MTERF3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
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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.
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.
引文
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[20]张林冰,韩艳艳,仇石,等.线粒体转录终止因子蛋白家族的表达以及在MPTP/MPP~+诱导下的表达变化[J].解剖学报,2017,48(1):22-27.
[21]SHUTT TE,SHADEL GS.A compendium of human mitochondrial gene expression machinery with links to disease[J].Environ Mol Mutagen,2010,51(5):360-397.
[22]自加吉,杨勇琴,孙美涛,等.非小细胞肺癌中人线粒体转录终止因子3蛋白的表达及临床病理学意义[J].医学研究生学报,2017,30(2):160-164.
[2]夏立丰,李义荣,谌南武.MDM2-p53通路在脑胶质瘤中的表达及其意义探讨[J].中国实用医药,2007,2(18):22-24.
[3]SHAPIRO JR.Genetics of nervous system tumors[J].Hematol Oncol Clin North Am,2001,15(6):961-977.
[4]MORENO-SANCHEZ R,RODRIQUEZ-ENRIQUEZ S,MARIN-HERNANDEZ A,et al.Energy metabolism in tumor cells[J].FEBS J,2007,274(6):1393-1418.
[5]柯超,陈忠平.胶质瘤细胞能量代谢研究进展[J].中国神经肿瘤杂志,2013,11(4):270-275.
[6]冯浩,张箴,高兴春,等.线粒体与肿瘤发生发展[J].生命科学,2014,26(8):800-803.
[7]秦晓燕,邓艳春.线粒体基因突变与脑胶质瘤的发生[J].中华神经外科疾病研究杂志,2009,8(1):89-91.
[8]高磊,肖慧蓉,李非,等.线粒体动力学与胶质瘤发生发展[J].生命的化学,2016,36(5):625-628.
[9]熊伟,余敏,左绍远.线粒体转录终止因子蛋白家族在线粒体基因表达中的调节作用[J].中国生物化学与分子生物学报,2015,31(3):223-231.
[10]孙美涛,梅雯,王唯斯,等.线粒体转录终止因子2对人子宫颈癌He La细胞线粒体基因表达的影响[J].生物技术,2018,28(1):36-43.
[11]PARK CB,ASIN-CAYUELA J,CAMARA Y,et al.MTERF3is a negative regulator of mammalian mtDNA transcription[J].Cell,2007,130(2):273-285.
[12]ZHANG C,WU N,GAO F,et al.MTERFD1 functions as an oncogene[J].Oncotarget,2016,8(9):1-8.
[13]熊伟,张晓娟,张海洋,等.基于生物信息学方法预测人线粒体转录终止因子3蛋白的结构与功能[J].生物技术通讯,2015,26(3):367-373.
[14]CHIURAZZI P,POMPONI MG,PIETROBONO R,et al.Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene[J].Hum Mol Genet,1999,8(12):2317-2323.
[15]LOUIS DN,OHGAKI H,WIESTLER OD,et al.The 2007 WHOclassification of tumours of the central nervous system[J].Acta Neuropathol,2007,114(2):97-109.
[16]COLMAN H,ALDAPE K.Molecular predictors in glioblastoma:toward personalized therapy[J].Arch Neurol,2008,65(7):877-883.
[17]ANDERSSON DC,FAUCONNIER J,PARK CB.Enhanced cardiomyocyte Ca(2+)cycling precedes terminal AV-block in mitochondrial cardiomyopathy Mterf3 KO mice[J].Antioxid Redox Signal,2011,15(9):2455-2464.
[18]HYVARINEN AK,POHJOISMAKI JL,HOLT IJ,et al.Overexpression of MTERFD1 or MTERFD3 impairs the completion of mitochondrial DNA replication[J].Mol Biol Rep,2011,38(2):1321-1328.
[19]WREDENBERG A,LAGOUGE M,BRATIC A,et al.MTERF3regulates mitochondrial ribosome biogenesis in invertebrates and mammals[J].PLoS Genet,2013,9(1):e1003178.
[20]张林冰,韩艳艳,仇石,等.线粒体转录终止因子蛋白家族的表达以及在MPTP/MPP~+诱导下的表达变化[J].解剖学报,2017,48(1):22-27.
[21]SHUTT TE,SHADEL GS.A compendium of human mitochondrial gene expression machinery with links to disease[J].Environ Mol Mutagen,2010,51(5):360-397.
[22]自加吉,杨勇琴,孙美涛,等.非小细胞肺癌中人线粒体转录终止因子3蛋白的表达及临床病理学意义[J].医学研究生学报,2017,30(2):160-164.