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
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摘要
GCF2是转录抑制因子,为了研究GCF2蛋白在肝癌细胞BEL-7404迁移中的作用,并根据芯片结果验证GCF2调控参与迁移的靶基因,我们采用siR NA沉默GCF2蛋白表达,然后通过transwell实验检测GCF2沉默前后BEL-7404细胞迁移的变化,最后荧光定量RT-PCR和Western blot验证芯片结果中与迁移相关基因的表达。研究结果显示siR NA干扰GCF2成功后进行迁移实验,si RNA-GCF2组,FAM-siR NA-NC组,脂质体组,和未处理野生型BEL-7404组24 h后细胞穿过8滋m微孔滤膜的数目(x±s)分别为41.66±1.52、41.66±1.15、41.33±1.5、18.66±0.577,差异均有统计学意义(p约0.01)。QRT-PCR验证9个候选基因中,MyD 88在si RNA-GCF2组内表达显著高于其余三组(p约0.05)。Western blot检测siR NA干扰GCF2后肝癌细胞BEL-7404的MyD 88的蛋白表达显著高于其余三组(p约0.05)。结果表明si RNA沉默转录因子GCF2蛋白表达可抑制肿瘤细胞BEL-7404迁移,推测GCF2作为转录因子可能通过抑制靶基因MyD 88的表达参与细胞的迁移。
GCF2 is a transcription inhibiting factor.In order to explore the function of GC-binding factor 2(GCF2) in migration of hepatocellular carcinoma cell BEL-7404,and identify migration related target genes,we used siR NA to silence the expression of GCF2 in BEL-7404,and analyzed cell migration by transwell assay.Then,fR eal-time fluorescent quantitative RT-PCR and western blot were used to identify nine migration related target genes screening from CHIP-chip method.Research findings showed that after siR NA silencing of GCF2 gene in BEL-7404 successfully,the number of the cells from the upper chamber into the lower chamber of siR NA-GCF2,FAM-si RNA-NC,lipo3000 and BEL-7404 were 41.66±1.52,41.66±1.15,41.33±1.5,18.66±0.577 respectively.Statistical analysis revealed that there was significant difference between siR NA-NC group and those other three groups(p<0.01),but no significant difference present among these three group.QRT-PCR results showed that only a significantly increase of MyD 88 expression in siR NA-GCF2 compared with other three groups(p<0.05),other 9 target genes assayed showed no significantly difference with other three groups.And western blot result showed that Myd88 protein in siR NA-GCF2 also significantly increased compared with other three group(p<0.05).Our results showed that GCF2 may promote cell migration in BEL-7404,and GCF2 may possible participate in cell migration through the key signal transduction pathway involving MyD 88.
GCF2 is a transcription inhibiting factor.In order to explore the function of GC-binding factor 2(GCF2) in migration of hepatocellular carcinoma cell BEL-7404,and identify migration related target genes,we used siR NA to silence the expression of GCF2 in BEL-7404,and analyzed cell migration by transwell assay.Then,fR eal-time fluorescent quantitative RT-PCR and western blot were used to identify nine migration related target genes screening from CHIP-chip method.Research findings showed that after siR NA silencing of GCF2 gene in BEL-7404 successfully,the number of the cells from the upper chamber into the lower chamber of siR NA-GCF2,FAM-si RNA-NC,lipo3000 and BEL-7404 were 41.66±1.52,41.66±1.15,41.33±1.5,18.66±0.577 respectively.Statistical analysis revealed that there was significant difference between siR NA-NC group and those other three groups(p<0.01),but no significant difference present among these three group.QRT-PCR results showed that only a significantly increase of MyD 88 expression in siR NA-GCF2 compared with other three groups(p<0.05),other 9 target genes assayed showed no significantly difference with other three groups.And western blot result showed that Myd88 protein in siR NA-GCF2 also significantly increased compared with other three group(p<0.05).Our results showed that GCF2 may promote cell migration in BEL-7404,and GCF2 may possible participate in cell migration through the key signal transduction pathway involving MyD 88.
