肝癌细胞hMTERF3基因启动子表达载体的构建及其受DNA甲基化的影响
|
摘要
为了筛选人线粒体转录终止因子3(human mitochondrial transcription termination factor 3,h MTERF3)基因各段启动子的活性,探讨肝癌细胞Hep G2和BEL-7402中h MTERF3基因启动子活性受DNA甲基化的影响,首先利用PCR技术扩增h MTERF3基因启动子区,克隆至荧光素酶基因报告载体中,构建p GL6-h MTERF3重组质粒;随后运用荧光素酶检测法检测肝癌细胞中各段h MTERF3启动子对荧光素酶报告基因的启动活性,并采用DNA甲基化酶SssⅠ处理肝癌细胞,观察各报告基因的启动子活性变化。结果显示,成功构建了7个携带h MTERF3基因启动子的重组荧光素酶报告基因载体,重组子经NheⅠ和BglⅡ双酶切及PCR鉴定正确。双荧光素酶报告基因检测法显示,与p GL6相比,含有P523的3个启动子区段P523、P866、P932有强启动子活性,组间比较差异无统计学意义(P>0.05)。此外,与对照组比较,SssⅠ甲基化酶处理的P523、P866和P932启动子活性显著降低(P<0.05)。以上研究提示,h MTERF3启动子活性受DNA甲基化的影响,其中P523可能是h MTERF3启动子核心,为进一步研究肝癌细胞中h MTERF3基因表达的表观遗传学机制奠定了实验基础。
To construct a reporter vector regulated by the h MTERF3 promoter to determine the effect of DNA methylation on the promoter activity of h MTERF3 gene, PCR amplification was performed to obtain seven potential h MTERF3 promoter fragments, which were then cloned into the vector to obtain recombinant constructs. Promoter activities of different h MTERF3 fragments in cancer cells were detected by luciferase assay to identify the potential promoter area. The activity of the h MTERF3 promoter with or without treatment of methylase M. Sss Ⅰ was also evaluated by luciferase assay. It showed that three fragments(P523,P866 and P932), all of which contained the P523 sequence, had stronger promoter activity than the p GL6 control. Their promoter activities decreased significantly after methylase treatment( P<0.05). A luciferase reporter gene system containing the h MTERF3 promoter was successfully constructed. P523 is the potential core area of the h MTERF3 promoter. These results provide a basis for studying the epigenetic mechanism of h MTERF3 gene expression in hepatoma cells.
To construct a reporter vector regulated by the h MTERF3 promoter to determine the effect of DNA methylation on the promoter activity of h MTERF3 gene, PCR amplification was performed to obtain seven potential h MTERF3 promoter fragments, which were then cloned into the vector to obtain recombinant constructs. Promoter activities of different h MTERF3 fragments in cancer cells were detected by luciferase assay to identify the potential promoter area. The activity of the h MTERF3 promoter with or without treatment of methylase M. Sss Ⅰ was also evaluated by luciferase assay. It showed that three fragments(P523,P866 and P932), all of which contained the P523 sequence, had stronger promoter activity than the p GL6 control. Their promoter activities decreased significantly after methylase treatment( P<0.05). A luciferase reporter gene system containing the h MTERF3 promoter was successfully constructed. P523 is the potential core area of the h MTERF3 promoter. These results provide a basis for studying the epigenetic mechanism of h MTERF3 gene expression in hepatoma cells.
引文
[1]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(Xiong Wei,Yu Min,Zuo Shao-yuan.Regulatory roles of mitochondrial transcription termination factor(MTERF)family proteins in mitochondrial gene expression[J].Chinese Journal of Biochemistry and Molecular Biology),2015,31(3):223-231.
[2]Linder T,Park C B,Asin-Cayuela J,et al.A family of putative transcription termination factors shared amongst metazoans and plants[J].Current Genetics,2005,48(4):265-269.
[3]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(Xiong Wei,Zhang Xiao-juan,Zhang Hai-yang,et al.Aanlysis of the structure and function of human mitochondrial transcription termination factor 3 based on bioinformatics methods[J].Letters in Biotechnology),2015,26(3):367-373.
[4]Roberti M,Polosa P L,Bruni F,et al.MTERF factors:a multifunction protein family[J].Biomolecular Concepts,2010,1(2):215-224.
