FOXC1蛋白基因shRNA干扰质粒对胃癌SGC-7901细胞增殖的影响
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摘要
目的探讨FOXC1蛋白基因RNA干扰质粒对胃癌SGC-7901细胞增殖的影响。方法采用RT-PCR及Westernblot法检测SGC-7901细胞和胃黏膜上皮细胞GES-1中FOXC1基因mRNA的转录水平和蛋白的表达水平。构建3个FOXC1基因shRNA真核表达质粒pshRNA-FOXC1a、pshRNA-FOXC1b、pshRNA-FOXC1c,分别转染SGC-7901细胞,并设pGenesil-1空质粒转染组和未转染的细胞对照组,RT-PCR及Western blot法分别检测转染细胞中FOXC1基因mRNA的转录水平和蛋白的表达水平;MTT法检测转染细胞的增殖活力;流式细胞术检测转染细胞的细胞周期。结果 SGC-7901细胞中FOXC1基因mRNA的转录水平及蛋白的表达水平均显著高于GES-1细胞(P<0.05)。pshRNA-FOXC1a组、pshRNA-FOXC1b组和pshRNA-FOXC1c组SGC-7901细胞中FOXC1基因mRNA的转录水平和蛋白的表达水平均显著低于pGenesil-1组和SGC-7901组(P<0.01);细胞增殖抑制率显著高于pGensil-1组(P<0.05)。处于G1、G2期的细胞比例显著高于SGC-7901组(P<0.01),S期细胞比例显著低于SGC-7901组(P<0.01)。结论成功构建了FOXC1蛋白基因shRNA真核表达质粒。FOXC1在胃癌细胞SGC-7901中呈高表达,抑制其表达后,细胞周期被阻滞在G1～G2期,细胞增殖受到抑制。
Objective To investigate the effect of shRNA interfering plasmid targeting FOXC1 gene on proliferation of gastric adenocarcinoma SGC-7901 cells.Methods The transcription level of FOXC1 mRNA and expression level of FOXC1 protein in SGC-7901 and gastric mucosal epithelium GES-1 cells were determined by RT-PCR and Western blot respectively.Three shRNA interfering plasmids targeting FOXC1 gene,i.e.pshRNA-FOXC1a,pshRNA-FOXC1b and pshRNA-FOXC1c,were constructed and transfected to SGC-7901 cells respectively,using the cells untransfected and those transfected with empty plasmid pGenesil as controls.The transfected SGC-7901 cells were determined for transcription level of FOXC1 mRNA and expression level of FOXC1 protein by RTPCR and Western blot respectively,for proliferation activity by MTT method,and for cell cycle by flow cytometry.Results Both the transcription level of FOXC1 mRNA and expression level of FOXC1 protein in SGC-7901 cells were significantly higher than those in GES-1 cells(P < 0.05).The transcription level of FOXC1 mRNA and expression level of FOXC1 protein in SGC-7901 cells transf ected with pshRNA-FOXC1a,pshRNA-FOXC1b and pshRNA-FOXC1c were significantly lower than those transfected with pGenesil-1 and those untransfected(P < 0.01),while the inhibition rate of proliferation was significantly higher than that transfected with pGensil-1(P < 0.05).However,the percentages at G1 and G2 phases of SGC-7901 cells transfected with pshRNA-FOXC1a,pshRNAFOXC1b and pshRNA-FOXC1c were significantly higher,while that at S phase was significantly lower,than those untransfected(P < 0.01).Conclusion The shRNA eukaryotic expression plasmid for FOXC1 gene was successfully constructed.FOXC1 was highly expressed in SGC-7901s,which arrested the cell cycle at G1 ~ G2 phase and inhibited the cell proliferation.
Objective To investigate the effect of shRNA interfering plasmid targeting FOXC1 gene on proliferation of gastric adenocarcinoma SGC-7901 cells.Methods The transcription level of FOXC1 mRNA and expression level of FOXC1 protein in SGC-7901 and gastric mucosal epithelium GES-1 cells were determined by RT-PCR and Western blot respectively.Three shRNA interfering plasmids targeting FOXC1 gene,i.e.pshRNA-FOXC1a,pshRNA-FOXC1b and pshRNA-FOXC1c,were constructed and transfected to SGC-7901 cells respectively,using the cells untransfected and those transfected with empty plasmid pGenesil as controls.The transfected SGC-7901 cells were determined for transcription level of FOXC1 mRNA and expression level of FOXC1 protein by RTPCR and Western blot respectively,for proliferation activity by MTT method,and for cell cycle by flow cytometry.Results Both the transcription level of FOXC1 mRNA and expression level of FOXC1 protein in SGC-7901 cells were significantly higher than those in GES-1 cells(P < 0.05).The transcription level of FOXC1 mRNA and expression level of FOXC1 protein in SGC-7901 cells transf ected with pshRNA-FOXC1a,pshRNA-FOXC1b and pshRNA-FOXC1c were significantly lower than those transfected with pGenesil-1 and those untransfected(P < 0.01),while the inhibition rate of proliferation was significantly higher than that transfected with pGensil-1(P < 0.05).However,the percentages at G1 and G2 phases of SGC-7901 cells transfected with pshRNA-FOXC1a,pshRNAFOXC1b and pshRNA-FOXC1c were significantly higher,while that at S phase was significantly lower,than those untransfected(P < 0.01).Conclusion The shRNA eukaryotic expression plasmid for FOXC1 gene was successfully constructed.FOXC1 was highly expressed in SGC-7901s,which arrested the cell cycle at G1 ~ G2 phase and inhibited the cell proliferation.
