胶质瘤中HuR通过上调PTBP1表达激活Akt通路对胶质瘤细胞的作用研究

目的探讨人类抗原R(HuR)在胶质瘤中的作用及其可能的作用机制。方法收集胶质瘤组织和正常对照组织各21例。体外培养正常胶质细胞株SVGp12和人胶质母细胞瘤来源的细胞株U251，qRT-PCR检测组织和细胞中HuR和多嘧啶结合蛋白1(PTBP1) mRNA表达；Western Blot检测组织和细胞中HuR、PTBP1、磷酸化蛋白激酶B(p-Akt)和蛋白激酶B(Akt)蛋白表达；RNA结合蛋白免疫沉淀实验检测HuR对PTBP1的调控作用；CCK-8检测细胞活力；EdU实验检测细胞增殖能力；流式细胞术检测细胞凋亡。结果与正常对照组相比，胶质瘤组HuR mRNA和蛋白表达、PTBP1的mRNA和蛋白表达显著升高（均P＜0.05）。与SVGp12组相比，U251组细胞中HuR mRNA和蛋白表达、PTBP1 mRNA和蛋白表达显著升高（均P＜0.05）。HuR通过结合PTBP1 mRNA调节PTBP1的表达。空白组和si-NC+oe-NC组细胞中各指标差异无统计学意义（均P＞0.05）。与si-NC+oe-NC组相比，si-HuR组+oe-NC组细胞中HuR、PTBP1 mRNA和蛋白表达均显著降低（均P＜0.05），48 h和72 h的细胞活力以及细胞增殖能力显著降低（均P＜0.05），细胞凋亡显著升高（P＜0.05），p-Akt/Akt蛋白表达显著降低（P＜0.05）；与si-HuR+oe-NC组相比，si-HuR+oe-PTBP1组细胞中HuR mRNA和蛋白表达差异无统计学意义（均P＞0.05），PTBP1 mRNA和蛋白表达显著升高（均P＜0.05），48 h和72 h的细胞活力以及细胞增殖能力显著升高（均P＜0.05），细胞凋亡显著降低（P＜0.05），p-Akt/Akt蛋白表达显著升高（P＜0.05）。结论HuR可能通过上调PTBP1表达激活Akt通路促进胶质瘤细胞增殖，抑制细胞凋亡。

Abstract: Objective To investigate the role of human antigen R(HuR) in glioma and its possible mechanism. Methods 21 cases of glioma tissue and normal control tissue were collected. Normal glioma cell line SVGp12 and human glioblastoma-derived cell line U251 were cultured in vitro, qRT-PCR was used to detect the mRNA expression of HuR and polypyrimidine tract-binding protein 1(PTBP1) in tissues and cells. Western Blot was used to detect the protein expression of HuR, PTBP1, phosphorylated protein kinase B(p-Akt) and protein kinase B(Akt) in tissues and cells. RNA-binding protein immunoprecipitation test was used to detect the regulatory effect of HuR on PTBP1. CCK-8 was used to detect cell viability. EdU test was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Results Compared with the normal control group, the mRNA and protein expression of HuR, and the mRNA and protein expression of PTBP1 in the glioma group were significantly increased(all P<0.05). Compared with the SVGp12 group, the mRNA and protein expression of HuR, and the mRNA and protein expression of PTBP1 in the U251 group were significantly increased(all P<0.05). HuR regulated the expression of PTBP1 by binding to PTBP1 mRNA. There was no statistically significant difference in the indicators between the blank group and the si-NC+oe-NC group(all P>0.05). Compared with the si-NC+oe-NC group, the mRNA and protein expressions of HuR and PTBP1 in the si-HuR group+oe-NC group were significantly reduced(all P＜0.05), and the cell viability at 48 h and 72 h and the proliferation ability of cell were significantly reduced(all P<0.05), cell apoptosis was significantly increased(P<0.05), and the protein expression of p-Akt/Akt was significantly reduced(P<0.05). Compared with the si-HuR+oe-NC group, the mRNA and protein expression of HuR in the cells of the si-HuR+oe-PTBP1 group was not statistically different(all P>0.05), while the expression of PTBP1 mRNA and protein was significantly increased(all P<0.05), cell viability at 48 h and 72 h and the proliferation ability of cell were significantly increased(all P＜0.05). Cell apoptosis was significantly reduced(P＜0.05), and the protein expression of p-Akt/Akt was significantly increased(P＜0.05). Conclusion HuR may activate Akt pathway by up-regulating the expression of PTBP1 to promote the proliferation of glioma cells and inhibit cell apoptosis.