NDUFA4过表达对人结直肠癌细胞体外生长的影响
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摘要
目的探讨过表达NDUFA4对人结直肠癌细胞体外生长的影响及其机制。方法将p-NDUFA4重组质粒(p-NDUFA4组)和p-Cont对照质粒(p-Cont组)体外分别转染人结直肠癌HCT116细胞;采用Real-timePCR和Westernblot检测细胞中NDUFA的表达;CCK-8法和克隆形成实验分别检测HCT116细胞增殖活性和克隆形成能力;实时荧光定量PCR法检测HCT116细胞中CDK2、CDK3、CDK4、CDK6的mRNA水平;Westernblot法检测细胞中蛋白总AKT、ERK1/2以及磷酸化AKT(p-AKT)、ERK1/2(p-ERK1/2)的表达水平。结果与p-Cont对照组相比,p-NDUFA4组中NDUFA4的表达水平显著升高(均P<0.01);细胞增殖和克隆形成能力明显增强(均P<0.05);CDK2、CDK3等的mRNA水平显著上调(均P<0.05);p-NDUFA4组中p-AKT和p-ERK1/2蛋白的表达水平明显上调(均P<0.05)。结论过表达NDUFA4能明显促进人结直肠癌细胞的体外生长,其机制可能与AKT和ERK信号通路的变化相关。
Objective To investigate the effects of NDUFA4 overexpression on the growth of human colon cancer cells in vitro and to explore its potential mechanism. Methods The recombinant plasmid p-NDUFA4(p-NDUFA4) and the control plasmid(p-Cont) were transiently transfected into human colon cancer HCT116 cells, respectively. Then, the relative expression level of NDUFA4 was determined by Real-time PCR and Western blot. The proliferation and colony formation ability of HCT116 cells were detected by CCK-8 assay and clone formation assay. The mRNA expression levels of cyclin-dependent kinase(CDK) 2, CDK3, CDK4 and CDK6 were determined by Real-time PCR. The expression levels of AKT(p-AKT) and ERK1/2(pERK1/2) were analyzed by Western blot. Results Compared with control group, the expression levels of NDUFA4 were increased remarkably in p-NDUFA4 group(all P<0.01). The proliferation and clone formation capacity of HCT116 cells in p-NDUFA4 group were obviously increased(all P<0.05). The expression of CDK2 and CDK3 mRNA were increased significantly(all P<0.05). The expression of p-AKT and p-ERK1/2 were increased obviously(all P<0.05). Conclusion The overexpression of NDUFA4 could significantly promote the growth of human colon cancer HCT116 cells in vitro, which might be closely related to the change of AKT and ERK signaling transduction.
Objective To investigate the effects of NDUFA4 overexpression on the growth of human colon cancer cells in vitro and to explore its potential mechanism. Methods The recombinant plasmid p-NDUFA4(p-NDUFA4) and the control plasmid(p-Cont) were transiently transfected into human colon cancer HCT116 cells, respectively. Then, the relative expression level of NDUFA4 was determined by Real-time PCR and Western blot. The proliferation and colony formation ability of HCT116 cells were detected by CCK-8 assay and clone formation assay. The mRNA expression levels of cyclin-dependent kinase(CDK) 2, CDK3, CDK4 and CDK6 were determined by Real-time PCR. The expression levels of AKT(p-AKT) and ERK1/2(pERK1/2) were analyzed by Western blot. Results Compared with control group, the expression levels of NDUFA4 were increased remarkably in p-NDUFA4 group(all P<0.01). The proliferation and clone formation capacity of HCT116 cells in p-NDUFA4 group were obviously increased(all P<0.05). The expression of CDK2 and CDK3 mRNA were increased significantly(all P<0.05). The expression of p-AKT and p-ERK1/2 were increased obviously(all P<0.05). Conclusion The overexpression of NDUFA4 could significantly promote the growth of human colon cancer HCT116 cells in vitro, which might be closely related to the change of AKT and ERK signaling transduction.
引文
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[14]李雪峰,陈临溪,伍尤华.生物信息学分析肾透明细胞癌相关分子标志物及其关键通路[J].中南医学科学杂志,2016,44(5):481-5,493.[Li XF,Chen LX,Wu YH.Analysis of the Molecular Markers and the Critical Pathway in Clear Cell Renal Cell Carcinoma Through Bioinformatics[J].Zhongnan Yi Xue Ke Xue Za Zhi,2016,44(5):481-5,493.]
