与ezrin基因表达相关的微小RNA的筛选及在肝细胞癌浸润转移中的作用  

Potential role of ezrin and its related microRNA in Liver cancer invasion and metastasis

作　　者：刘碧英[1] 李坚[1] 

机构地区：[1]中南大学卫生部肝胆肠外科研究中心,湖南长沙410008

出　　处：《中国现代医学杂志》2013年第36期5-10,共6页China Journal of Modern Medicine

摘　　要：目的筛选肝癌细胞中与ezrin基因表达相关的微小RNA(miRNA),并分析其在肝癌浸润转移中的作用。方法①实时逆转录(RT)PCR技术和蛋白印迹法分别检测两对具有不同侵袭转移能力的肝癌细胞系HepG2和HepG2-X细胞及SF7721和SMMC-7721细胞中ezrin mRNA和蛋白的表达差异,选取其中ezrin mRNA和蛋白表达差异相对较大的l对细胞系(即SF7721和SMMC-7721细胞)用于以下实验。②用miRNA芯片筛选SF7721和SMMC-7721细胞中差异表达miRNA,然后用生物信息学工具[三大数据库,即TARGETSCAN(http://www.targetscan.org)、MICROCOSM(http://www.ebi.ac.Uk/enright-srv/microcosm/htdocs/targets/vS/)和PICTAR(http://www.pictar.mdc-berlin.de)数据库]预测并比对miRNA芯片的筛选结果,筛选出与ezrin基因表达相关的miRNA,并采用实时RT-PCR技术验证。将筛选出来的miRNA转染SF7721细胞,用蛋白印迹法和实时RT-PCR技术检测其对SF7721细胞中ezrin mBNA和蛋白表达的调控作用,以未做任何处理者为空白对照。结果①SF7721细胞中ezrin mRNA的表达水平是SMMC-7721细胞的(62.53±4.37)倍,HepG2-X细胞中ezrin mRNA的表达水平是HepG2细胞的(2.61±0.23)倍,两者分别比较,差异均有显著性(P<0.01);蛋白印迹法检测显示,SF7721细胞中ezrin蛋白的表达强度明显高于SMMC-7721细胞,HepG2-X细胞中ezrin蛋白的表达强度明显高于HepG2细胞。选取其中ezrin mRNA和蛋白表达差异相对较大的l对细胞系,即SF7721和SMMC-7721细胞用于以下实验。②将miRNA芯片筛选结果与生物信息学工具预测结果对比分析,筛选出两个可能调控ezrin基因表达的特异miRNA,即miR-183和miR-22;实时RT-PCR技术检测显示,miR-183和miR-22在SF7721和SMMC-7721细胞中的表达差异,与miRNA芯片筛选结果致,SMMC-7721细胞中miR-183和miR-22的表达水平分别是SF7721细胞的(7.21±1.16)和(8.52±1.43)倍,两者分别比较,差异均有显著性(P<0.01)。miRNA转染SF7721细胞后,其ezrin mRNA的表达水平是空白对照的([ Objective ] To screen microRNA （miRNA） that inhibit expression of the metastasis related gene ezfin in liver cancer cells and explore their correlation to the invasion and metastasis of oliver cancer. [Methods ] Thedift^rential expression of ezrin in two paired high- metastatic and low-metastatic cell lines were examined by real time reverse transcription （RT）-PCR and western blot. A functional screen with microarray was employed to identify miRNA that were differentially expressed between SF7721 and SMMC-7721 cell lines. Three programs, TARGETSCAN（http：//www.targetscarL org）, MICROCOSM （http：//www.ebi.ac.Uk/enright-srv/micracosm/htdocs/targets/ vS/） and PICTAR （http：//www.pictar.Mdc-berlin.de）, were employed to identify all miRNA, which may inhibit the expression of ezrin and were differentially expressed between SF7721 and SMMC-7721 cells. To test the repressive potential of these miRNA, synthetic mimetics were transfected individually into SF7721 cells and endogenous ezrin expression levels monitored by western blot and real-time RT-PCR. [Results] The mRNA average level of ezrin were （62.53±4.37）-fold higher expression level in SF7721 versus SMMC-7721 ceils （P 〈0.01）, while （2.61±0.23）- fold in HepG2 versus HepG2-X cells （P 〈0.01）. Elevated protein level of ezrin were observed in SF7721 ceils compared with that in SMMC-7721 cells, and the same that in HepG2-X cells compared with HepG2 cells. Paired SF7721 and SMMC-7721 cells were employed to study the more significant difference in ezrin expression between them. By a functional screen using miRNA microarray combined with bioinformatics analysis, the miR-183 and rniR-22 were indentified as two candidate miRNA which may have the potential regulatory role in ezrin expression. Real time RT-PCR assays revealed that miR-183 and miR-22 were,respectively, an average of （7.21±l.16）-fold and （8.52 ±l.43） -fold higher expression level in SSF7721 versus SMMC-7721 cells （P 〈0.01） which were in agreement with

关 键 词：肝癌 微RNAS 细胞支架蛋白质类 肿瘤转移 

分 类 号：R735.7[医药卫生—肿瘤]