沉默线粒体核糖体蛋白L35基因对人食管癌TE-1细胞生长的影响
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摘要
目的:探讨慢病毒介导沉默线粒体核糖体蛋白L35 (MRPL35)基因对人食管癌TE-1细胞生长的影响,并阐明其机制。方法:选择3种人食管癌TE-1、ECA109和KYSE150细胞,采用实时定量PCR法检测3种细胞中MRPL35mRNA相对表达水平。食管癌TE-1细胞分为shMRPL35组和shCtrl组,分别加入沉默靶基因带有嘌呤霉素抗性的si-RNA慢病毒和带有嘌呤霉素抗性的阴性对照si-RNA慢病毒进行感染,建立稳定沉默MRPL35基因的食管癌细胞系,实时定量PCR及Western blotting法检测各组细胞中MRPL35基因沉默效率,采用CCK-8法检测各组细胞生长曲线,AnnexinⅤ-PE/7AAD双染色后流式细胞术检测细胞凋亡率。结果:3种食管癌细胞均表达MRPL35基因,3种细胞中MRPL35mRNA表达水平比较差异无统计学意义(P>0.05)。实时定量PCR法和Western blotting法检测,shMRPL35组TE-1细胞中MRPL35mRNA和蛋白表达水平明显低于shCtrl组(P<0.05)。与shCtrl组比较,shMRPL35组TE-1细胞生长速度明显降低(P<0.05),凋亡率明显升高(P<0.01)。结论:沉默MRPL35基因可抑制食管癌细胞TE-1增殖,并通过凋亡途径发挥作用。
Objective:To investigate the effect of lentivirus-mediated silencing of mitochondrial ribosomal protein L35(MRPL35)gene on the growth of human esophageal cancer TE-1 cells,and to clarify its mechanism.Methods:Three kinds of human esophageal cancer cells,TE-1,ECA109 and KYSE150,were selected.The relative expression levels of MRPL35 mRNA in three kinds of cells by real-time quantitative PCR.The esophageal cancer TE-1 cells were divided into shMRPL35 group and shCtrl group,and the cells were infected with si-RNA lentivirus and si-RNA lentivirus;the esophageal cancer cell line stably silenting the MRPL35 gene was established.Real-time quantitative PCR and Western blotting methods were used to detect the efficiency of MRPL35 gene silencing.The cell growth curves in various groups were detected by CCK-8 method,and the apoptotic rates were detected by flow cytometry after AnnexinⅤ-PE/7 AAD double staining.Results:Three kinds of esophageal cancer cells expressed MRPL35 gene,and the expression levels were not statistically significant between them(P>0.05).The results of real-time quantitative PCR and Western blotting methods showed that the mRNA and protein levels of MRPL35 in the TE-1 cells in shMRPL35 group were significantly lower than those in shCtrl group(P<0.05).Compared with shCtrl group,the cell growth speed in shMRPL35 group was decreased(P<0.05),and the apoptotic rate was significantly increased(P<0.01).Conclusion:Silencing MRPL35 gene can inhibit the proliferation of esophageal cancer TE-1 cells and plays a role through the apoptotic pathway.
Objective:To investigate the effect of lentivirus-mediated silencing of mitochondrial ribosomal protein L35(MRPL35)gene on the growth of human esophageal cancer TE-1 cells,and to clarify its mechanism.Methods:Three kinds of human esophageal cancer cells,TE-1,ECA109 and KYSE150,were selected.The relative expression levels of MRPL35 mRNA in three kinds of cells by real-time quantitative PCR.The esophageal cancer TE-1 cells were divided into shMRPL35 group and shCtrl group,and the cells were infected with si-RNA lentivirus and si-RNA lentivirus;the esophageal cancer cell line stably silenting the MRPL35 gene was established.Real-time quantitative PCR and Western blotting methods were used to detect the efficiency of MRPL35 gene silencing.The cell growth curves in various groups were detected by CCK-8 method,and the apoptotic rates were detected by flow cytometry after AnnexinⅤ-PE/7 AAD double staining.Results:Three kinds of esophageal cancer cells expressed MRPL35 gene,and the expression levels were not statistically significant between them(P>0.05).The results of real-time quantitative PCR and Western blotting methods showed that the mRNA and protein levels of MRPL35 in the TE-1 cells in shMRPL35 group were significantly lower than those in shCtrl group(P<0.05).Compared with shCtrl group,the cell growth speed in shMRPL35 group was decreased(P<0.05),and the apoptotic rate was significantly increased(P<0.01).Conclusion:Silencing MRPL35 gene can inhibit the proliferation of esophageal cancer TE-1 cells and plays a role through the apoptotic pathway.
