HOTAIRM1靶向miR-129-5p对胶质瘤细胞增殖、迁移、侵袭的影响

The effect of HOTAIRM1 targeting miR-129-5p on the proliferation,migration and invasion of glioma cells

目的:研究长链非编码RNA HOTAIRM1(lncRNA HOTAIRM1)与微小RNA-129-5p(miR-129-5p)的靶向关系及其对胶质瘤细胞增殖、迁移、侵袭的影响。方法:荧光定量PCR(qPCR)检测HOTAIRM1和miR-129-5p在人正常脑组织和胶质瘤组织中的表达。建立抑制HOTAIRM1表达细胞株,研究其对U251细胞增殖、凋亡、迁移及侵袭的影响。MTT法检测细胞增殖;流式细胞仪检测细胞凋亡;Transwell小室法检测细胞迁移和侵袭;蛋白质印迹法(Western blot)检测细胞周期蛋白D1(Cyclin D1)、p21、B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)、上皮钙黏素(E-cadherin)、基质金属蛋白酶-2(MMP-2)水平。生物学信息预测和双荧光素酶报告基因法分析HOTAIRM1和miR-129-5p之间的靶向关系。共转染si-HOTAIRM1和anti-miR-129-5p,观察抑制miR-129-5p表达对抑制HOTAIRM1表达诱导的U251细胞增殖、凋亡、迁移及侵袭的影响。结果:HOTAIRM1在胶质瘤组织中的表达明显上调(P<0.05),miR-129-5p表达下调(P<0.05)。抑制HOTAIRM1表达显著降低U251细胞24 h、48 h、72 h的细胞活性、迁移细胞数、侵袭细胞数及Cyclin D1、Bcl-2、MMP-2蛋白表达量(P<0.05),明显增加U251细胞凋亡率和p21、Bax、E-cadherin蛋白表达量(P<0.05)。miR-129-5p是HOTAIRM1的靶基因。上调或下调HOTAIRM1表达明显调控miR-129-5p表达(P<0.05)。抑制miR-129-5p表达逆转了抑制HOTAIRM1表达对U251细胞24 h、48 h、72 h的细胞活性、迁移细胞数、侵袭细胞数及Cyclin D1、MMP-2、Bcl-2蛋白表达的抑制作用,并逆转了抑制HOTAIRM1表达对U251细胞p21、E-cadherin、Bax蛋白表达和细胞凋亡率的促进作用。结论:lncRNA HOTAIRM1通过靶向miR-129-5p影响胶质瘤细胞增殖、凋亡、迁移和侵袭。

Objective:To study the targeting relationship of lncRNA HOTAIRM1 to miR-129-5p and its effect on proliferation,migration and invasion of glioma cells.Methods:qPCR was used to detect the expression of HOTAIRM1 and miR-129-5p in human normal brain tissues and glioma tissues.The inhibiting of HOTAIRM1 expression cell lines were established to investigate its effects on U251 cell proliferation,apoptosis,migration and invasion.MTT assay was used for cell proliferation.Flow cytometry detected cell apoptosis.Transwell cell assay detected cell migration and invasion.Western blot detected Cyclin D1,p21,Bcl-2,Bax,E-cadherin and MMP-2 level.Bioinformatics prediction and dual luciferase reporter gene assay were used to analyze the targeting relationship between HOTAIRM1 and miR-129-5p.si-HOTAIRM1 and anti-miR-129-5p were co-transfected to observe the influences of inhibiting miR-129-5p expression on the proliferation,apoptosis,migration and invasion of U251 cells induced by inhibiting HOTAIRM1 expression.Results:The expression of HOTAIRM1 was significantly up-regulated in glioma tissues(P<0.05),and the expression of miR-129-5p was down-regulated(P<0.05).Inhibition of HOTAIRM1 expression obviously decreased the cell activity at 24 h,48 h and 72 h,migration cell number,invasive cell number and the expression of Cyclin D1,Bcl-2 and MMP-2 in U251 cells(P<0.05),and significantly increased the apoptotic rate of U251 cells and the expression of p21,Bax and E-cadherin(P<0.05).miR-129-5p was the target gene of HOTAIRM1.Up-regulation or down-regulation of HOTAIRM1 expression remarkably regulated the expression of miR-129-5p(P<0.05).Inhibiting miR-129-5p expression reversed the suppressive effects of inhibiting HOTAIRM1 expression on the activity of U251 cells at 24 h,48 h and 72 h,the expression of Cyclin D1,MMP-2,Bcl-2,the number of migrating cells and invasive cells,and reversed the promotion effects of p21,E-cadherin,Bax protein expression and apoptotic rate of U251 cells.Conclusion:lncRNA HOTAIRM1 affects the proliferation,apoptosis,migration and invasion of glioma cells by targeting miR-129-5p.