人SND1基因慢病毒载体构建及稳定表达SND1蛋白的人卵巢癌SKOV3细胞株筛选
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摘要
目的构建人SND1基因慢病毒载体,筛选稳定表达SND1蛋白的人卵巢癌SKOV3细胞株,为探讨SND1基因对卵巢癌的调控作用提供细胞系模型。方法采用PCR法从pCMV-FLAG-SND1质粒中扩增FLAG-SND1基因片段,连接到慢病毒载体表达质粒pLVX-IRES-Hyg中,获得重组慢病毒载体pLVX-FLAG-SND1。以pLVX-FLAGSND1瞬时转染293T细胞48 h,采用Western blotting法检测SND1蛋白表达量。pLVX-FLAG-SND1重组质粒通过与包装质粒共转染293T细胞,获得携带FLAG-SND1的重组慢病毒。以慢病毒感染SKOV3细胞48 h,在细胞培养基中加入1μg/mL潮霉素B,筛选稳定表达SND1蛋白的细胞株,采用Western blotting法检测该细胞株内SND1蛋白表达量。结果重组慢病毒载体经双酶切和基因测序比对鉴定正确。重组慢病毒载体在瞬时转染的293T细胞中SND1蛋白表达量高于野生型SKOV3细胞(P<0.01)。SKOV3细胞经慢病毒感染、药物筛选后获得的稳定表达株中SND1蛋白表达量高于野生型SKOV3细胞(P<0.01)。结论成功构建了SND1基因慢病毒载体pLVXFLAG-SND1,并筛选出稳定表达SND1蛋白的SKOV3细胞株,为进一步明确卵巢癌发生、发展机制奠定了基础。
Objective To construct the lentivirus vector of human SND1 gene and to screen the human ovarian cancer cell line SKOV3 which can stably expresses SND1 protein. Methods The fragment of FLAG-SND1 was cloned from pCMV-FLAG-SND1 plasmid,and then was inserted into lentivirus vector pLVX-IRES-Hyg. The lentivirus expression vector pLVX-FLAG-SND1 was established and was transiently transfected into 293T cells for 48 h. Western blotting was employed to determine SND1 expression. Then the lentivirus expression vector was co-transfected into 293T cells with packaging plasmids to obtain the recombinant lentivirus carrying FLAG-SND1. The lentivirus was collected to infect SKOV3 cells. After48 h of infection,1 μg / mL hygromycin B was used to screen the infected cells,so as to obtain a cell line with stable expression of SND1 protein. The expression level of SND1 was detected by Western blotting. Results Double restriction enzyme digestion and DNA sequencing demonstrated the lentivirus expression vector pLVX-FLAG-SND1 was constructed. The recombinant plasmids were transiently transfected into 293T cells,and the expression level was significantly higher than that of the wild-type SKOV3 cells( P < 0. 01). In the SKOV3 cells which stably expressed SND1 after infection and drug screening,the expression of SND1 was significantly higher than that of the wild-type SKOV3 cells( P < 0. 01). Conclusion The SND1 gene lentivirus vector pLVX-FLAG-SND1 is constructed successfully and we screen the SKOV3 cell line stably overexpressing SND1 via lentivirus system.
Objective To construct the lentivirus vector of human SND1 gene and to screen the human ovarian cancer cell line SKOV3 which can stably expresses SND1 protein. Methods The fragment of FLAG-SND1 was cloned from pCMV-FLAG-SND1 plasmid,and then was inserted into lentivirus vector pLVX-IRES-Hyg. The lentivirus expression vector pLVX-FLAG-SND1 was established and was transiently transfected into 293T cells for 48 h. Western blotting was employed to determine SND1 expression. Then the lentivirus expression vector was co-transfected into 293T cells with packaging plasmids to obtain the recombinant lentivirus carrying FLAG-SND1. The lentivirus was collected to infect SKOV3 cells. After48 h of infection,1 μg / mL hygromycin B was used to screen the infected cells,so as to obtain a cell line with stable expression of SND1 protein. The expression level of SND1 was detected by Western blotting. Results Double restriction enzyme digestion and DNA sequencing demonstrated the lentivirus expression vector pLVX-FLAG-SND1 was constructed. The recombinant plasmids were transiently transfected into 293T cells,and the expression level was significantly higher than that of the wild-type SKOV3 cells( P < 0. 01). In the SKOV3 cells which stably expressed SND1 after infection and drug screening,the expression of SND1 was significantly higher than that of the wild-type SKOV3 cells( P < 0. 01). Conclusion The SND1 gene lentivirus vector pLVX-FLAG-SND1 is constructed successfully and we screen the SKOV3 cell line stably overexpressing SND1 via lentivirus system.
