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目的分析miR-222在胃癌中的表达水平并鉴定miR-222的靶基因。方法选取第三军医大学新桥医院普外科10例患者的胃癌及远癌组织,匀浆法提取总RNA,用Real-time PCR法检测miR-222表达。利用miRNA靶基因预测数据库TargetScan,MICROCOSM及PicTar对miR-222的靶基因进行预测。化学合成包含有miR-222结合位点的靶基因3′非编码区(3′UTR)退火引物,插入荧光素酶报告载体pMIR-REPORT及绿色荧光蛋白(GFP)报告载体pMIR-GFP-RE-PORT,检测荧光素酶的活性变化及观察GFP表达量,并在蛋白水平用Western blot检测miR-222对靶基因的抑制。结果 miR-222在70%(7/10)的胃癌组织样本中表达上调。生物信息学(TargetScan,MICROCOSM及PicTar 3个miRNA靶基因预测数据库)分析可得在包括人类在内的不同物种中,FOS基因3′UTR区都有miR-222的保守结合种子序列。通过荧光素酶活性检测和GFP抑制试验可得miR-222在mRNA水平可以与FOS基因结合。Western blot结果显示miR-222可以抑制c-fos蛋白表达。结论 miR-222在胃癌样本中表达上调,证明FOS为miR-222的一个靶基因。
Objective To analyze the expression level of miR-222 in gastric cancer and to identify the target gene of miR-222. Methods Ten cases of gastric cancer and distant cancer tissues from General Surgery, Xinqiao Hospital, Third Military Medical University were selected. Total RNA was extracted by homogenization. The expression of miR-222 was detected by Real-time PCR. Target genes of miR-222 were predicted using TargetScan, MICROCOSM, and PicTar, a miRNA target prediction database. The 3 ’untranslated region (3’UTR) annealed primer containing the miR-222 binding site was chemically synthesized and inserted into luciferase reporter vector pMIR-REPORT and green fluorescent protein (GFP) reporter vector pMIR-GFP-RE- PORT was used to detect the changes of luciferase activity and to observe the expression of GFP. Western blot was used to detect the inhibition of target gene by miR-222 at the protein level. Results miR-222 was up-regulated in 70% (7/10) gastric cancer tissue samples. Bioinformatics (TargetScan, MICROCOSM and PicTar 3 miRNA Target Gene Prediction Databases) Analysis There are conserved binding sequences of miR-222 in the 3’UTR region of FOS gene in different species including human. Through luciferase activity assay and GFP inhibition assay, miR-222 can bind FOS gene at mRNA level. Western blot results showed that miR-222 can inhibit c-fos protein expression. Conclusion miR-222 is up-regulated in gastric cancer samples and FOS is a target gene of miR-222.