[5]Xiong W,Luo Y H,Zhang C X,et al.Expression,purification of recombinant human mitochondrial transcription termination factor 3(h MTERF3)and preparation of polyclonal antibody against h MTERF3[J].Applied Biochemistry and Biotechnology,2012,167(8):2318-2329.
[6]Park C B,Asin-Cayuela J,Cámara Y,et al.MTERF3 is a negative regulator of mammalian mt DNA transcription[J].Cell,2007,130(2):273-285.
[7]康鲁东,崔福爱,吴伟芳,等.人PSMA基因启动子表达载体p GL3-PSMP的构建[J].山东大学学报(医学版)(Kang Ludong,Cui Fu-ai,Wu Wei-fang,et al.Construction of the expression vector p GL3-PSMP containing prostate specific membrane atigen promoter[J].Journal of Shandong Univerisity(Health Sciences),2005,43(7):567-569.
[8]杨勇琴,孙美涛,李月,等.基于生物信息学分析人类线粒体转录终止因子1基因启动子[J].医学研究生学报(Yang Yongqin,Sun Mei-tao,Li Yue,et al.Bioinformatics analysis of the human MTERF1 gene promoter[J].Journal of Medical Postgraduates),2016,29(4):348-353.
[9]Li L C,Dahiya R.Meth Primer:designing primers for methylation PCRs[J].Bioinformatics,2002,18(11):1427-1431.
[10]Ma J,Liu Q H,Wang B B,et al.Construction of a reporter vector regulated by the Pdx1 promoter and evaluation of the effect of DNA methylation on Pdx1 promoter activity in gastric cancer cells[J].World Chinese Journal of Digestology,2011,19(31):3222-3228.
[11]Wredenberg A,Lagouge M,Bratic A,et al.MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals[J].PLo S Genetics,2013,9(1):e1003178.
[12]Andersson D C,Fauconnier J,Park C B,et al.Enhanced cardiomyocyte Ca(2+)cycling precedes terminal AV-block in mitochondrial cardiomyopathy Mterf3 KO mice[J].Antioxidants&Redox Signaling,2011,15(9):2455-2464.
[13]陈衍,刘文超,贾军,等.人脂肪酸合成酶FAS启动子的克隆及其在几种肿瘤细胞中的转录活性分析[J].现代肿瘤医学(Chen Yan,Liu Wen-chao,Jia Jun,et al.Clone of human fatty acid synthase promoter and analysis of its transcriptional activity in tumor cells[J].Modern Oncology),2006,14(12):1485-1488.
[14]Xu Y Z,Kannagaratham C,Jancik S,et al.Promoter deletion analysis using a dual-luciferase reporter system[J].Methods in Molecular Biology,2013,977(1):79-93.
[15]Solberg N,Krauss S.Luciferase assay to study the activity of a cloned promoter DNA fragment[J].Methods in Molecular Biology,2013,977(1):65-78.
[16]朱建冬,梁庄严,王森,等.IL-37基因启动子的克隆及其荧光素酶报告基因载体的构建[J].生物技术(Zhu Jian-dong,Liang Zhuang-yan,Wang Sen,et al.Construction of promoter region and constructions of the luciferase reporter gene vectors containing IL-37 promoter[J].Biotechnology),2015,25(6):520-523.
[17]Feng Q,Stern J E,Hawes S E,et al.DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection[J].Experimental and Molecular Pathology,2010,88(2):287-292.
[18]Kim B H,Cho N Y,Shin S H,et al.Cp G island hypermethylation and repetitive DNA hypomethylation in premalignant lesion of extrahepatic cholangiocarcinoma[J].Virchows Archiv,2009,455:343.
[19]周雯,王青松.人claudin-10基因启动子转录调控的初步分析[J].生物技术(Zhou Wen,Wang Qing-song.Preliminary analysis of transcriptional regulation of human claudin-10 gene promoter[J].Biotechnology),2015,25(3):205-211.
[2]Linder T,Park C B,Asin-Cayuela J,et al.A family of putative transcription termination factors shared amongst metazoans and plants[J].Current Genetics,2005,48(4):265-269.
