肿瘤蛋白 53靶基因 1靶向微小 RNA-33a对胶质瘤细胞增殖、凋亡、迁移、侵袭的影响

目的 探讨肿瘤蛋白53靶基因1(TP53TG1)对胶质瘤细胞增殖、凋亡及迁移侵袭的影响和机制.方法 本研究起止时间为2019年1—6月,人正常神经胶质细胞(HA)和神经胶质瘤细胞U251购于上海斯信生物科技有限公司.实时定量PCR(qRT-PCR)检测HA和U251细胞中TP53TG1的表达水平.构建过表达TP53TG1的U251细胞株,噻唑蓝(MTT)法用于评估细胞增殖能力,流式细胞术分析细胞凋亡,Transwell法测定迁移和侵袭数,Western blotting检测细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白激酶抑制剂(P21)、B细胞淋巴瘤-2(Bcl-2)、Bcl相关x蛋白(Bax)、基质金属蛋白酶2(MMP-2)、E钙黏蛋白(E-cad?herin)表达水平.双荧光素酶报告基因实验和Western blotting验证TP53TG1和微小RNA-33a(miR-33a)的靶向关系.结果 与人正常神经胶质细胞HA比较,神经胶质瘤细胞U251中TP53TG1的表达水平(1.03±0.10)比(0.28±0.03)]显著降低.过表达TP53TG1能够显著抑制U251细胞活力,下调Cyclin D1、Bcl-2和MMP-2蛋白水平,减少细胞迁移数(138±14.01)个比(72±7.26)个]和侵袭数(126±12.54)个比(65±6.63)个],上调P21、Bax和E-cadherin蛋白表达,促进细胞凋亡(8.16±0.86)％比(22.57±2.65)％].TP53TG1能够靶向负性调控miR-33a的表达.过表达miR-33a能够逆转miR-33a对U251细胞增殖、凋亡(22.68±2.69)％比(11.36±1.20)％]、迁移(70±7.05)个比(108±11.37)个]和侵袭(70±7.05)个比(108±11.37)个]的影响.结论 TP53TG1通过靶向miR-33a抑制胶质瘤细胞增殖、迁移和侵袭,促进细胞凋亡.

Effects of tumor protein 53 target gene 1 on proliferation, apoptosis, migration and invasion of glioma cells by targeting microRNA-33a

Objective To investigate the effect of tumor protein 53 target gene 1 (TP53TG1) on proliferation, apoptosis and migra-tion of glioma cells and its mechanism.Methods The research started and ended from January to June 2019. Human normal glial cells(HA) and glioma cells U251 were purchased from Shanghai Sixin Biotechnology Co., Ltd. Real-time quantitative PCR (qRT-PCR) wasused to detect the expression level of TP53TG1 in HA and U251 cells. The U251 cell line with the overexpression of TP53TG1 was con-structed. Cell proliferation ability was determined by Methyl Thiazolyl Tetrazolium (MTT) methods, apoptosis was calculated by flow cy-tometry, cell migration and invasion numbers were assessed by Transwell assay, expression of Cyclin D1, cyclin kinase inhibitor (P21),B-cell lymphoma-2 (Bcl-2), Bcl-associated x protein (Bax), matrix metalloproteinase 2 (MMP-2) and E-cadherin were detected by West-ern blotting. The dual luciferase reporter gene assay and Western blotting were to confirm the targeting regulatory relationship betweenTP53TG1 and miR-33a.Results Compared with human normal glial cells HA, the expression level of TP53TG1 (1.03±0.10) vs. (0.28±0.03)] in glioma cell line U251 was significantly decreased. Over-expression of TP53TG1 significantly inhibited the viability of U251 cells, down-regulated the levels of Cyclin D1, Bcl-2 and MMP-2 proteins, reduced the number of cell migration (138±14.01) vs. (72±7.26)] and invasion (126±12.54) vs. (65±6.63)], up-regulated the expression of P21, Bax and E-cadherin proteins, and promoted apoptosis (8.16±0.86)% vs. (22.57±2.65)%]. TP53TG1 negatively regulated miR-33a expression. Over-expression of miR-33a reversed the effects of miR-33a on proliferation, apoptosis (22.68±2.69)% vs. (11.36±1.20)%], migration (70±7.05) vs. (108±11.37)] and inva-sion (70±7.05) vs. (108±11.37)] of U251 cells.Conclusion TP53TG1 inhibits the proliferation, migration and invasion of glioma cells and promotes apoptosis by targeting miR-33a.