摘要
目的观察人卵巢癌(OC)细胞系细胞微小RNA-211-5p(miR-211-5p)表达、转染miR-211-5p模拟物(miR-211-5p mimics)后的增殖及迁移侵袭和上皮间质转化(EMT)。方法体外培养人OC细胞系(SKOV3、A2780、HO8910细胞)及人正常卵巢上皮IOSE80细胞,采用qRT-PCR法检测上述细胞miR-211-5p。取对数生长期的SKOV3细胞,并随机分为两组,高表达组转染miR-211-5p mimics,对照组转染miR-NC,采用CCK-8实验、细胞划痕实验、Transwell实验、Western blotting法分别测算两组细胞OD值、划痕移动距离百分比、侵入细胞数、EMT相关蛋白及壳多糖酶3样蛋白1(CHI3L1)蛋白灰度值比值。将miR-211-5p mimics或miR-NC与CHI3L1野生型双荧光素酶报告载体CHI3L1-wt或CHI3L1突变型双荧光素酶报告载体CHI3L1-mut共转染入SKOV3细胞中,分为记为miR-211-5p mimics+CHI3L1-wt组、miR-NC+CHI3L1-wt组、miR-211-5p mimics+CHI3L1-mut组、miR-NC+CHI3L1-mut组,转染24 h后收集并裂解细胞,应用荧光素酶试剂盒检测各组细胞的荧光素酶活性。结果与IOSE80细胞比较,SKOV3、A2780、HO8910细胞miR-211-5p相对表达量降低(P均<0.05)。与对照组比较,高表达组OD值、划痕移动距离百分比、侵入细胞数、N-Cadherin和Fibronectin蛋白灰度值比值降低,E-Cadherin蛋白灰度值比值升高(P均<0.05)。与对照组比较,高表达组CHI3L1蛋白灰度值比值降低(P<0.05)。与miR-NC+CHI3L1-wt组比较,miR-211-5p mimics+CHI3L1-wt组细胞相对荧光素酶活性降低(P<0.05)。结论miR-211-5p在OC各细胞系中表达降低;转染miR-211-5p mimics可抑制SKOV3细胞增殖、迁移、侵袭及EMT,机制是通过负向调控CHI3L1表达实现的。
Objective To observe the expression of microRNA-211-5p(miR-211-5p)in human ovarian cancer(OC)cell lines and the cell proliferation,migration,invasion,and epithelial mesenchymal transition(EMT)in cells after transfection of miR-211-5p mimics.Methods Human OC cell lines(SKOV3,A2780,HO8910 cells)and human normal ovarian epithelial IOSE80 cells were cultured in vitro,and the miR-211-5p in these cells was detected using qRTPCR.SKOV3 cells in the logarithmic growth phase were selected and randomly divided into two groups.Cells in the high expression group were transfected with miR-211-5p mimics,while cells in the control group were transfected with miRNC.The OD value,percentage of scratch movement distance,the number of invading cells,gray value ratio of EMT-related protein and chitosanase 3-like protein 1(CHI3L1)protein were measured by CCK-8 experiment,cell scratch experiment,Transwell experiment,and Western blotting,respectively.The miR-211-5p mimics or miR-NC and CHI3L1 wildtype dual luciferase reporter vector CHI3L1-wt or CHI3L1 mutant dual luciferase reporter vector CHI3L1-mut were cotransfected into SKOV3 cells,which were taken as the miR-211-5p mimics+CHI3L1-wt group,miR-NC+CHI3L1-wt group,miR-211-5p mimics+CHI3L1 mut group,and miR-NC+CHI3L1 mut group,respectively.After 24 h of transfection,cells were collected and lysed,and the luciferase activity of cells in each group was detected using the luciferase as-say kit.Results Compared with the IOSE80 cells,the relative expression of miR-211-5p in SKOV3,A2780,and HO8910 cells decreased(all P<0.05).Compared with the control group,the OD value,the percentage of scratch move-ment distance,the number of invading cells,and gray value ratio of N-Cadherin and Fibronectin protein in the high expres-sion group decreased,while the gray value ratio of E-Cadherin protein increased(all P<0.05).Compared with the control group,the gray value ratio of CHI3L1 protein in the high expression group decreased(P<0.05).Compared with the miR-NC+CHI3L1-wt group,the relative luciferase activity decreased in the miR-211-5p mimics+CHI3L1-wt group(P<0.05).Conclusion The expression of miR-211-5p is reduced in OC cell lines;transfection with high expression miR-211-5p can inhibit SKOV3 cell proliferation,migration,invasion,and EMT,and the mechanism is achieved through negative reg-ulation of CHI3L1 expression.
作者
杨立芬
何建清
尹立新
YANG Lifen;HE Jianqing;YIN Lixin(Department of Obstetrics and Gynecology,Tangshan Maternal and Child Health Hospital,Tangshan 063000,China;不详)
出处
《山东医药》
CAS
2023年第36期5-9,共5页
Shandong Medical Journal
基金
河北省医学科学研究课题计划(20191534)。