引文
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[3]O’BRIEN T W.The general occurrence of 55Sribosomes in mammalian liver mitochondria[J].J Biol Chem,1971,246(10):3409-3417.
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[5]ZHANG Z,GERSTEIN M.Identification and characterization of over 100 mitochondrial ribosomal protein pseudogenes in the human genome[J].Genomics,2003,81(5):468-480.
[6]KOC EC,BURKHART W,BLACKBURN K,et al.The large subunit of the mammalian mitochondrial ribosome.Analysis of the complement of ribosomal proteins present[J].J Biol Chem,2001,276(47):43958-43969.
[7]BOX J M,KAUR J,STUART R A.MrpL35,a mitospecific component of mitoribosomes,plays a key role in cytochrome c oxidase assembly[J].Mol Biol Cell,2017,28(24):3489-3499.
[8]MENEZES M J,GUO Y,ZHANG J,et al.Mutation in mitochondrial ribosomal protein S7(MRPS7)causes congenital sensorineural deafness,progressive hepatic and renal failure and lactic acidemia[J].Hum Mol Genet,2015,24(8):2297-2307.
[9]ARNOLD M,SOERJOMATARAM I,FERLAY J,et al.Global incidence of oesophageal cancer by histological subtype in 2012[J].Gut,2015,64(3):381-387.
[10]SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2017[J].CA Cancer J Clin,2017,67(1):7-30.
[11]SDRALIS E I K,PETOUSIS S,RASHID F,et al.Epidemiology,diagnosis,and management of esophageal perforations:systematic review[J].Dis Esophagus,2017,30(8):1-6.
[12]NAKATSU T,MOTOYAMA S,MARUYAMA K,et al.Tumoral CRP expression in thoracic esophageal squamous cell cancers is associated with poor outcomes[J].Surg Today,2012,42(7):652-658.
[13]SUROVTSEVA Y V,SHUTT T E,COTNEY J,et al.Mitochondrial ribosomal protein L12selectively associates with human mitochondrial RNA polymerase to activate transcription[J].Proc Natl Acad Sci U S A,2011,108(44):17921-17926.
[14]NOUWS J,GOSWAMI A V,BESTWICK M,et al.Mitochondrial ribosomal protein L12is required for POLRMTstability and exists as two forms generated by alternative proteolysis during import[J].J Biol Chem,2016,291(2):989-997.
[15]SERRE V,ROZANSKA A,BEINAT M,et al.Mutations in mitochondrial ribosomal protein MRPL12leads to growth retardation,neurological deterioration and mitochondrial translation deficiency[J].Biochim Biophys Acta,2013,1832(8):1304-1312.
[16]HUTTLIN E L,BRUCKNER R J,PAULO J A,et al.Architecture of the human interactome defines protein communities and disease networks[J].Nature,2017,545(7655):505-509.
[17]SEGURA M M,MANGION M,GAILLET B,et al.New developments in lentiviral vector design,production and purification[J].Expert Opin Biol Ther,2013,13(7):987-1011.
[18]DAREKAR S D,MUHAMMAD M,GURRAPU S,et al.Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts[J].Oncotarget,2015,6(25):21016-21018.
[19]SNOPOK B,YURCHENKO M,SZEKELY L,et al.SPR-based immunocapture approach to creating an interfacial sensing architecture:Mapping of the MRS18-2binding site on retinoblastoma protein[J].Anal Bioanal Chem,2006,386(7/8):2063-2073.
[20]KASHUBA E,YURCHENKO M,YENAMANDRA S P,et al.EBV-encoded EBNA-6binds and targets MRS18-2to the nucleus,resulting in the disruption of pRb-E2F1complexes[J].Proc Natl Acad Sci U S A,2008,105(14):5489-5494.
[21]KASHUBA E,YENAMANDRA S P,DAREKAR S D,et al.MRPS18-2protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties[J].Proc Natl Acad Sci U S A,2009,106(47):19866-19871.