摘要
目的探讨MTERF3基因过表达对脑胶质瘤U251细胞线粒体基因表达及细胞增殖和迁移的影响。方法以U251细胞作为实验对象,构建MTERF3过表达的U251细胞稳定转染株。实验分为3组:空白对照组(无转染的野生型U251细胞)、阴性对照组(转染EGFP-Flag质粒的U251细胞)、实验组(转染MTERF3-Flag质粒的U251细胞)。采用半定量PCR和Western blot分别检测MTERF3过表达对U251细胞线粒体DNA拷贝量及线粒体编码蛋白的表达水平的影响;采用流式细胞术和XTT法分别检测MTERF3过表达对U251细胞周期及细胞增殖的影响;采用细胞划痕和Transwell小室迁移实验检测U251细胞迁移能力的变化。结果半定量PCR结果显示,与对照组比较,MTERF3过表达显著抑制U251细胞线粒体DNA的拷贝量(P<0.05);Western blot结果显示,与对照组比较,MTERF3过表达显著降低线粒体DNA编码ND1、ND6和CYB蛋白表达水平(P<0.05);XTT结果显示,与对照组比较,MTERF3过表达明显促进U251细胞的增殖(P<0.05);流式细胞术结果显示,与对照组比较,MTERF3过表达能促进U251细胞G1/S转换,未出现细胞周期阻滞和细胞凋亡。Transwell小室迁移实验结果显示,实验组在每个视野下迁移的细胞数[(435.00±42.26)个]显著多于空白对照组[(259.00±30.22)个]和阴性对照组[(252.00±27.58)个],组间差异极显著(P<0.01)。结论 MTERF3过表达对人脑胶质瘤U251细胞的线粒体DNA拷贝量和线粒体蛋白质表达有负调控作用,且上调U251细胞MTERF3的表达使细胞增殖和迁移能力显著提高,并且未出现细胞周期阻滞和细胞凋亡。
Objective To investigate the effect of overexpression MTERF3 on mitochondrial gene expression as well as cell proliferation and migration of brain glioma U251 cells. Methods A stably transfected U251 cell line with overexpression MTERF3 was established,and the U251 cells were divided into three groups:blank control group(wild-type U251 cells without transfection),negative control group(U251 cells transfected with egfp-flag plasmid),experimental groups(U251 cells transfected with MTERF3-flag plasmid). Semi-quantitative PCR and Western blot were used to detect the changes in the copy number of mitochondrial DNA and the expression level of mitochondrial coding proteins in the cells of glioma U251. Flow cytometry and XTT assay were used to detect the effect of MTERF3 overexpression on the cell cycle and cell proliferation. Transwell assay was used to measure the cell migration ability. Results Semi-quantitative PCR detection showed that overexpression of MTERF3 has significant inhibition effect on mitochondrial DNA copy number;Western blot detection showed that MTERF3 overexpression decrease the expression levels of mitochondrial ND1,ND6,and CYB protein. The results of XTT assay showed that MTERF3 overexpression promote the proliferation of U251 cells;Flow cytometry analysis of cell cycle found that the MTERF3 overexpression can promote U251 cells from G1 to S phase,and cell cycle was not arrested and cell apoptosis was not induced. The count of migrated cells were remarkalby higher in the experimental group[(435.00±42.26)] than in the blank control group[(259.00±30.22)]and negative control group[(252.00±27.58)](P<0.05). Conclusion Overexpression of MTERF3 has a negative regulatory effect on the copy number of mitochondrial DNA and mitochondrial gene expression in human glioma U251 cells,and the overexpression of MTERF3 can accelerate the proliferation and migration of the U251 cells,but without inducing cell cycle arrest and cell apoptosis.
作者
孙美涛
梅雯
王唯斯
李素芬
自加吉
张晓娟
熊伟
SUN Mei-tao;MEI Wen;WANG Wei-si(Preclinical School,Dali University,Dali,Yunnan 671000,China)
出处
《实用医药杂志》
2019年第5期454-459,共6页
Practical Journal of Medicine & Pharmacy
基金
国家自然科学基金(81560458
31601155)
