miR-7靶向调控KLF4对结肠癌干细胞生物学行为的影响  

Effects of miR-7 regulating KLF4 expression on biological behavior of colon cancer stem cells

作　　者：常云丽 胥明 陈国裕[1] 汤杰 陈玲玲 季洁如[1] CHANG Yunli;XU Ming;CHEN Guoyu;TANG Jie;CHEN Lingling;JI Jieru(Department of Gastroenterology,Shanghai Pudong New Area People's Hospital,Shanghai 201299,China)

机构地区：[1]上海市浦东新区人民医院消化内科,上海201299

出　　处：《山东医药》2024年第1期11-15,共5页Shandong Medical Journal

基　　金：上海市浦东新区科技发展基金事业单位民生科研专项(医疗卫生)项目(PKJ2021-Y24)。

摘　　要：目的探讨微小核糖核酸7(miR-7)靶向调控Krüppe样因子4(KLF4)对结肠癌干细胞生物学行为的影响。方法体外传代培养结肠癌干细胞。取传15代、对数生长期、生长状态良好的结肠癌干细胞,随机分为对照组、NC mimics组、miR-7 mimics组,NC mimics组和miR-7 mimics组分别转染空载质粒、miR-7 mimics质粒,对照组不予转染。采用RT-qPCR法验证转染效率。收集各组转染48 h结肠癌干细胞,采用CCK-8法检测细胞活力,采用细胞划痕实验检测细胞迁移能力,采用改良Boyden小室法检测细胞侵袭能力,采用流式细胞术检测细胞凋亡率。通过TargetScan在线网站预测miR-7与KLF4的结合位点,并通过双荧光素酶报告基因实验验证miR-7对KLF4的靶向调控作用。收集各组转染48 h结肠癌干细胞,分别采用RT-qPCR法、Western blotting法检测KLF4 mRNA和蛋白表达。结果miR-7 mimics组miR-7相对表达量高于对照组和NC mimics组(P均<0.05),而对照组与NC mimics组比较差异无统计学意义(P>0.05)。miR-7 mimics组细胞存活率、细胞迁移率、穿膜细胞数均低于NC mimics组和对照组,细胞凋亡率高于NC mimics组和对照组(P均<0.05);NC mimics组与对照组细胞存活率、细胞迁移率、穿膜细胞数、细胞凋亡率比较差异均无统计学意义(P均>0.05)。经TargetScan在线网站预测,KLF4基因的3′非翻译区存在miR-7的结合位点;经双荧光素酶报告基因实验验证,miR-7对KLF4存在靶向调控作用。miR-7 mimics组KLF4mRNA与蛋白相对表达量均低于NC mimics组和对照组(P均<0.05),而NC mimics组与对照组比较差异均无统计学意义(P均>0.05)。结论miR-7可通过靶向下调KLF4表达而抑制结肠癌干细胞的增殖、侵袭、迁移并促进其凋亡。Objective To investigate the effect of microRNA 7(miR-7)on the biological behavior of colon cancer stem cells by targetedly regulating Krüppe-like factor 4(KLF4)expression.Methods Colon cancer stem cells were cultured in vitro.Colon cancer stem cells of the 15th generation,which were in the logarithmic growth stage and had good growth state,were taken and randomly divided into the control group,mimics NC group and miR-7 mimics group.Cells in the mimics NC group and miR-7 mimics group were transfected with empty plasmid and miR-7 mimics plasmid,respectively,while cells in the control group were not transfected.The transfection efficiency was verified by RT-qPCR.Colorectal cancer stem cells transfected for 48 h in each group were collected,and cell viability was detected by CCK-8,cell migration ability was detected by cell scratch assay,cell invasion ability was detected by modified Boyden cell assay,and apoptosis rate was detected by flow cytometry.The binding site of miR-7 and KLF4 gene was predicted by TargetScan online website,and the regulatory effect of miR-7 on KLF4 was verified by double luciferase reporter gene assay.Colon cancer stem cells transfected for 48 h in each group were collected,and the expression levels of KLF4 mRNA and protein were detected by RT-qPCR and Western blotting,respectively.Results The relative expression of miR-7 was higher in miR-7 mimics group than in the control group and mimics NC group(both P<0.05),but there was no statistically significant difference between the control group and mimics NC group(P>0.05).The cell survival rate,cell mobility and number of transmembrane cells in the miR-7 mimics group were lower than those in the mimics NC group and control group,and the apoptosis rate was higher than those in the mimics NC group and control group(all P<0.05);there were no significant differences in cell survival rate,cell mobility,number of transmembrane cells or apoptosis rate between the mimics NC group and control group(all P>0.05).As predicted by TargetScan online,miR-7 bi

关 键 词：结肠癌 微小核糖核酸7 Krüppe样因子4 

分 类 号：R735.3[医药卫生—肿瘤]