下调lncRNA TTTY15靶向miR-4500抑制胶质瘤细胞A172生物学特性的实验研究

Down-regulation of lncRNA TTTY15 targeting miR-4500 to inhibit the biological characteristics of A172 gliomacells

目的探讨下调lncRNA TTTY15靶向miR-4500对胶质瘤细胞A172增殖、凋亡、迁移、侵袭的影响及机制。方法用qRT-PCR法检测胶质瘤细胞和组织中TTTY15表达的差异。用Starbase软件预测TTTY15的靶基因, 发现其与miR-4500存在互补结合位点, 用荧光素酶报告系统鉴定二者的靶向关系。将胶质瘤细胞A172分为对照组、si-NC(转染siRNA对照组)、si-TTTY15(转染TTTY15 siRNA)、si-TTTY15+Anti-miR-NC(共转染TTTY15 siRNA、inhibitor对照组)和si-TTTY15+Anti-miR-4500组(共转染TTTY15 siRNA、miR-4500 inhibitor), 分别用CCK-8法和流式细胞术检测细胞增殖和凋亡, 用Transwell小室检测细胞的迁移和侵袭, 用Western印迹法检测Bax、Bcl-2、MMP-2、MMP-9蛋白的表达。结果胶质瘤细胞和组织的TTTY15表达均升高。TTTY15与miR-4500的表达呈负相关。TTTY15与miR-4500互为靶向关系。与对照组、si-NC组相比, si-TTTY15组胶质瘤细胞miR-4500表达升高, A172细胞存活率降低, 凋亡率升高, 细胞迁移数目和侵袭数目增多, Bax蛋白表达升高, Bcl-2、MMP-2、MMP-9蛋白表达降低(P < 0.05)。与si-TTTY15+Anti-miR-NC组相比, si-TTTY15+Anti-miR-4500组胶质瘤细胞中miR-4500表达降低, 细胞存活率升高, 凋亡率降低, 细胞迁移和侵袭数目增加, 细胞中Bax蛋白表达降低, Bcl-2、MMP-2、MMP-9蛋白表达升高(P < 0.05)。结论下调TTTY15靶向miR-4500可抑制胶质瘤细胞A172增殖、迁移、侵袭并诱导其凋亡。

Objective To explore the effect and mechanism of down-regulating lncRNA TTTY15targeting miR-4500 on the proliferation,apoptosis,migration and invasion of A172 glioma cells,MethodsThe difference in TTTY15 expression between the glioma cells and tissue was determined with a qRT-PCRmethod,Complementary binding sites of TTTY15 and miR-4500 were predicted with Starbase software,andthe targeting relationship was validated with a luciferase reporter system.A172 glioma cells were dividedinto Control,si-NC(transfected with control siRNA),si-TTTY15(transfected with TTTY15 siRNA),si-TTTY15+Anti-miR-NC(co-transfected with TTTY15 siRNA and inhibitor control)and si-TTTY15+Anti-miR-4500(co-transfected with TTTY15 siRNA and miR-4500 inhibitor)groups.Proliferation,apoptosis,migration and invasion,and the expression of Bax,Bc-2,MMP-2 and MMP-9 proteins of theA172 glioma cells were respectively detected with CCK-8,flow cytometry,Transwell chamber and Westernblotting assays.Results The expression of TTTY15 in glioma cells and glioma tissues have both increased.The expression levels of TTTY15 and miR-4500 in glioma tissues were inversely correlated.TTTY15 andmiR-4500 are mutually targeted.Compared with those of the Control and si-NC groups,the glioma cells inthe si-TTTY15 group showed increased level of miR-4500,decreased survival rate,increased apoptosis rate,enhanced cell migration and invasion,increased expression of Bax protein,and decreased expression of Bcl-2,MMP-2 and MMP-9 proteins(P<0.05).Compared with those of the si-TTTY15+Anti-miR-NC group,the A172 glioma cells in the si-TTTY15+Anti-miR-4500 group showed decreased level of miR-4500,increased cell survival rate,decreased apoptosis rate,enhanced cell migration and invasion,decreased expression of Bax protein,and increased expression of Bcl-2,MMP-2,and MMP-9 proteins(P<0.05).Conclusion Down regulating TTTY15 targeting miR-4500 can inhibit the proliferation,migration,invasion and induce apoptosis of the A172 glioma cells.