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[16]陆仁飞,周敏,肖峰,等.探讨NDUFA13抑制结肠癌细胞增殖的机制[J].江苏医药,2012,38(24):2972-4.[Lu RF,Zhou M,Xiao F,et al.An investigation on the mechanism for NDUFA13to inhibit proliferation of human Colon cancer[J].Jiangsu Yi Yao,2012,38(24):2972-4.]
[17]Lv Y,Nie SL,Zhou JM,et al.Overexpression of NDUFA4L2is associated with poor prognosis in patients with colorectal cancer[J].ANZ J Surg,2016,87(12):E251-R5.
[18]Liu L,Lan G,Peng L,et al.NDUFA4L2 expression predicts poor prognosis in clear cell renal cell carcinoma patients[J].Ren Fail,2016,38(8):1199-205.
[19]Zhang YJ,Xu ZG,Li SQ,et al.Benzimidazoisoquinoline derivatives inhibit glioblastoma cell proliferation through downregulating Raf/MEK/ERK and PI3K/AKT pathways[J].Cancer Cell Int,2018,18:90.
[20]Wang X,Zhu Y,Zhu L,et al.Eupatilin inhibits the proliferation of human esophageal cancer TE1 cells by targeting the AKT-GSK3βand MAPK/ERK signaling cascades[J].Oncol Rep,2018,39(6):2942-50.
[21]Zhu J,Yao J,Huang R,et al.Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/AKT/m TOR/P70S6K and ERK signaling pathways[J].Biochem Biophy Res Commun,2018,498(3):616-20.
[22]Trotta AP,Gelles JD,Serasinghe MN,et al.Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition[J].J Biol Chem,2017,292(28):11727-39.
[23]Song HP,Chu ZG,Zhang DX,et al.PI3K-AKT Pathway Protects Cardiomyocytes Against Hypoxia-Induced Apoptosis by MitoKATP-Mediated Mitochondrial Translocation of pAKT[J].Cell Physiol Biochem,2018,49(2):717-27.
[24]Fang R,Zhang LL,Zhang LZ,et al.Sphingosine 1-Phosphate Postconditioning Protects Against Myocardial Ischemia/reperfusion Injury in Rats via Mitochondrial Signaling and AKT-Gsk3βPhosphorylation[J].Arch Med Res,2017,48(2):147-55.
[2]孟庆彬,吴彪,肖新波,等.结直肠癌组织中Krüppel样因子17的表达及其与预后的关系[J].肿瘤防治研究,2015,42(3):256-60.[Meng QB,Wu B,Xiao XB,et al.Expression of KLF17 Protein in Human Colorectal Cancer Tissue and Its Correlation with Prognosis[J].Zhong Liu Fang Zhi Yan Jiu,2015,42(3):256-60.]
[3]Wu J,Yang X,Lu H,et al.Inhibitory effect and mechanism of exogenous mammalian sterile 20-like kinase 1 on the growth of human colorectal cancer[J].Oncol Lett,2017,13(4):2656-64.
[4]Liu T,Zhang X,Gao S,et al.Exosomal long noncoding RNACRNDE-h as a novel serum-based biomarker for diagnosis and prognosis of colorectal cancer[J].Oncotarget,2016,7(51):85551-63.
[5]武雪亮,王立坤,薛军,等.结直肠癌组织中DNA结合分化抑制蛋白1和MMP-9的表达及其与LMVD的相关性[J].肿瘤防治研究,2016,43(11):959-63.[Wu XL,Wang LK,Xue J,et al.Expression and Correlation of Id-1,MMP-9 and LMVD in Colorectal Adenocarcinoma tissues[J].Zhong Liu Fang Zhi Yan Jiu,2016,43(11):959-63.]
[6]Balsa E,Marco R,Perales-Clemente E,et al.NDUFA4 Is a Subunit of Complex IV of the Mammalian Electron Transport Chain[J].Cell Metab,2012,16(3):378-86.
[7]Hayashi T,Ishimori C,Takahashi-Niki K,et al.DJ-1 binds to mitochondrial complex I and maintains its activity[J].Biochem Biophys Res Commun,2009,390(3):667-72.
