RNA干扰GCF2基因沉默对人肝癌BEL-7404细胞周期的影响
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摘要
目的 GC结合因子2(GCF2)是一种转录抑制因子,通过特异性结合富含GC序列的基因启动子而抑制该基因转录。文中通过靶向沉默GCF2基因表达观察GCF2对人肝癌细胞BEL-7404细胞周期的影响。方法将细胞分为空白对照组、阴性对照组及GCF2-siRNA组。空白对照组不转染任何序列,阴性对照组转染阴性对照序列,GCF2-siRNA组转染靶向GCF2基因的GCF2-siRNA序列以沉默GCF2在肝癌BEL-7404细胞中的表达。CCK-8法和流式细胞术分别检测沉默GCF2对细胞增殖和周期的影响,Western blot检测细胞周期蛋白cyclin D1及cyclin E、细胞周期蛋白依赖性激酶Cdk2及Cdk4蛋白表达。结果 GCF2下调后,GCF2-siRNA组细胞增殖在转染24、48、72和96 h受到不同程度抑制,抑制率分别为27.3%,43.6%,33.5%和29.7%,以转染48 h抑制率最高。与GCF2-siRNA组G1期百分比[(61.50±0.47)%]比较,空白对照组、阴性对照组[(45.50±0.75)%、(47.30±2.44)%]均下降(P<0.05);与GCF2-siRNA组S期百分比[(21.30±1.23)%]比较,空白对照组、阴性对照组[(37.00±1.80)%、(34.50±1.86)%]均升高(P<0.05)。转染48 h后,GCF2-siRNA组cyclin E和Cdk2蛋白相对表达量明显低于阴性对照组和空白对照组(P<0.05)。结论下调GCF2表达引起BEL-7404细胞周期阻滞,提示GCF2对肝癌细胞周期影响可能是其参与肝癌发生发展的重要途径。
Objective GC binding factor 2(GCF2) is a transcriptional factor that negatively regulates the transcription of PDGF-A,EGFR and TNF-α by specially binding totheir promoters enriching GC sequences.The goal of this study was to observe the effect of targeted GCF2 gene silencing on cell cycle of human hepatocellular carcinoma cell line BEL-7404.Methods BEL-7404 cells were divided into three groups:MOCK group(transfected with nothing),NC-siRNA group(transfected with negative control siRNA) and GCF2-siRNA group(transfected with the siRNA targeting GCF2 mRNA to inhibit the expression of GCF2 in the hepatoma cell BEL-7404).Cell proliferation was determined by Cell Counting Kit-8,cell cycle by flow cytometry,and protein expression of cyclin D1,cyclin E and Cdk2/4 by western blotting.Results After GCF2 down-regulation,cell proliferation was inhibited at varying degrees in GCF2-siRNA group at 24,48,72 and 96 h after transfected.The inhibition rates were 27.3%,43.6%,33.5% and 29.7% and the rate was highest at 48 h.The percentage of cells in G1 phase increased in GCF2-siRNA group compared with that in NC-siRNA group and MOCK group[(61.5±0.47) %,(47.3±2.44) % and(45.5±0.75) %,respectively](P < 0.05),and the percentage of cells in S phase decreased in GCF2-siRNA group compared with the NC-siRNA group and MOCK group [(21.30 ± 01.23) %,(34.50 ± 1.86) % and(37.00±1.80) %,respectively](P< 0.05) The cyclin E and Cdk2 protein expression reduced remarkably in GCF2-siRNA group at 48 h after transfection compared with NC-siRNA group and MOCK group(P<0.05).Conclusion Down-regulation of GCF2 could suppress cell proliferation and cause cell cycle arrest in BEL-7404,which suggest that GCF2 plays a vital role in the development of HCC by affecting cell cycle.
Objective GC binding factor 2(GCF2) is a transcriptional factor that negatively regulates the transcription of PDGF-A,EGFR and TNF-α by specially binding totheir promoters enriching GC sequences.The goal of this study was to observe the effect of targeted GCF2 gene silencing on cell cycle of human hepatocellular carcinoma cell line BEL-7404.Methods BEL-7404 cells were divided into three groups:MOCK group(transfected with nothing),NC-siRNA group(transfected with negative control siRNA) and GCF2-siRNA group(transfected with the siRNA targeting GCF2 mRNA to inhibit the expression of GCF2 in the hepatoma cell BEL-7404).Cell proliferation was determined by Cell Counting Kit-8,cell cycle by flow cytometry,and protein expression of cyclin D1,cyclin E and Cdk2/4 by western blotting.Results After GCF2 down-regulation,cell proliferation was inhibited at varying degrees in GCF2-siRNA group at 24,48,72 and 96 h after transfected.The inhibition rates were 27.3%,43.6%,33.5% and 29.7% and the rate was highest at 48 h.The percentage of cells in G1 phase increased in GCF2-siRNA group compared with that in NC-siRNA group and MOCK group[(61.5±0.47) %,(47.3±2.44) % and(45.5±0.75) %,respectively](P < 0.05),and the percentage of cells in S phase decreased in GCF2-siRNA group compared with the NC-siRNA group and MOCK group [(21.30 ± 01.23) %,(34.50 ± 1.86) % and(37.00±1.80) %,respectively](P< 0.05) The cyclin E and Cdk2 protein expression reduced remarkably in GCF2-siRNA group at 48 h after transfection compared with NC-siRNA group and MOCK group(P<0.05).Conclusion Down-regulation of GCF2 could suppress cell proliferation and cause cell cycle arrest in BEL-7404,which suggest that GCF2 plays a vital role in the development of HCC by affecting cell cycle.
