miR-21靶向调控Bcl-2对肠道上皮HT29细胞凋亡的影响及机制研究  

Study on effects and mechanism of microRNA-21 on apoptosis of intestinal epithelial cells through targeting Bcl-2

作　　者：王卫卫[1] 邢文韬 魏思忱[1] 高巧营 魏新亮[1] 石亮[4] 孔郁[1] WANG Wei-Wei;XING Wen-Tao;WEI Si-Chen;GAO Qiao-Ying;WEI Xin-Liang;SHI Liang;KONG Yu(Department of Gastroenterology Two,Cangzhou Central Hospital,Cangzhou 061001,China)

机构地区：[1]沧州市中心医院消化内二科,沧州061001 [2]沧州市中心医院消化内一科,沧州061001 [3]南开医院急腹症研究所,天津300100 [4]沧州市中心医院普外窥镜室,沧州061001

出　　处：《中国免疫学杂志》2019年第20期2457-2463,共7页Chinese Journal of Immunology

基　　金：河北省自然科学基金项目(20151666)资助

摘　　要：目的:探究微小型RNA-21(microRNA-21,miR-21)对肠道上皮HT29细胞凋亡的影响及作用机制。方法:利用RT-PCR检测B细胞淋巴瘤/白血病基因-2(Bcl-2)与miR-21在结肠组织和HT29细胞系中的mRNA水平。利用生物信息预测Bcl-2与miR-21的结合位点,通过荧光素酶报告进一步验证。将细胞分为HT29组、miR-21 inhibitor组、Bcl-2 siRNA(si-Bcl-2)组和si-Bcl-2+miR-21 inhibitor组,miR-21 inhibitor组和si-Bcl-2组分别或同时转染经TNF-α预处理的HT29细胞后,用MTT检测细胞存活情况,流式检测细胞凋亡情况,免疫印迹检测Bcl-2、增殖细胞核抗原(PCNA)、Ki67、Cleaved Caspase-3和Cleaved Caspase-9蛋白表达情况,ELISA检测炎症细胞因子IL-6、IL-1β和IL-8的表达水平。结果:溃疡性结肠炎组织和TNF-α处理后的HT29细胞中miR-21 mRNA水平显著升高,Bcl-2 mRNA水平显著降低。miR-21 inhibitor可显著降低miR-21 mRNA水平,升高Bcl-2 mRNA水平。miR-21mimic可显著降低野生型Bcl-2质粒(Bcl-2 wt)的荧光素酶活性。与HT29组比较,miR-21 inhibitor组中Bcl-2表达显著增加,si-Bcl-2组中Bcl-2表达显著减少。同时,与HT29组比较,miR-21 inhibitor组细胞存活率及PCNA、Ki67的表达水平显著上升,细胞凋亡率及Cleaved Caspase-3、Cleaved Caspase-9的表达水平显著降低,si-Bcl-2组细胞存活率及PCNA、Ki67的表达水平显著下降,细胞凋亡率及Cleaved Caspase-3、Cleaved Caspase-9的表达水平显著上升。与miR-21 inhibitor组比较,si-Bcl-2+miR-21 inhibitor组细胞存活率及PCNA、Ki67的表达水平显著下降,细胞凋亡率及Cleaved Caspase-3、Cleaved Caspase-9的表达水平显著上升。此外,与HT29组比较,miR-21 inhibitor组中IL-6、IL-1β和IL-8的浓度显著降低,si-Bcl-2组中IL-6、IL-1β和IL-8的浓度显著升高。与miR-21 inhibitor组比较,si-Bcl-2+miR-21 inhibitor组中IL-6、IL-1β和IL-8的浓度显著升高。结论:miR-21通过靶向下调Bcl-2促进肠道上皮HT29细胞的凋亡。Objective:To investigate the effect and mechanism of MicroRNA-21(miR-21) on cell apoptosis in intestinal epithelial cell line HT29.Methods: The mRNA levels of B-cell lymphoma/leukemia gene-2(Bcl-2)and miR-21 in colon tissues or HT29 cell line were measured by reverse transcription PCR.The binding site between Bcl-2 and miR-21 was predicted by bioinformatics and conformed by luciferase reporter assay.Cells were divided into HT29 group,miR-21 inhibitor group,Bcl-2 siRNA(si-Bcl-2) group and si-Bcl-2+miR-21 inhibitor groups.And after HT29 and miR-21 inhibitor respectively or meanwhile transferred with the HT29 cells preprocessed by TNF-α,MTT was performed to evaluate HT29 cell viability,flow cyometry was performed to evaluate cell apoptosis,Western blot were performed to evaluate the protein expression of Bcl-2,proliferating cell nuclear antigen(PCNA),Ki67,Cleaved Caspase-3 and Cleaved Caspase-9,the expression level of interleukin-6(IL-6),IL-1β and IL-8 were measured by ELISA.Results: The mRNA levels of miR-21 were increased in ulcerative colitis tissues and the HT29 cell processed by TNF-α,but the mRNA levels of Bcl-2 were decreased.The mRNA level of miR-21 was decreased by miR-21 inhibitor,while the mRNA level of Bcl-2 was increased.MiR-21 mimic decreased the luciferase activity of wild type Bcl-2 plasmid(Bcl-2 wt).Compared with HT29 group,the protein level of Bcl-2 in miR-21 inhibitor group was increased significantly,while it was decreased in si-Bcl-2 group.Moreover,compared with HT29 group,the cell viability rate and the expression of PCNA,Ki67 were increased in miR-21 inhibitor group,but the cell apoptosis rate and the expression of Cleaved Caspase-3,Cleaved Caspase-9 were decreased notably.While in si-Bcl-2 group,the cell viability rate and the expression of PCNA,Ki67 were decreased,the cell apoptosis rate and the expression of Cleaved Caspase-3,Cleaved Caspase-9 were increased signally.Compared with miR-21 inhibitor group,the cell viability rate and the expression of PCNA,Ki67 were decreased in si-Bcl-2+mi

关 键 词：MIR-21 BCL-2 溃疡性结肠炎 凋亡 

分 类 号：R574.62[医药卫生—消化系统]