RNA干扰GCF2基因沉默对人肝癌BEL-7404细胞侵袭能力的影响
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摘要
目的探讨沉默GCF2基因表达对人肝癌BEL-7404细胞侵袭能力的影响。方法实验分为GCF2-siRNA组、NC-siRNA组及MOCK组,GCF2-siRNA组转染靶向GCF2基因的RNA干扰序列GCF2-siRNA,阴性对照组转染阴性对照序列NC-siRNA,MOCK组不转染任何序列。将靶向GCF2基因的RNA干扰序列(GCF2-siRNA)瞬时转染BEL-7404细胞以沉默GCF2表达,采用CCK-8法检测细胞增殖活性,体外侵袭实验检测细胞侵袭能力,Western blot检测基质金属蛋白酶(MMP)-9及GCF2靶基因MMP-23蛋白表达。结果转染GCF2-siRNA使GCF2表达下调,后者导致BEL-7404增殖受阻;GCF2-siRNA组的穿膜细胞数较阴性对照组(NC-siRNA)及未转染组(MOCK)明显减少(穿膜细胞数分别为41.5±0.95、61.3±1.57、64.5±1.65,P<0.05);MMP-9和MMP-23蛋白表达较两个对照组明显下降(P<0.05)。结论下调GCF2表达能抑制BEL-7404侵袭能力,提示GCF2促进肝癌细胞的侵袭可能是其参与肝癌发展进程的重要途径。
Objective To observe the effect of GCF2 on invasion of human hepatocellular carcinoma cell line BEL-7404. Methods The siRNA targeting GCF2 mRNA( named as GCF2-siRNA) was transiently transfected into BEL-7404 cells. Forty-eight hours after transfection,cell proliferation was determined by cell counting Kit-8,and cell invasion ability was determined by Tran-swell invasion assay. MMP-9 and MMP-23 protein expression was assessed by western blotting. Results GCF2-siRNA effectively down-regulated GCF2 expression. Compared with the negative control group and MOCK group,cell proliferation and invasion abilities in siRNA-GCF2 group were remarkably suppressed. Meanwhile,MMP-9 and MMP-23 protein expression was also obviously reduced. Conclusion Down-regulation of GCF2 by siRNA suppresses cell invasion abilities of BEL-7404,which suggests that GCF2 plays a crucial role in the progression of HCC by affecting cell invasion.
Objective To observe the effect of GCF2 on invasion of human hepatocellular carcinoma cell line BEL-7404. Methods The siRNA targeting GCF2 mRNA( named as GCF2-siRNA) was transiently transfected into BEL-7404 cells. Forty-eight hours after transfection,cell proliferation was determined by cell counting Kit-8,and cell invasion ability was determined by Tran-swell invasion assay. MMP-9 and MMP-23 protein expression was assessed by western blotting. Results GCF2-siRNA effectively down-regulated GCF2 expression. Compared with the negative control group and MOCK group,cell proliferation and invasion abilities in siRNA-GCF2 group were remarkably suppressed. Meanwhile,MMP-9 and MMP-23 protein expression was also obviously reduced. Conclusion Down-regulation of GCF2 by siRNA suppresses cell invasion abilities of BEL-7404,which suggests that GCF2 plays a crucial role in the progression of HCC by affecting cell invasion.
引文
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[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]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.
[4]Suriano AR,Sanford AN,Kim N,et al.GCF2/LRRFIP1 represses tumor necrosis factor alpha expression[J].Cell Biol,2005,25(20):9073-9081.
[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]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.
[7]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].Cancer Letter,2015,365(1):132-140.
[8]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.
[9]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.
[10]吴博,晁耐霞,赵飞兰,等.转录抑制因子GCF2促进肝癌细胞BEL-7404迁移相关靶基因的初步鉴定[J].基因组学与应用生物学,2015,34(1):35-40.
[11]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.
[12]曹建平,龙晓兰,龚勇,等.RNA干扰Cofilin-1基因沉默对人肝癌细胞株Huh-7侵袭和转移能力的影响[J].医学研究生学报,2015,28(5):465-469.
[13]Song B,Liu X,Wang Q,et al.Adenovirus-mediated shRNA interference against HSV-1 replication in vitro[J].J Neurovirol,2016,22(6):799-807.
[14]Wu A,Li J,Wu K,Mo Y,et al.LATS2 as a poor prognostic marker regulates non-small cell lung cancer invasion by modulating MMPs expression[J].Biomed Pharmacother,2016,82:290-297.
[15]Grunwald B,Vandooren J,Gerg M,et al.Systemic ablation of MMP-9 triggers invasive growth and metastasis of pancreatic cancer via deregulation of IL-6 expression in the bone marrow[J].Mol Cancer Res,2016.[Epub ahead of print]
[16]Li JP,Cao NX,Wu B,et al.Ch IP-chip analysis for target genes of GCF2transcription factor in a hepatocellular carcinoma cell line Hep G2[J].Ann Anat,2014,196(Suppl):281-286.
[17]Pei D,Kang T,Qi H.Cysteine Array Matrix metalloproteinase(CA-MMP)/MMP-23 is tapeⅡtransmembrane Matrix metalloproteinase regulated by a single cleavage for both secretion and activation[J].J Biol Chem,2000,275(43):33988-33997.
[18]Galea CA,Nguyen HM,George Chandy K,et al.Domain Structure and function of matrix metalloprotease 23(MMP23):role in potassium channel trafficking[J].Cell Mol Life Sci,2014,71(7):1191-1210.
[19]Gencer S,Cebeci A,Irmak Yazicioglu MB.Matrix metalloproteinase gene expressions might be oxidative stress targets in gastric cancer cell lines[J].Chin J Cancer Res,2013,25(3):322-333.
[20]Moogk D,da Silva IP,Ma MW,et al.Melanoma expression of matrixmetalloproteinase-23 is associated with blunted tumor immunity and poor responses to immunotherapy[J].J Transl Med,2014,12:342-352.
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