miR-342-5p调控靶基因Merlin表达促进肝细胞癌侵袭转移的实验研究  

Enhancing effect of miR-342-5p on invasion and metastasis of hepatocellular carcinoma via regulating its target gene Merlin expression

作　　者：胡宽[1] 陶一明[1] 黄云[1] 李新营[1] 王志明[1] 

机构地区：[1]中南大学湘雅医院普外肝脏外科,湖南长沙410008

出　　处：《中国普通外科杂志》2014年第9期1200-1206,共7页China Journal of General Surgery

基　　金：国家自然科学基金面上基金资助项目(81372631)

摘　　要：目的:探讨miR-342-5p及其可疑靶基因Merlin在肝细胞癌(HCC)中表达及意义。方法:用qRT-PCR检测miR-342-5p和Merlin mRNA在HCC组织与癌旁组织,以及正常肝细胞系与各种不同HCC细胞系中的表达;用重组慢病毒包装质粒pGCSIL-GFP-miR-342-5p或空载体(阴性对照)转染HCCLM3细胞,以无处理的HCCLM3细胞作为空白对照,划痕愈合及Transwell实验观察细胞侵袭运动能力;Western blot法检测细胞中Merlin蛋白的表达。采用双荧光素酶基因报告系统,将含野生型或突变型Merlin基因3'UTR质粒(psiCHECK-Merlin)分别与pGCSIL-GFP-miR-342-5p、空载体(阴性对照)共转染或单独转染(空白对照)HCCLM3细胞后,检测各组细胞荧光素酶活性。结果:与癌旁组织比较,HCC组织中miR-342-5p表达明显上调,Merlin mRNA表达明显下调(均P<0.05);HCC组织中,血管侵犯组较无血管侵犯组miR-342-5p表达明显上调,Merlin mRNA表达明显下调(均P<0.05),且miR-342-5p与Merlin mRNA表达呈负相关(r2=5.364,P<0.05)。各HCC细胞系中miR-342-5p表达均明显高于正常肝细胞系,且miR-342-5p的表达随HCC细胞系的侵袭性增高而上调,而Merlin mRNA表达则呈相反趋势(均P<0.05)。与空白对照组及阴性对照组比较,HCCLM3细胞转染pGCSIL-GFP-miR-342-5p质粒后,细胞侵袭运动能力明显下降(均P<0.05),而Merlin蛋白表达明显上调。psiCHECK-Merlin野生型与pGCSIL-GFP-miR-342-5p质粒共转染HCCLM3细胞后,细胞的荧光素酶活性较其阴性对照组或空白对照组明显降低(P<0.05),而psiCHECK-Merlin突变型与pGCSIL-GFP-miR-342-5p质粒共转染HCCLM3细胞后,细胞的荧光素酶活性与其空白对照组或阴性对照组无统计学差异(P>0.05)。结论:Merlin是miR-342-5p的靶基因,miR-342-5p可能通过调控MerlinmRNA的表达促进肝细胞癌侵袭转移。Objective： To investigate the expressions of miR-342-5p and its putative target gene Merlin in hepatocellular carcinoma （HCC） and the significance. Methods： The expressions of miR-342-5p and Merlin mRNA in HCC and tumor-adjacent tissues as well as in normal hepatic cell line and different HCC cell lines were determined by qRT-PCR method. HCCLM3 ceils were transfected with lentiviral packaging plasmid pGCSIL-GFP-miR-342-5p or empty vector （negative control） with the untreated HCCLM3 cells as blank control, and then the invasion potential and migration activity were assessed by scratch-wound healing and Transwell migration assa）5 and the Merlin protein expression was measured byWestern blot analysis, respectively. Using a dual-luciferase reporter assay system, the plasmids tagged with the 3＇UTR of the wild- or mutant-type Merlin gene （psiCHECK-Merlin） was co-transfected with pGCSIL- GFP-miR-342-5p or empty vectors （negative control）, or transfected alone （blank control） into the HCCLM3 cells, and then the luciferase activity in each group of cells was detected. Results： Compared with tumor-adjacent tissue, the miR-342-5p expression was significantly up-regulated while Merlin mRNA expression was significantly down-regulated in HCC tissue （both P〈0.05）; in HCC tissues, the miR-342-5p expression was siguificantly higher while Merlin mRNA expression was significantly lower than that in non-vascular invasion group （P〈0.05）, in addition, the miR-342-5p and Merlin mRNA expression showed a negative correlation （r2=5.364, P〈0.05）. The miR-342-5p expression was siguificanfly increased in all the studied HCC cell lines compared with the normal hepatic cell line, and was up-regulated with the increase of the invasion ability of the HCC cells, while the Merlin mRNA expression showed the exact opposite pattern （all P〈0.05）. In HCCLM3 cells transfected with pGCSIL-GFP-miR-342-5p, the invasion potential and migration activity were significantly reduced （both P〈0.05） and t

关 键 词：癌 肝细胞 微RNAS miR-342-5p MERLIN 肿瘤侵润 

分 类 号：R735.7[医药卫生—肿瘤]