摘要
目的分析miR-625靶向高迁移率族蛋白A1(HMGA1)对食管癌细胞生物学行为的影响及机制。方法收集2018年3月—2019年3月经手术切除的食管癌组织及癌旁组织(距癌组织≥5 cm)标本,体外培养食管癌细胞系(KYSE150、KYSE30、ECA109、EC9706)和正常食管上皮细胞HET-1A,采用RT-qPCR检测不同组织和细胞中miR-625的表达。将miR-625 mimic、miR-625 inhibitor转染ECA109细胞,采用CCK-8实验、流式细胞仪、Transwell小室实验和划痕实验检测miR-625对ECA109细胞增殖、周期、侵袭和迁移的影响,Western blot检测增殖、侵袭和迁移相关蛋白的表达。经生物信息学软件在线预测miR-625靶基因,通过RT-qPCR、Western blot及双荧光素酶报告基因检测验证靶向关系。同时干预miR-625和HMGA1,检测ECA109细胞增殖、周期、侵袭和迁移。结果miR-625相对表达量,食管癌组织低于癌旁组织,食管癌细胞系KYSE150、KYSE30、ECA109、EC9706低于正常食管上皮细胞HET-1A(P<0.05)。与mimic NC组比较,miR-625 mimic组miR-625相对表达量、处于G1期细胞比例及p21、E-cadherin表达升高,细胞增殖、侵袭、迁移能力及处于S期细胞比例、Cyclin D1、N-cadherin、Vimentin表达降低(P<0.05);miR-625 inhibitor组可逆转上述作用(P<0.05)。miR-625与HMGA13'端非编码区存在结合位点。与mimic NC组比较,miR-625 mimic组细胞中相对荧光素酶活性和HMGA1表达降低(P<0.05)。与pcDNA3.1-HMGA1组比较,miR-625+HMGA1组HMGA1表达及细胞增殖、侵袭、迁移能力降低,处于G1期细胞比例升高(P<0.05)。结论miR-625在食管癌中低表达,miR-625过表达可靶向HMGA1抑制食管癌细胞的增殖、侵袭与迁移。
Objective To investigate the effect of miR-625 targeting high mobility group protein A1(HMGA1)on the biological behavior of esophageal cancer cells and its mechanism.Methods In this study,esophageal cancer tissue and adjacent normal tissue samples(equal or more than 5 cm from the cancerous tissues)removed by surgeries between March 2018 and March 2019 were collected.Esophageal cancer cell lines(KYSE150,KYSE30,ECA109,EC9706)and esophageal epithelial cell line(HET-1A)were cultured in vitro.Real time quantitative polumerase chain reaction(RT-qPCR)was used to detect miR-625 expressions in different tissues and cells.The miR-625 mimic and miR-625 inhibitor were transfected into ECA109 cells.Effects of miR-625 on proliferation,cell cycle,invasion and migration of ECA109 cells were examined by using CCK-8 assay,flow cytometry,Transwell chamber assay and scratch assay.Meantime,expressions of proliferation,invasion and migration-related proteins were detected by Western blot.The target genes of miR-625 were predicted by bioinformatics software online,and the targeting relationships were verified by RT-qPCR,Western blot and dual luciferase reporter gene assays.The proliferation,cell cycle,invasion and migration abilities of ECA109 cells were detected after simultaneous intervention of miR-625 and HMGA1.Results The relative expressions of miR-625 in esophageal cancer tissues were lower than those in adjacent normal tissues,and the expressions in esophageal cancer cell lines KYSE150,KYSE30,ECA109 and EC9706 were lower than those in esophageal epithelial cell line HET-1A(P<0.05).Compared with those in mimic NC group,relative expression of miR-625,proportion of cells in G1 phase,and expressions of p21 and E-cadherin were increased,while cell proliferation,invasion,migration abilities,proportion of cells in S phase,and expressions of Cyclin D1,N-cadherin and Vimentin were decreased in miR-625 mimic group(P<0.05),while miR-625 inhibitor group could reverse the above increase and decrease effects(P<0.05).A binding site was found
作者
李大伟
刘磊
LI Da-wei;LIU Lei(Department of Cardiothoracic Surgery,the Affiliated Hospital of Hubei University of Arts and Science Xiangyang Central Hospital,Xiangyang,Hubei 441021,China;Department of Oncology,the Affiliated Hospital of Hubei University of Arts and Science Xiangyang Central Hospital,Xiangyang,Hubei 441021,China)
出处
《临床误诊误治》
CAS
2021年第11期107-112,116,共7页
Clinical Misdiagnosis & Mistherapy
基金
湖北省自然基金资助项目(2017CCV063)。