引文
Ariake K.,Ohtsuka H.,Motoi F.,Douchi D.,Oikawa M.,Rikiyama T.,Fukase K.,Katayose Y.,Egawa S.,and Unno M.,2012,GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin-dependent Rho A activation,Cancer Lett.,325(1):99-107
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Jiang R.T.,Cao N.X.,Ma P.,Li J.P.,and Luo G.R.,2013,Screening of human hepatocellurar carcinoma cell lines withhigh expression of GCF2 gene,Guangdong Yixue(Guangdong Medical Journal),33(11):1551-1553(蒋日婷,晁耐霞,马平,李金平,罗国容,2013,肿瘤相关抗原GCF2高水平表达的肝癌细胞株的筛选,广东医学,33(11):1551-1553)
Li J.P.,Cao N.X.,Jiang R.T.,He S.J.,Huang T.M.,Wu B.,Chen D.F.,Ma P.,Chen L.,Zhou S.F.,Xie X.X.,and Luo G.R.,2014,Knockdown of GCF2/LRRFIP1 by RNAi causes cell growthinhibition and increased apoptosis in human hepatomaHep G2 cells,Asian Pac.J.Cancer Prev.,15(6):2753-2758
Naugler W.E.,Sakurai T.,Kim S.,Maeda S.,Kim K.,ElsharkawyA.M.,and Karin M.,2007,Gender disparity in liver cancerdue to sex differences in MyD 88-dependent IL-6 production,Science,317(5834):121-124
Ohtsuka H.,Oikawa M.,Ariake K.,Rikiyama T.,Motoi F.,Katayose Y.,Unno M.,and Johnson A.C.,2011,GCF2 interacts with dishevelled and regulates Wnt signaling pathways inhuman carcinoma cell lines,Int.J.Cancer,129(7):1599-1610
Rikiyama T.,Curtis J.,Oikawa M.,Zimonjic D.B.,Popescu N.,Murphy B.A.,Wilson M.A.,and Johnson A.C.,2003,GCF2:expression and molecular analysis of repression,Biochim.Biophys.Acta,1629(1-3):15-25
Shimizu Y.,Hishiki T.,Ujino S.,Sugiyama K.,Funami K.,andShimotohno K.,2011,Lipoprotein component associatedwith hepatitis C virus is essential for virus infectivity,Curr.Opin.Virol.,1(1):19-26
Suriano A.R.,Sanford A.N.,Kim N.,Oh M.,Kennedy S.,Henderson M.J.,Dietzmann K.,and Sullivan K.E.,2005,GCF2/LRRFIP1 represses tumor necrosis factor alpha expression,Mol.Cell Biol.,25(20):9073-9081
Wang J.Q.,Jeelall Y.S.,Ferguson L.L.,and Horikawa K.,2014,Toll-like receptors and cancer:MYD88 mutation and inflammation,Front.Immunol.,31(5):367
Wu J.,Li J.,Salcedo R.,Mivechi N.F.,Trinchieri G.,and Horuzsko A.,2012,The proinflammatory myeloid cell receptorTREM-1 controls Kupffer cell activation and developmentof hepatocellular carcinoma,Cancer Res.,72(16):3977-3986
Boucher E.,Forner A.,Reig M.,and Bruix J.,2009,New drugsfor t he treatment of hepatocellular carcinoma,Liver Int.,(S1):148-158
Jiang R.T.,Cao N.X.,Ma P.,Li J.P.,and Luo G.R.,2013,Screening of human hepatocellurar carcinoma cell lines withhigh expression of GCF2 gene,Guangdong Yixue(Guangdong Medical Journal),33(11):1551-1553(蒋日婷,晁耐霞,马平,李金平,罗国容,2013,肿瘤相关抗原GCF2高水平表达的肝癌细胞株的筛选,广东医学,33(11):1551-1553)
Li J.P.,Cao N.X.,Jiang R.T.,He S.J.,Huang T.M.,Wu B.,Chen D.F.,Ma P.,Chen L.,Zhou S.F.,Xie X.X.,and Luo G.R.,2014,Knockdown of GCF2/LRRFIP1 by RNAi causes cell growthinhibition and increased apoptosis in human hepatomaHep G2 cells,Asian Pac.J.Cancer Prev.,15(6):2753-2758
Naugler W.E.,Sakurai T.,Kim S.,Maeda S.,Kim K.,ElsharkawyA.M.,and Karin M.,2007,Gender disparity in liver cancerdue to sex differences in MyD 88-dependent IL-6 production,Science,317(5834):121-124
Ohtsuka H.,Oikawa M.,Ariake K.,Rikiyama T.,Motoi F.,Katayose Y.,Unno M.,and Johnson A.C.,2011,GCF2 interacts with dishevelled and regulates Wnt signaling pathways inhuman carcinoma cell lines,Int.J.Cancer,129(7):1599-1610
Rikiyama T.,Curtis J.,Oikawa M.,Zimonjic D.B.,Popescu N.,Murphy B.A.,Wilson M.A.,and Johnson A.C.,2003,GCF2:expression and molecular analysis of repression,Biochim.Biophys.Acta,1629(1-3):15-25
Shimizu Y.,Hishiki T.,Ujino S.,Sugiyama K.,Funami K.,andShimotohno K.,2011,Lipoprotein component associatedwith hepatitis C virus is essential for virus infectivity,Curr.Opin.Virol.,1(1):19-26
Suriano A.R.,Sanford A.N.,Kim N.,Oh M.,Kennedy S.,Henderson M.J.,Dietzmann K.,and Sullivan K.E.,2005,GCF2/LRRFIP1 represses tumor necrosis factor alpha expression,Mol.Cell Biol.,25(20):9073-9081
Wang J.Q.,Jeelall Y.S.,Ferguson L.L.,and Horikawa K.,2014,Toll-like receptors and cancer:MYD88 mutation and inflammation,Front.Immunol.,31(5):367
Wu J.,Li J.,Salcedo R.,Mivechi N.F.,Trinchieri G.,and Horuzsko A.,2012,The proinflammatory myeloid cell receptorTREM-1 controls Kupffer cell activation and developmentof hepatocellular carcinoma,Cancer Res.,72(16):3977-3986
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