干扰PRMT5表达肝癌细胞稳转细胞株的建立及鉴定
|
摘要
目的探讨干扰蛋白质精氨酸甲基转移酶5(PRMT5)表达肝癌细胞稳转细胞株的建立及鉴定。方法针对PRMT5设计出该基因的有效短发夹RNA(shRNA)片段,选择合适慢病毒载体,通过基因工程技术,构建出PRMT5慢病毒载体介导的shRNA重组载体,并通过PCR以及测序检测构建序列的正确性。将重组病毒载体在病毒包装细胞中大量生产后,用病毒原液感染PRMT5表达量相对较高的SMMC-7721、BEL-7402细胞,2 d后,用最低杀死浓度的嘌呤霉素进行筛选,得到克隆样生长的细胞,扩大培养后分别提取蛋白和RNA,Western印迹和q PCR检测PRMT5的表达。结果成功构建出由人源性PRMT5慢病毒载体介导的shRNA重组载体p LKO.1-sh-PRMT5,PCR及测序均证明构建序列的正确。Western印迹法和q PCR检测,p LKO.1-sh-PRMT5干扰组的PRMT5蛋白及mRNA水平明显低于p LKO.1-sh-GFP组(P<0.05),p LKO.1-sh-PRMT5载体具有良好的干扰效果。结论运用慢病毒感染并筛选出稳转细胞株,在干扰效率及经济角度均优越于瞬时转染,为今后研究PRMT5在肝癌细胞中作用功能提供了良好的科研工具。
Objective To investigate the establishment and identification of a stable cell line expressing protein arginine methyltransferase 5(PRMT5)interference in hepatocellular carcinoma cells.Methods The effective short hairpin RNA(shRNA)fragment of the PRMT5 gene was designed,By selecting the suitable lentiviral vector,the recombinant vector of PRMT5 lentiviral vector mediated by shRNA was constructed by gene engineering technology.The correctness of the sequence was detected by PCR and sequencing.Recombinant viral vectors were produced in a large number in viral packaging cells.SMMC-7721 and BEL-7402 cells with high expression of PRMT5 were infected with virus solution for 2 d.The cells presented with clone-like growth were cloned by using the minimum kill concentration of puromycin.The expression of PRMT5 was detected by Western blotting and qP CR after extracting the protein and RNA.Results ShRNA recombinant vector pL KO.1-shPRMT5 was successfully constructed by human derived PRMT5 lentiviral vector,and the sequence of PCR was proved to be correct.Western blotting and qP CR detection showed that the expressions of PRMT5 and mRNA in the pL KO.1-sh-PRMT5 interference group were significantly lower than those of the pL KO.1-sh-GFP group(P<0.05).pL KO.1-sh-PRMT5 vector had good interference effect of PRMT5 protein.Conclusions The lentiviral vector which used to screen the stable cell line,was superior to the transient transfection in the interferenceefficiency and economic point of view,which provided a good research tool for studying the function of PRMT5in hepatoma cells in the future.
Objective To investigate the establishment and identification of a stable cell line expressing protein arginine methyltransferase 5(PRMT5)interference in hepatocellular carcinoma cells.Methods The effective short hairpin RNA(shRNA)fragment of the PRMT5 gene was designed,By selecting the suitable lentiviral vector,the recombinant vector of PRMT5 lentiviral vector mediated by shRNA was constructed by gene engineering technology.The correctness of the sequence was detected by PCR and sequencing.Recombinant viral vectors were produced in a large number in viral packaging cells.SMMC-7721 and BEL-7402 cells with high expression of PRMT5 were infected with virus solution for 2 d.The cells presented with clone-like growth were cloned by using the minimum kill concentration of puromycin.The expression of PRMT5 was detected by Western blotting and qP CR after extracting the protein and RNA.Results ShRNA recombinant vector pL KO.1-shPRMT5 was successfully constructed by human derived PRMT5 lentiviral vector,and the sequence of PCR was proved to be correct.Western blotting and qP CR detection showed that the expressions of PRMT5 and mRNA in the pL KO.1-sh-PRMT5 interference group were significantly lower than those of the pL KO.1-sh-GFP group(P<0.05).pL KO.1-sh-PRMT5 vector had good interference effect of PRMT5 protein.Conclusions The lentiviral vector which used to screen the stable cell line,was superior to the transient transfection in the interferenceefficiency and economic point of view,which provided a good research tool for studying the function of PRMT5in hepatoma cells in the future.