引文
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[11]Tsuchiya N,Ochiai M,Nakashima K,et al.SND1,a component of RNA-induced silencing complex,is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis[J].Cancer Res,2007,67(19):9568-9576.
[12]Wang N,Du X,Zang L,et al.Prognostic impact of MetadherinSND1 interaction in colon cancer[J].Mol Biol Rep,2012,39(12):10497-10504.
[13]Yin J,Ding J,Huang L,et al.SND1 affects proliferation of hepatocellular carcinoma cell line SMMC-7721 by regulating IGFBP3expression[J].Anat Rec(Hoboken),2013,296(10):1568-1575.
[14]Cappellari M,Bielli P,Paronetto MP,et al.The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells[J].Oncogene,2014,33(29):3794-3802.
[15]Ho J,Kong JW,Choong LY,et al.Novel breast cancer metastasisassociated proteins[J].J Proteome Res,2009,8(2):583-594.
[2]Tsuchiya N,Nakagama H.MicroRNA,SND1,and alterations in translational regulation in colon carcinogenesis[J].Mutat Res,2010,693(1-2):94-100.
[3]Gao X,Shi X,Fu X,et al.Human Tudor staphylococcal nuclease(Tudor-SN)protein modulates the kinetics of AGTR1-3'UTR granule formation[J].FEBS Lett,2014,588(12):2154-2161.
[4]Duan Z,Zhao X,Fu X,et al.Tudor-SN,a novel coactivator of peroxisome proliferator-activated receptor gamma protein,is essential for adipogenesis[J].J Biol Chem,2014,289(12):8364-8374.
[5]Yu L,Liu X,Cui K,et al.SND1 acts downstream of TGFbeta1and upstream of smurf1 to promote breast cancer metastasis[J].Cancer Res,2015,75(7):1275-1286.
[6]Yoo BK,Santhekadur PK,Gredler R,et al.Increased RNA-induced silencing complex(RISC)activity contributes to hepatocellular carcinoma[J].Hepatology,2011,53(5):1538-1548.
[7]Kuruma H,Kamata Y,Takahashi H,et al.Staphylococcal nuclease domain-containing protein 1 as a potential tissue marker for prostate cancer[J].Am J Pathol,2009,174(6):2044-2050.
[8]Tong X,Drapkin R,Yalamanchili R,et al.The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE[J].Mol Cell Biol,1995,15(9):4735-4744.
[9]Callebaut I,Mornon JP.The human EBNA-2 coactivator p100:multidomain organization and relationship to the staphylococcal nuclease fold and to the tudor protein involved in Drosophila melanogaster development[J].Biochem J,1997,321(Pt 1):125-132.
[10]Murashov AK,Chintalgattu V,Islamov RR,et al.RNAi pathway is functional in peripheral nerve axons[J].Faseb J,2007,21(3):656-670.
[11]Tsuchiya N,Ochiai M,Nakashima K,et al.SND1,a component of RNA-induced silencing complex,is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis[J].Cancer Res,2007,67(19):9568-9576.
[12]Wang N,Du X,Zang L,et al.Prognostic impact of MetadherinSND1 interaction in colon cancer[J].Mol Biol Rep,2012,39(12):10497-10504.
[13]Yin J,Ding J,Huang L,et al.SND1 affects proliferation of hepatocellular carcinoma cell line SMMC-7721 by regulating IGFBP3expression[J].Anat Rec(Hoboken),2013,296(10):1568-1575.
[14]Cappellari M,Bielli P,Paronetto MP,et al.The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells[J].Oncogene,2014,33(29):3794-3802.
[15]Ho J,Kong JW,Choong LY,et al.Novel breast cancer metastasisassociated proteins[J].J Proteome Res,2009,8(2):583-594.