引文
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[11]富志民,刘亚伦,唐国基.人体胃癌细胞株SGC7901培养及其特性[J].肿瘤临床,1984,3(1):57-60.
[12]Berry FB,Skarie JM,Mirzayans F,et al.FOXC1 is required forcell viability and resistance to oxidative stress in the eye throughthe transcriptional regulation of FOXO1A[J].Hum Mol Genet,2008,17(4):490-505.
[13]Paik JH,Kollipara R,Chu G,et al.FoxOs are lineage-restrictedredundant tumor suppressors and regulate endothelial cell home-ostasis[J].Cell,2007,128(2):309-323.
[14]Ray PS,Wang J,Qu Y,et al.FOXC1 is a potential prognosticbiomarker with functional significance in basal-like breast cancer[J].Cancer Res,2010,70(10):3870-3876.
[15]Egeblad M,Werb Z.New functions for the matrix metallopro-teinases in cancer progression[J].Nat Rev Cancer,2002,2(3):161-174.
[16]Skarie JM,Link BA.FoxC1 is essential for vascular basementmembrane integrity and hyaloid vessel morphogenesis[J].InvestOphthalmol Vis Sci,2009,50(11):5026-5034.
[2]Seo S,Fujita H,Nakano A,et al.The forkhead transcriptionfactors,Foxc1and Foxc2,are required for arterial specificationand lymphatic sprouting during vascular development[J].DevBiol,2006,294(2):458-470.
[3]Zhou Y,Kato H,Asanoma K,et al.Identification of FOXC1 asa TGF-beta1 responsive gene and its involvement in negative reg-ulation of cell growth[J].Genomics,2002,80(5):465-472.
[4]Van Der Heul-Nieuwenhuijsen L,Dits NF,Jenster G.Gene ex-pression of forkhead transcription factors in the normal and dis-eased human prostate[J].BJU Int,2009,103(11):1574-1580.
[5]罗欣,刘丹丹,漆洪波,等.靶向人Gadd45α基因的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞生物学功能损伤的影响[J].中国生物制品学杂志,2010,23(11):1168-1172.
[6]邓玮,易永芬,文雪,等.RNA干扰P115基因对胃癌细胞巨噬细胞移动抑制因子表达的抑制作用[J].中国生物制品学杂志,2011,24(4):404-409.
[7]柯大智,陈庆伟,李桂琼,等.小鼠胰岛素诱导因子1siRNA稳定表达细胞株的建立及其对细胞胆固醇代谢的影响[J].中国生物制品学杂志,2011,24(12):1445-1449.
[8]Cha JY,Birsoy B,Kofron M,et al.The role of FoxC1 in earlyXenopus development[J].Dev Dyn,2007,236(10):2731-2741.
[9]Wotton KR,Mazet F,Shimeld SM.Expression of FoxC,FoxF,FoxL1,and FoxQ1 genes in the dogfish Scyliorhinus caniculadefines ancient and derived roles for fox genes in vertebrate de-velopment[J].Dev Dyn,2008,237(6):1590-1603.
[10]柯杨,宁涛.人胃粘膜上皮细胞GES-1的建立及其生物学特性[J].中华肿瘤杂志,1994,16(1):7-9.
[11]富志民,刘亚伦,唐国基.人体胃癌细胞株SGC7901培养及其特性[J].肿瘤临床,1984,3(1):57-60.
[12]Berry FB,Skarie JM,Mirzayans F,et al.FOXC1 is required forcell viability and resistance to oxidative stress in the eye throughthe transcriptional regulation of FOXO1A[J].Hum Mol Genet,2008,17(4):490-505.
[13]Paik JH,Kollipara R,Chu G,et al.FoxOs are lineage-restrictedredundant tumor suppressors and regulate endothelial cell home-ostasis[J].Cell,2007,128(2):309-323.
[14]Ray PS,Wang J,Qu Y,et al.FOXC1 is a potential prognosticbiomarker with functional significance in basal-like breast cancer[J].Cancer Res,2010,70(10):3870-3876.
[15]Egeblad M,Werb Z.New functions for the matrix metallopro-teinases in cancer progression[J].Nat Rev Cancer,2002,2(3):161-174.
[16]Skarie JM,Link BA.FoxC1 is essential for vascular basementmembrane integrity and hyaloid vessel morphogenesis[J].InvestOphthalmol Vis Sci,2009,50(11):5026-5034.