LncRNA RGMB-AS1靶向miR-574-3p影响人前列腺癌细胞株DU145增殖、迁移与侵袭

LncRNA RGMB⁃AS1 targeting miR⁃574⁃3p affects the proliferation,migration and invasion of human prostate cancer cells(DU145)

目的观察长链非编码核糖核酸(LncRNA)排斥导向分子B-反义链(RGMB-AS1)对人前列腺癌细胞株DU145增殖、迁移与侵袭的影响,并探讨其对微小核糖核酸-574-3p(miR-574-3p)的靶向作用。方法取人前列腺癌细胞株DU145,培养传代。设RGMB-AS1上调组(转染pcDNA3.1-RGMB-AS1)、RGMB-AS1上调对照组(转染pcDNA3.1-control)、RGMB-AS1下调组(转染si-RGMB-AS1)、RGMB-AS1下调对照组(转染si-NC)、miR-574-3p上调组(转染miR-574-3p mimics)、miR-574-3p上调对照组(转染miR mimics-NC)、miR-574-3p下调组(转染miR-574-3p inhibitor)、miR-574-3p下调对照组(转染miR inhibitor-NC)、空白组,每组3个复孔。48 h后细胞增殖实验(CCK-8)法检测增殖活性并计算增殖抑制率;细胞划痕愈合实验检测迁移活性;Transwell小室检测侵袭活性;实时逆转录聚合酶链反应(RT-qPCR)检测LncRNA RGMB-AS1、miR-574-3p表达及细胞周期蛋白D1(Cyclin D1)、P21、锌指E盒结合蛋白1(ZEB1)、N-钙黏蛋白(N-cadherin)、E-钙黏蛋白(E-cadherin)、波形蛋白(VIM)基因表达;免疫印迹法检测Cyclin D1、P21、ZEB1、N-cadherin、E-cadherin、VIM蛋白表达;双荧光素酶基因报告实验检测LncRNA RGMB-AS1是否靶向miR-574-3p。结果与空白组、RGMB-AS1上调对照组、RGMB-AS1下调对照组、miR-574-3p上调对照组、miR-574-3p下调对照组比较,RGMB-AS1上调组、miR-574-3p上调组D值、划痕愈合率、侵袭细胞数、Cyclin D1、ZEB1、N-cadherin、VIM表达均下降(P<0.05),增殖抑制率、P21、E-cadherin表达均升高(P<0.05),RGMB-AS1下调组、miR-574-3p下调组D值、划痕愈合率、侵袭细胞数、Cyclin D1、ZEB1、N-cadherin、VIM表达均升高(P<0.05),增殖抑制率、P21、E-cadherin表达均下降(P<0.05)。与miR-NC组相比,miR-574-3p mimics组Wt-RGMB-AS1荧光素酶活性降低(P<0.05),Mut-RGMB-AS1荧光素酶活性无统计学意义改变(P>0.05)。结论上调LncRNA RGMB-AS1可靶向上调miR-574-3p抑制人前列腺癌细胞株DU145增殖、迁移与侵袭,推测与抑制Cyclin D1、ZEB1、N-cadherin、VIM表达,上调P21、E-cadherin表达有关。

Objective To investigate the effects of long non⁃coding ribonucleic acid(LncRNA)repulsive guidance molecules B⁃antisense chain(RGMB⁃AS1)on the proliferation,migration and invasion of human prostate cancer cells(DU145),and explore its targeting effect on miR⁃574⁃3p.Methods Human prostate cancer cells(DU145)were cultured and used in the study.Cell tests were divided into 9 groups including RGMB⁃AS1 upregulation group(transfected with pcDNA3.1⁃RGMB⁃AS1),RGMB⁃AS1 upregulation control group(transfected with pcDNA3.1 control),RGMB⁃AS1 downregulation group(transfected with si⁃RGMB⁃AS1),RGMB⁃AS1 downregulation control group(transfected with si⁃NC),miR⁃574⁃3p upregulation group(transfected with miR⁃574⁃3p mimics),miR⁃574⁃3p upregulation control group(transfected with miR mimics NC),miR⁃574⁃3p downregulation group(transfected with miR⁃574⁃3p inhibitor),miR⁃574⁃3p downregulated control group(transfected with miR inhibitor NC)and blank group,and 3 wells in each group.After 48 hours culture,cell proliferation activity was detected by CCK⁃8 assay.Cell migration ability was evaluated by wound healing assay.Cell invasive ability was assessed by Matrigel invasion assay(Transwell chamber).The expression of LncRNA RGMB⁃AS1,the expression of miR⁃574⁃3p,and the gene expressions of Cyclin D1,P21,ZEB1,N⁃cadherin,E⁃cadherin,and Vimentin were detected by LncRNA⁃RT⁃qPCR,miRNA⁃RT⁃qPCR and RT⁃qPCR,respectively.The protein expressions of Cyclin D1,P21,ZEB1,N⁃cadherin,E⁃cadherin,and VIM were measured by western blot.The dual luciferase gene reporter assay was used to determine whether LncRNA RGMB⁃AS1 targets miR⁃574⁃3p.Results Compared with those in the blank group,RGMB⁃AS1 up⁃regulated control group,RGMB⁃AS1 down⁃regulated control group,miR⁃574⁃3p up⁃regulated control group,and miR⁃574⁃3p down⁃regulated control group,the D value,scratch healing rates,invasive cell counts,Cyclin D1,ZEB1,N⁃cadherin,and VIM expressions in the RGMB⁃AS1 up⁃regulated group and miR⁃574⁃3p up⁃regulated group were all decreased(P<0.05),while the proliferation inhibition rates,P21,and E⁃cadherin expressions were all increased(P<0.05).Simultaneously,the D value,scratch healing rates,invasive cell counts,Cyclin D1,ZEB1,N⁃cadherin,and VIM expressions in the RGMB⁃AS1 down⁃regulated group and miR⁃574⁃3p down⁃regulated group were all increased(P<0.05),while the proliferation inhibition rates,P21,and E⁃cadherin expressions were all decreased(P<0.05).Compared with the miR⁃NC group,the miR⁃574⁃3p mimics group showed a decrease in Wt⁃RGMB⁃AS1 luciferase activity(P<0.05),but little change in Mut RGMB⁃AS1⁃luciferase activity(P>0.05).Conclusion Upregulation of LncRNA RGMB⁃AS1 can target upregulation of miR⁃574⁃3p to inhibit the proliferation,migration,and invasion of human prostate cancer cells,which may be attributed to downregulation of Cyclin D1,ZEB1,N⁃cadherin,VIM expressions,and upregulation of P21 and E⁃cadherin expressions.