[3]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(Xiong Wei,Zhang Xiao-juan,Zhang Hai-yang,et al.Aanlysis of the structure and function of human mitochondrial transcription termination factor 3 based on bioinformatics methods[J].Letters in Biotechnology),2015,26(3):367-373.
[4]Roberti M,Polosa P L,Bruni F,et al.MTERF factors:a multifunction protein family[J].Biomolecular Concepts,2010,1(2):215-224.
[5]Xiong W,Luo Y H,Zhang C X,et al.Expression,purification of recombinant human mitochondrial transcription termination factor 3(h MTERF3)and preparation of polyclonal antibody against h MTERF3[J].Applied Biochemistry and Biotechnology,2012,167(8):2318-2329.
[6]Park C B,Asin-Cayuela J,Cámara Y,et al.MTERF3 is a negative regulator of mammalian mt DNA transcription[J].Cell,2007,130(2):273-285.
[7]康鲁东,崔福爱,吴伟芳,等.人PSMA基因启动子表达载体p GL3-PSMP的构建[J].山东大学学报(医学版)(Kang Ludong,Cui Fu-ai,Wu Wei-fang,et al.Construction of the expression vector p GL3-PSMP containing prostate specific membrane atigen promoter[J].Journal of Shandong Univerisity(Health Sciences),2005,43(7):567-569.
[8]杨勇琴,孙美涛,李月,等.基于生物信息学分析人类线粒体转录终止因子1基因启动子[J].医学研究生学报(Yang Yongqin,Sun Mei-tao,Li Yue,et al.Bioinformatics analysis of the human MTERF1 gene promoter[J].Journal of Medical Postgraduates),2016,29(4):348-353.
[9]Li L C,Dahiya R.Meth Primer:designing primers for methylation PCRs[J].Bioinformatics,2002,18(11):1427-1431.
[10]Ma J,Liu Q H,Wang B B,et al.Construction of a reporter vector regulated by the Pdx1 promoter and evaluation of the effect of DNA methylation on Pdx1 promoter activity in gastric cancer cells[J].World Chinese Journal of Digestology,2011,19(31):3222-3228.
[11]Wredenberg A,Lagouge M,Bratic A,et al.MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals[J].PLo S Genetics,2013,9(1):e1003178.
[12]Andersson D C,Fauconnier J,Park C B,et al.Enhanced cardiomyocyte Ca(2+)cycling precedes terminal AV-block in mitochondrial cardiomyopathy Mterf3 KO mice[J].Antioxidants&Redox Signaling,2011,15(9):2455-2464.
[13]陈衍,刘文超,贾军,等.人脂肪酸合成酶FAS启动子的克隆及其在几种肿瘤细胞中的转录活性分析[J].现代肿瘤医学(Chen Yan,Liu Wen-chao,Jia Jun,et al.Clone of human fatty acid synthase promoter and analysis of its transcriptional activity in tumor cells[J].Modern Oncology),2006,14(12):1485-1488.
[14]Xu Y Z,Kannagaratham C,Jancik S,et al.Promoter deletion analysis using a dual-luciferase reporter system[J].Methods in Molecular Biology,2013,977(1):79-93.
[15]Solberg N,Krauss S.Luciferase assay to study the activity of a cloned promoter DNA fragment[J].Methods in Molecular Biology,2013,977(1):65-78.
[16]朱建冬,梁庄严,王森,等.IL-37基因启动子的克隆及其荧光素酶报告基因载体的构建[J].生物技术(Zhu Jian-dong,Liang Zhuang-yan,Wang Sen,et al.Construction of promoter region and constructions of the luciferase reporter gene vectors containing IL-37 promoter[J].Biotechnology),2015,25(6):520-523.
[17]Feng Q,Stern J E,Hawes S E,et al.DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection[J].Experimental and Molecular Pathology,2010,88(2):287-292.
[18]Kim B H,Cho N Y,Shin S H,et al.Cp G island hypermethylation and repetitive DNA hypomethylation in premalignant lesion of extrahepatic cholangiocarcinoma[J].Virchows Archiv,2009,455:343.
[19]周雯,王青松.人claudin-10基因启动子转录调控的初步分析[J].生物技术(Zhou Wen,Wang Qing-song.Preliminary analysis of transcriptional regulation of human claudin-10 gene promoter[J].Biotechnology),2015,25(3):205-211.