乳酸诱导PLEKHA4的上调参与胶质瘤细胞增殖和凋亡的生物学进程

Lactate-induced up-regulation of PLEKHA4 promotes proliferation and apoptosis of human glioma cells

目的探究乳酸诱导的PLEKHA4基因表达对胶质瘤细胞的生物学功能的影响以及潜在分子机制。方法通过GEO数据库以及GEPIA2在线网站分析PLEKHA4表达水平与胶质瘤病理分级的关系,通过RNA小干扰技术设计PLEKHA4 siRNA后分别转染胶质瘤U251和T98G细胞处理1 d,通过实时细胞分析技术以及Edu实验检测胶质瘤细胞的增殖能力,平板克隆实验检测细胞的克隆形成能力,流式分析技术检测胶质瘤细胞的周期和凋亡;PCR检测临床收集的胶质瘤样本和对照以及乳酸和葡萄糖治疗下胶质瘤细胞中PLEKHA4的mRNA表达量的变化;体内异种移植小鼠实验检测裸鼠成瘤能力;Westernblot检测cyclinD1、CDK2、Bcl2、β-catenin表达和MAPK信号通路关键蛋白的磷酸化表达水平。结果GEO数据库和在线网站分析结果显示PLEKHA4在胶质瘤组织中高表达且与不良预后相关;RTCA以及Edu实验显示,敲降PLEKHA4抑制胶质瘤细胞的增殖(P<0.05);平板克隆实验显示,敲降组的细胞克隆数目明显低于对照组(P<0.01);流式细胞术分析结果显示敲低PLEKHA4后细胞周期受到阻滞G1期细胞增加,S期细胞减少,凋亡细胞增加(P<0.01);胶质瘤样本和乳酸和葡萄糖治疗后胶质瘤细胞的PLEKHA4基因的mRNA表达升高(P<0.01);敲降PLEKHA4基因后,裸鼠成瘤能力降低;Westernblot结果显示敲减PLEKHA4后能够抑制cyclinD1、CDK2、Bcl2等功能蛋白的表达,且显著抑制ERK、p38的磷酸化和β-catenin蛋白表达(P<0.01)。结论敲减PLEKHA4基因通过抑制MAPK信号通路的活化和β-Catenin的表达,抑制胶质瘤细胞的增殖促进凋亡,同时糖酵解产生的乳酸诱导PLEKHA4表达上调。

Objective To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism.Methods GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma.A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells,and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment.The colony-forming ability of the cells was evaluated using plate cloning assay,and cell cycle changes and cell apoptosis were analyzed with flow cytometry.The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose.Xenograft mice in vivo was used to detect tumor formation in nude mice;Western blotting was used to detect the expressions of cyclinD1,CDK2,Bcl2,β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway.Results The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis;PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells(P<0.05).Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells(P<0.01).PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment(P<0.01).PLEKHA4 knockdown,tumor formation ability of nude mice decreased;PLEKHA4 knockdown obviously lowered the expression of cyclinD1,CDK2,Bcl2 and other functional proteins,inhibited the phosphorylation of ERK and p38 and reduced the expression ofβ-catenin protein(P<0.01).Conclusion PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression ofβ-catenin.Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.