与ezrin基因表达相关的微小RNA的筛选及在卵巢上皮性癌浸润转移中的作用  

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

作　　者：李俊[1] 梁山辉[1] 鹿欣[1] 

机构地区：[1]复旦大学附属妇产科医院妇科,上海200011

出　　处：《中华妇产科杂志》2010年第10期787-792,共6页Chinese Journal of Obstetrics and Gynecology

摘　　要：目的 筛选卵巢上皮性癌（卵巢癌）细胞中与ezrin基因表达相关的微小RNA（miRNA）,并分析其在卵巢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卵巢癌细胞系SKOV3和SKOV3ip细胞、HO-8910和HO-8910PM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1对细胞系（即SKOV3和SKOV3ip细胞）用于以下实验.（2）用miRNA芯片筛选SKOV3和SKOV3ip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/v5/）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SKOV3ip细胞,用蛋白印迹法和实时RT-PCR技术检测其对SKOV3ip细胞中ezrin mRNA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 （1）SKOV3ip细胞中ezrin mRNA的表达水平是SKOV3细胞的（81.74±5.34）倍,HO-8910PM细胞中ezrin mRNA的表达水平是HO-8910细胞的（2.61±0.14）倍,两者分别比较,差异均有统计学意义（P＜0.01）;蛋白印迹法检测显示,SKOV3ip细胞中ezrin蛋白的表达强度明显高于SKOV3细胞,HO-8910PM细胞中ezrin蛋白的表达强度明显高于HO-8910细胞.选取其中ezrin mRNA和蛋白表达差异相对较大的1对细胞系,即SKOV3和SKOV3ip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SKOV3和SKOV3ip细胞中的表达差异,与miRNA芯片筛选结果一致,SKOV3细胞中miR-183和miR-22的表达水平分别是SKOV3ip细胞的（5.84±0.66）和（6.67±0.67）倍,两者分别比较,差异均有统Objective To screen microRNA （miRNA） that inhibit expression of the metastasisrelated gene ezrin in ovarian cancer cells and explore their correlation to the invasion and metastasis of ovarian cancer. Methods The differential expression of ezrin in two paired high-metastatic and lowmetastatic 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 SKOV3 and SKOV3ip cell lines. Three programs, TARGETSCAN （ http：//www. targetscan. org ）,MICROCOSM （ http ：//www. ebi. ac. uk/enright-srv/microcosm/htdocs/targets/v5/） 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 SKOV3 and SKOV3ip cells. To test the repressive potential of these miRNA, synthetic mimetics were transfected individually into SKOV3ip cells and endogenous ezrin expression levels monitored by western blot and real-time RT-PCR. Results （ 1 ） The mRNA average level of ezrin were （81.74 ± 5.34） -fold higher expression level in SKOV3ip versus SKOV3 cells （ P ＜ 0. 01 ）, while （2. 61 ±0. 14）-fold in HO-8910PM versus HO-8910 cells （P ＜0. 01 ）. Elevated protein level of ezrin were observed in SKOV3ip cells compared with that in SKOV3 cells, and the same that in HO-8910PM cells compared with HO-8910 cells. Paired SKOV3 and SKOV3ip cells were employed to study the more significant difference in ezrin expression between them. （2） By a functional screen using miRNA microarray combined with bioinformatics analysis,the miR-183 and miR-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 （5.84 ± 0.66）-fold and（6.67 ± 0.67）-fold higher expression level in SKOV3ip versus SKOV3 cells （P ＜0. 01 ）, whic

关 键 词：卵巢肿瘤 微RNAS 细胞支架蛋白质类 肿瘤转移 

分 类 号：R686[医药卫生—骨科学]