miR-325-3p通过靶向FOXM1调控乳腺癌细胞增殖、侵袭、迁移及上皮-间质转化的实验研究

Experimental study of miR-325-3p regulating the proliferation,invasion,migration,and epithelial mesenchymal transformation of breast cancer cells by targeting FOXM1

目的探讨miR-325-3p通过靶向叉头框蛋白(FOX)M1调控乳腺癌细胞增殖、侵袭、迁移及上皮间质转化(EMT)。方法培养MCF-7细胞,根据转染质粒不同分为NC组(转染miRNA mimic阴性对照)、miR-325-3p组(转染miR-325-3p mimic)、miR-NC组(转染miRNA inhibitor阴性对照)、miR-325-3p inhibitor组(转染miR-325-3p inhibitor)、pcDNA3.1组(转染pcDNA3.1空质粒)、FOXM1组(转染pcDNA3.1-FOXM1)、si-NC组(转染si-NC无意义序列)、si-FOXM1组(转染si-FOXM1质粒)、miR-325-3p inhibitor+si-FOXM1组(转染miR-325-3p inhibitor+si-FOXM1质粒)及miR-325-3p inhibitor+si-NC组(转染miR-325-3p inhibitor+si-NC无意义序列)。CCK-8检测细胞24、48、72 h增殖能力,Transwell实验检测细胞侵袭能力,划痕实验检测细胞迁移能力,RT-qPCR检测细胞miR-325-3p、FOXM1 mRNA水平,Western blot检测细胞FOXM1、E-cadherin、N-cadherin及Vimentin蛋白水平,萤光素酶实验检测miR-325-3p与FOXM1的靶向关系。结果miR-325-3p组miR-325-3p表达水平高于NC组(P<0.01),miR-325-3p inhibitor组miR-325-3p水平低于miR-NC组(P<0.01)。48、72 h,miR-325-3p组光密度(OD)值、侵袭细胞数及迁移率低于NC组(P<0.01),miR-325-3p inhibitor组OD值,侵袭细胞数及迁移率高于miR-NC组(P<0.01)。miR-325-3p组E-cadherin蛋白表达水平高于NC组,Vimentin、N-cadherin蛋白表达水平低于NC组(P<0.01);miR-325-3p inhibitor组E-cadherin蛋白表达水平低于miR-NC组,Vimentin、N-cadherin蛋白表达水平高于miR-NC组(P<0.01)。FOXM1组细胞FOXM1蛋白、mRNA表达水平高于pcDNA3.1组(P<0.01);si-FOXM1组FOXM1蛋白、mRNA表达水平低于si-NC组(P<0.01)。48、72 h,FOXM1组OD值、侵袭细胞数及迁移率高于pcDNA3.1组(P<0.01);si-FOXM1组OD值,侵袭细胞数及迁移率低于si-NC组(P<0.01)。FOXM1组E-cadherin蛋白表达水平低于pcDNA3.1组,Vimentin、N-cadherin蛋白表达水平高于pcDNA3.1组(P<0.01);si-FOXM1组E-cadherin蛋白表达水平高于si-NC组,Vimentin、N-cadherin蛋白表达水平低于si-NC组(P<0.01)。48、72 h,miR-325-3p inhibitor+si-FOXM1组OD值、侵袭细胞数及迁移率低于miR-325-3p inhibitor+si-NC组(P<0.01)。miR-325-3p inhibitor+si-FOXM1组E-cadherin蛋白表达水平高于miR-325-3p inhibitor+si-NC组,Vimentin、N-cadherin蛋白表达水平低于miR-325-3p inhibitor+si-NC组(P<0.01)。miR-325-3p组FOXM1蛋白、mRNA表达水平低于NC组(P<0.01);miR-325-3p inhibitor组FOXM1蛋白、mRNA表达水平高于miR-NC组(P<0.01)。NC+WT-FOXM1组与NC+MUT-FOXM1组萤光素酶活性比较,差异无统计学意义(P>0.05);miR-325-3p+WT-FOXM1组萤光素酶活性低于miR-325-3p+MUT-FOXM1组(P<0.01)。结论miR-325-3p在乳腺癌组织中低表达,过表达miR-325-3p可靶向FOXM1抑制乳腺癌细胞的增殖、侵袭、迁移及EMT。

