的靶向关系。将HCC Huh7细胞根据转染质粒不同分为模拟物阴性对照(mimic NC)组、miR-150-5p模拟物(miR-150-5p mimic)组、miR-150-5p模拟物+过表达阴性对照(miR-150-5p mimic+ovNC)组和miR-150-5p模拟物+过表达NCAPG(miR-150-5p mimic+ovNCAPG)组。实时荧光定量PCR(RT-qPCR)法检测各组细胞中NCAPG mRNA表达水平,Western blotting法检测各组细胞中NCAPG蛋白表达水平,5-乙炔基-2'-脱氧尿嘧啶核苷(EdU)实验检测各组细胞EdU阳性率,流式细胞术检测各组细胞凋亡率,Transwell实验检测各组细胞中迁移和侵袭细胞数。将裸鼠分为agomiR-NC组、agomiR-150-5p组、agomiR-150-5p+过表达阴性对照(agomiR-150-5p+ovNC)组和agomiR-150-5p+过表达NCAPG(agomiR-150-5p+ovNCAPG)组,Huh7细胞皮下成瘤,检测各组裸鼠肿瘤体积和质量。结果:双荧光素酶报告基因实验证实NCAPG是miR-150-5p的靶基因。与mimic NC组比较,miR-150-5p mimic组Huh7细胞中NCAPG mRNA和蛋白表达水平及EdU阳性率明显降低(P<0.05),细胞凋亡率明显升高(P<0.05),迁移细胞数和侵袭细胞数明显减少(P<0.05);与miR-150-5p mimic+ovNC组比较,miR-150-5p mimic+ovNCAPG组Huh7细胞中NCAPG mRNA和蛋白表达水平及EdU阳性率明显升高(P<0.05),细胞凋亡率明显降低(P<0.05),迁移细胞数和侵袭细胞数明显增加(P<0.05)。与agomiR-NC比较,agomiR-150-5p组裸鼠Huh7细胞移植瘤体积和质量明显减少(P<0.05);与agomiR-150-5p+ovNC组比较,agomiR-150-5p+ovNCAPG组裸鼠Huh7细胞移植瘤体积和质量明显增加(P<0.05)。结论:miR-150-5p通过靶向结合抑制NCAPG表达进而抑制HCC细胞增殖、迁移、侵袭和肿瘤生长,促进细胞凋亡。Objective:To explore the targeting relationship between miR-150-5p and non-SMC condensateⅠcomplex subunit G(NCAPG)and its effect on the biological function of hepatocellular carcinoma(HCC)cells,and to provide the potential targets for the clinical diagnosis and treatment of HCC.Methods:The targeting relationship between miR-150-5p and NCAPG was verified by dualluciferase reporter gene experiment.The Huh7 cells were divided into mimic negative control(mimic NC)group,miR-150-5p mimic group,miR-150-5p mimic+overexpression negative control(miR-150-5p mimic+ovNC)group and miR-150-5p mimic+overexpression NCAPG(miR-150-5p mimic+ovNCAPG)group.The expression levels of NCAPG mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method,and the expression levels of NCAPG protein in the cells in various groups were detected by Western blotting method;5-ethynyl-2'-deoxyuridine(EdU)assay was used to detect the EdU positive rates of cells in various groups,flow cytometry was used to detect the apoptotic rates,and the numbers of migration and invasion cells were detected by Transwell assay.The nude mice were divided into agomiR-NC group,agomiR-150-5p group,agomiR-150-5p+overexpression negative control(agomiR-150-5p+ovNC)group and agomiR-150-5p+overexpression NCAPG(agomiR-150-5p+ovNCAPG)group.The Huh7 cells formed subcutaneous tumors,and the tumor volumes and tumor weights of nude mice in various groups were measured.Results:The dual luciferase reporter gene assay results confirmed that NCAPG was the target gene of miR-150-5p.Compared with mimic NC group,the expression levels of NCAPG mRNA and protein and the EdU positive rate in the Huh7 cells in miR-150-5p mimic group were significantly decreased(P<0.05),the apoptotic rate was significantly increased(P<0.05),and the numbers of migration and invasion cells were significantly decreased(P<0.05);compared with miR-150-5p mimic+ovNC group,the expression levels of NCAPG mRNA and protein and the EdU positive rate in the Huh7 cells in miR-

关 键 词：非SMC凝聚体Ⅰ复合物亚基G 肝细胞肝癌 miR-150-5p 细胞增殖 细胞迁移 细胞侵袭 

分 类 号：R735.7[医药卫生—肿瘤]