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  

Identify Target Genes Involved in Transcription Factor GCF2 that Promotes Cell Migration in Tumor Cell BEL-7404

作　　者：吴博[1] 晁耐霞[2] 赵飞兰[3] 李金平[4] 陈力[1] 陈德凤[1] 黄天明[1] 莫发荣[1] 张琪惠 罗国容[1] 

机构地区：[1]广西医科大学组织胚胎学教研室,南宁530021 [2]广西医科大学生物化学与分子生物学教研室,南宁530021 [3]广西中医药大学组织胚胎学教研室,南宁530021 [4]广西桂林医学院组织胚胎学教研室,桂林541004

出　　处：《基因组学与应用生物学》2015年第1期35-40,共6页Genomics and Applied Biology

基　　金：国家自然科学基金地区基金(81060168;81260313);广西自然科学基金青年基金项目(2014GXN-SFBA118157);广西医科大学青年科学基金(No.GXMUYSF201343)共同资助

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.

关 键 词：原发性肝细胞肝癌 GCF2 细胞迁移 小干扰RNA MYD88 

分 类 号：R735.7[医药卫生—肿瘤]