Objective To investigate the regulation of miR-325-3p on proliferation,invasion,migration,and epithelial mesenchymal transformation(EMT)of breast cancer cells by targeting forkhead box protein(FOXM1).Methods MCF-7 cells were cultured and divided into NC group(transfected miRNA mimic negative control),miR-325-3p group(transfected miR-325-3p mimic),miR-NC group(transfected miRNA inhibitor negative control),miR-325-3p inhibitor group(transfected with miR-325-3p inhibitor),pcDNA3.1 group(transfected with pcDNA3.1 empty plasmid),FOXM1 group(transfected with PCDNA3.1-FoxM1),si-NC group(transfected with si-NC meaningless sequence),si-FOXM1 group(transfected with si-FOXM1 plasmid),miR-325-3p inhibitor+si-FOXM1 group(transfected with miR-325-3p inhibitor+si-FOXM1 plasmid),and miR-325-3p inhibitor+si-NC group(transfected with miR-325-3p inhibitor+si-NC meaningless sequence).CCK-8 was used to detect cell proliferation at 24,48 h,and 72 h,Transwell assay was used to detect cell invasion,scratch assay was used to detect cell migration,the levels of miR-325-3p and FOXM1 mRNA were detected by RT-qPCR,the levels of FOXM1,E-cadherin,N-cadherin,and Vimentin protein were detected by Western blot,the targeting relationship between miR-325-3p and FOXM1 was detected by luciferase assay.Results The expression level of miR-325-3p in miR-325-3p group was higher than that in NC group(P<0.01),and the level of miR-325-3p in miR-325-3p inhibitor group was lower than that in miR-NC group(P<0.01).At 48 h and 72 h,optical density(OD)value,invasion cell number,and migration of miR-325-3p group were lower than those of NC group(P<0.01),while OD value,invasion cell number,and migration of miR-325-3p inhibitor group were higher than those of miR-NC group(P<0.01).The expression level of E-cadherin protein in miR-325-3p group was higher than that in NC group,while the expression levels of Vimentin and N-cadherin protein in miR-325-3P group were lower than those in NC group(P<0.01);the expression level of E-cadherin protein in miR-325-3p inhibitor group was lower than that in miR-NC group,while the expression levels of Vimentin and N-cadherin protein in miR-NC group were higher than those in miR-NC group(P<0.01).The expression levels of FOXM1 protein and mRNA in FOXM1 group were higher than those in pcDNA3.1 group(P<0.01);the expression levels of FOXM1 protein and mRNA in si-FOXM1 group were lower than those in si-NC group(P<0.01).At 48 h and 72 h,OD value,invasion cell number,and migration in FOXM1 group were higher than those in pcDNA3.1 group(P<0.01);OD value,invasion cell number,and migration of si-FOXM1 group were lower than those of si-NC group(P<0.01).The expression level of E-cadherin protein in FOXM1 group was lower than that in pcDNA3.1 group,while the expression levels of Vimentin and N-cadherin protein in FXOM1 group were higher than those in pcDNA3.1 group(P<0.01);the expression level of E-cadherin protein in si-FOXM1 group was higher than that in si-NC group,while the expression levels of Vimentin and N-cadherin protein were lower than those in Si-NC group(P<0.01).At 48 h and 72 h,OD value,invasion cell number,and migration of miR-325-3p inhibitor+si-FOXM1 group were lower than those of miR-325-3p inhibitor+si-NC group(P<0.01).The expression level of E-cadherin protein in miR-325-3p inhibitor+si-FOXM1 group was higher than that of miR-325-3p inhibitor+si-NC group,while the expression levels of Vimentin and N-cadherin protein were lower than those of miR-325-3p inhibitor+si-NC group(P<0.01).The expression levels of FOXM1 protein and mRNA in miR-325-3p group were lower than those in NC group(P<0.01);the expression levels of FOXM1 protein and mRNA of miR-325-3p inhibitor group were higher than those of miR-NC group(P<0.01).There was no significant difference in luciferase activity between NC+WT-FOXM1 group and NC+MUT-FOXM1 group(P>0.05);the luciferase activity of miR-325-3p+WT-FOXM1 group was lower than that of miR-325-3p+MUT-FOXM1 group(P<0.01).Conclusion miR-325-3p is underexpressed in breast cancer tissues,and overexpression of miR-325-3p can inhibit proliferation,invasion,migration,and EMT of breast cancer cells by targeting FOXM1.