引文
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[12]吴海卫,沈中华,王军,等.RNA干扰抑制c-myc基因对体外培养大鼠血管平滑肌细胞活性的影响[J].医学研究生学报,2012,25(1):11-16.
[13]Song B,Liu X,Wang Q,et al.Adenovirus-mediated shRNA interference against HSV-1 replication in vitro[J].J Neurovirol,2016.[Epub ahead of print]
[14]Lin SL,Chang DC,Ying SY,et al.MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of the CDK2 and CDK4/6 cell cycle pathways[J].Cancer Res,2010,70(22):9473-9482.
[15]Narasimha AM,Kaulich M,Shapiro GS,et al.Cyclin D activates the Rb tumor suppressor by mono-phosphorylation[J].elife,2014,3:e02872.
[2]Rikiyama T,Curtis J,Oikawa M,et al.GCF2:expression and molecular analysis of repression[J].Biochim Biophys Acta,2003,1629(1-3):15-25.
[3]Suriano AR,Sanford AN,Kim N,et al.GCF2/LRRFIP1 represses tumor necrosis factor alpha expression[J].Cell Biol,2005,25(20):9073-9081.
[4]Cohen G,Ettinger K,Lecht S,et al.Transcriptional down-regulation of epidermal growth factor(EGF)receptors by nerve growth factor(NGF)in PC12 cells[J].J Mol Neurosci,2014,54(3):574-585.
[5]Ohtsuka H,Oikawa M,Ariake K,et al.GC-binding factor 2 interacts with dishevelled and regulates Wnt signaling pathways in human carcinoma cell lines[J].Int J Cancer,2011,129(7):1599-1610.
[6]Ariake K,Ohtsuka H,Motoi F,et al.GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrindependent Rho A activation[J].Cancer Letter,2012,325(1):99-107.
[7]Shen DW,Pouliot LM,Gillet JP,et al.The transcription factor GCF2 is an upstream repressor of the small GTPAse Rho A,regulating membrane protein trafficking,sensitivity to doxorubicin,and resistance to cisplatin[J].Mol Pharm,2012,9(6):1822-1833.
[8]Douchi D,Ohtsuka H,Ariake K,et al.Silencing of LRRFIP1 reverses the epithelial-mesenchymal transition via inhibition of the Wnt/β-catenin signaling pathway[J].2015,365(1):132-140.
[9]蒋日婷,晁耐霞,马平,等.肿瘤相关抗原GCF2高水平表达的肝癌细胞株的筛选[J].广东医学,2012,33(11):1551-1553.
[10]李金平,蒋日婷,晁耐霞,等.siRNA沉默GC结合因子2表达对BEL-7404细胞增殖和凋亡的影响[J].医学研究生学报,2013,26(9):912-915.
[11]Li JP,Cao NX,Jiang RT,et al.Knockdown of GCF2/LRRFIP1by RNAi causes cell growth inhibition and increased apoptosis in human hepatoma Hep G2 cells[J].Asian Pac J Cancer Prev,2014,15(6):2753-2758.
[12]吴海卫,沈中华,王军,等.RNA干扰抑制c-myc基因对体外培养大鼠血管平滑肌细胞活性的影响[J].医学研究生学报,2012,25(1):11-16.
[13]Song B,Liu X,Wang Q,et al.Adenovirus-mediated shRNA interference against HSV-1 replication in vitro[J].J Neurovirol,2016.[Epub ahead of print]
[14]Lin SL,Chang DC,Ying SY,et al.MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of the CDK2 and CDK4/6 cell cycle pathways[J].Cancer Res,2010,70(22):9473-9482.
[15]Narasimha AM,Kaulich M,Shapiro GS,et al.Cyclin D activates the Rb tumor suppressor by mono-phosphorylation[J].elife,2014,3:e02872.
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