[8]Zong NC,Li H,Li H,et al.Integration of cardiac proteome biology and medicine by a specialized knowledgebase[J].Circ Res,2013,113(9):1043-53.
[9]Pitceathly RDS,Taanman JW.NDUFA4(Renamed COXFA4)Is a Cytochrome-c,Oxidase Subunit[J].Trends Endocrinol Metab,2018,29(7):452-4.
[10]Fu F,Li Y,Li R,et al.NDUFA4 enhances neuron growth by triggering growth factors and inhibiting neuron apoptosis through Bcl-2 and cytochrome C mediated signaling pathway[J].Am JTransl Res,2018,10(1):164-74.
[11]Kadenbach B.Regulation of Mammalian 13-Subunit Cytochrome c Oxidase and Binding of other Proteins:Role of NDUFA4[J].Trends Endocrin Met,2017,28(11):761-70.
[12]Müller FE,Braun M,Syring I,et al.NDUFA4 expression in clear cell renal cell carcinoma is predictive for cancer-specific survival[J].Am J Cancer Res,2015,5(9):2816-22.
[13]Lei L,Chen C,Zhao J,et al.Targeted Expression of miR-7Operated by TTF-1 Promoter Inhibited the Growth of Human Lung Cancer through the NDUFA4 Pathway[J].Mol Ther Nucleic Acids,2017,6:183-97.
[14]李雪峰,陈临溪,伍尤华.生物信息学分析肾透明细胞癌相关分子标志物及其关键通路[J].中南医学科学杂志,2016,44(5):481-5,493.[Li XF,Chen LX,Wu YH.Analysis of the Molecular Markers and the Critical Pathway in Clear Cell Renal Cell Carcinoma Through Bioinformatics[J].Zhongnan Yi Xue Ke Xue Za Zhi,2016,44(5):481-5,493.]
[15]Chen Y,Lu H,Liu Q,et al.Function of GRIM-19,a mitochondrial respiratory chain complex I protein,in innate immunity[J].J Biol Chem,2012,287(32):27227-35.
[16]陆仁飞,周敏,肖峰,等.探讨NDUFA13抑制结肠癌细胞增殖的机制[J].江苏医药,2012,38(24):2972-4.[Lu RF,Zhou M,Xiao F,et al.An investigation on the mechanism for NDUFA13to inhibit proliferation of human Colon cancer[J].Jiangsu Yi Yao,2012,38(24):2972-4.]
[17]Lv Y,Nie SL,Zhou JM,et al.Overexpression of NDUFA4L2is associated with poor prognosis in patients with colorectal cancer[J].ANZ J Surg,2016,87(12):E251-R5.
[18]Liu L,Lan G,Peng L,et al.NDUFA4L2 expression predicts poor prognosis in clear cell renal cell carcinoma patients[J].Ren Fail,2016,38(8):1199-205.
[19]Zhang YJ,Xu ZG,Li SQ,et al.Benzimidazoisoquinoline derivatives inhibit glioblastoma cell proliferation through downregulating Raf/MEK/ERK and PI3K/AKT pathways[J].Cancer Cell Int,2018,18:90.
[20]Wang X,Zhu Y,Zhu L,et al.Eupatilin inhibits the proliferation of human esophageal cancer TE1 cells by targeting the AKT-GSK3βand MAPK/ERK signaling cascades[J].Oncol Rep,2018,39(6):2942-50.
[21]Zhu J,Yao J,Huang R,et al.Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/AKT/m TOR/P70S6K and ERK signaling pathways[J].Biochem Biophy Res Commun,2018,498(3):616-20.
[22]Trotta AP,Gelles JD,Serasinghe MN,et al.Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition[J].J Biol Chem,2017,292(28):11727-39.
[23]Song HP,Chu ZG,Zhang DX,et al.PI3K-AKT Pathway Protects Cardiomyocytes Against Hypoxia-Induced Apoptosis by MitoKATP-Mediated Mitochondrial Translocation of pAKT[J].Cell Physiol Biochem,2018,49(2):717-27.
[24]Fang R,Zhang LL,Zhang LZ,et al.Sphingosine 1-Phosphate Postconditioning Protects Against Myocardial Ischemia/reperfusion Injury in Rats via Mitochondrial Signaling and AKT-Gsk3βPhosphorylation[J].Arch Med Res,2017,48(2):147-55.
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