引文
[1]Baldwin RM,Morettin A,C8téJ.Role of PRMTs in cancer:Could minor isoforms be leaving a mark[J].World J Biol Chem,2014,5(2):115-129.
[2]Ren J,Wang Y,Liang Y,et al.Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis[J].J Biol Chem,2010,285(17):12695-12705.
[3]Zhou Z,Sun X,Zou Z,et al.PRMT5 regulates Golgi apparatus structure through methylation of the golgin GM130[J].Cell Res,2010,20(9):1023-1033.
[4]Dacwag CS,Ohkawa Y,Pal S,et al.The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling[J].Mol Cell Biol,2007,27(1):384-394.
[5]Dacwag CS,Bedford MT,Sif S,et al.Distinct protein arginine methyltransferases promote ATP-dependent chromatin remodeling function at different stages of skeletal muscle differentiation[J].Mol Cell Biol,2009,29(7):1909-1921.
[6]Mallappa C,Hu YJ,Shamulailatpam P,et al.The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase(Prmt)5 but directly requires Prmt4[J].Nucleic Acids Res,2011,39(4):1243-1255.
[7]Ancelin K,Lange UC,Hajkova P,et al.Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells[J].Nat Cell Biol,2006,8(6):623-630.
[8]Eckert D,Biermann K,Nettersheim D,et al.Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells,CIS/IGCNU and germ cell tumors[J].BMC Dev Biol,2008,8:106.
[9]Diehl JA.Cycling to cancer with cyclin D1[J].Cancer Biol Ther,2002,1(3):226-231.
[10]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
[11]Barik S.Development of gene-specific double-stranded RNA drugs[J].Ann Med,2004,36(7):540-551.
[12]Crombie C,Fraser A.Treating genetic disease through RNA interference[J].Lancet,2005,365(9467):1288-1290.
[13]Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2001,15(2):188-200.
[14]Tomar RS,Matta H,Chaudhary PM.Use of adeno-associated viral vector for delivery of small interfering RNA[J].Oncogene,2003,22(36):5712-5715.
[2]Ren J,Wang Y,Liang Y,et al.Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis[J].J Biol Chem,2010,285(17):12695-12705.
[3]Zhou Z,Sun X,Zou Z,et al.PRMT5 regulates Golgi apparatus structure through methylation of the golgin GM130[J].Cell Res,2010,20(9):1023-1033.
[4]Dacwag CS,Ohkawa Y,Pal S,et al.The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling[J].Mol Cell Biol,2007,27(1):384-394.
[5]Dacwag CS,Bedford MT,Sif S,et al.Distinct protein arginine methyltransferases promote ATP-dependent chromatin remodeling function at different stages of skeletal muscle differentiation[J].Mol Cell Biol,2009,29(7):1909-1921.
[6]Mallappa C,Hu YJ,Shamulailatpam P,et al.The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase(Prmt)5 but directly requires Prmt4[J].Nucleic Acids Res,2011,39(4):1243-1255.
[7]Ancelin K,Lange UC,Hajkova P,et al.Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells[J].Nat Cell Biol,2006,8(6):623-630.
[8]Eckert D,Biermann K,Nettersheim D,et al.Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells,CIS/IGCNU and germ cell tumors[J].BMC Dev Biol,2008,8:106.
[9]Diehl JA.Cycling to cancer with cyclin D1[J].Cancer Biol Ther,2002,1(3):226-231.
[10]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
[11]Barik S.Development of gene-specific double-stranded RNA drugs[J].Ann Med,2004,36(7):540-551.
[12]Crombie C,Fraser A.Treating genetic disease through RNA interference[J].Lancet,2005,365(9467):1288-1290.
[13]Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2001,15(2):188-200.
[14]Tomar RS,Matta H,Chaudhary PM.Use of adeno-associated viral vector for delivery of small interfering RNA[J].Oncogene,2003,22(36):5712-5715.