iTRAQ技术筛选人黑素瘤A375细胞系let-7a靶标蛋白  

Screening for and identification of targets of let-7a microRNA in A375 melanoma cells by using iTRAQ technology

作　　者：王焱[1] 张倩[1] 周晓伟 王震英[4] 方方[1] 孙建方 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所江苏省皮肤病与性病分子生物学重点实验室皮肤外科,南京210042 [2]中国医学科学院中心实验室,南京210042 [3]中国医学科学院中心实验室病理科,南京210042 [4]山东大学附属省立医院皮肤科

出　　处：《中华皮肤科杂志》2016年第5期342-347,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81272992）;江苏省自然科学基金（BK20131063）;江苏省临床医学科技专项（BL2012003）;山东省科技发展计划（2011GSF11847）;江苏省皮肤病号性病分子生物学重点实验室培育课题（2012ZD011）

摘　　要：目的同位素标记相对和绝对定量（iTRAQ）技术在人黑素瘤A375细胞系中筛选let-7a潜在的靶标蛋白，探讨let-7a的抑癌机制。方法100nmol／Llet-7a模拟物及其对照转染A375细胞，提取细胞总蛋白，运用同位素标记的iTRAQ技术分析和鉴定差异表达的蛋白质，运用生物信息学分析let-7a靶标蛋白及其功能，采用双荧光素酶报告系统验证靶标。结果let-7a模拟物组和对照组相比，质谱共分析鉴定到327个显著差异蛋白，其中表达量上调〉1．2倍的蛋白有151种，下调〈0．8倍的蛋白有176种。下调的176种蛋白中，软件预测到结合靶标有47个，双荧光素酶报告系统验证表明，let-7a模拟物组与对照组相比，HMGA2和THOC2野生型3’UTR比值分别降低64．3％和46．4％。结论iTRAQ技术和生物信息学方法在人黑素瘤A375细胞系中筛选出47个let-7a候选靶标蛋白，其中HMGA2和THOC2分别为let-7a的靶标。Objective To screen for and identify targets of let-7a microRNA （miRNA） in A375 melanoma cells by using isobaric tags for relative and absolute quantitation （iTRAQ） technology, and to explore mechanisms underlying the tumor-suppressing effect of let-7a. Methods Cultured A375 cells were classified into two groups to be transfected with 100 nmol/L hsa-let-7a mimics （hsa-let-7a mimics group） or negative control mimic （NC group）. After 54-hour incubation, A375 cells were collected and total proteins were collected, iTRAQ technology was used to analyze and identify differentially expressed proteins, bioinformatic analysis was performed to assess let-7a candidate targets and their functions, and a dual-luciferase reporter system was utilized to verify let-7a targets. Results As mass spectrometry showed, a total of 327 differentially expressed proteins were identified in the hsa-let-Ta mimics group compared with the NC group, including 151 up-regulated proteins with iTRAQ ratio 〉 1.2 and 176 down-regulated proteins with iTRAQ ratio 〈 0.8. Of 176 down-regulated proteins, 47 were predicted as miRNA targets by the miRWalk software. The dual-luciferase reporter system showed that the relative luciferase activity of the 3＇ untranslated region （UTR） of the wild-type HMGA2 and THOC2 genes were reduced by 64.3% and 46.4%, respectively, in the hsa-let-7a mimics group compared with the NC group. Conclusion A total of 47 candidate let-7a targets were screened out in A375 melanoma cells by using iTRAQ technology and bioinformatic analysis, and HMGA2 and THOC2 genes were identified as direct targets of let-7a.

关 键 词：黑色素瘤 细胞系 肿瘤 微RNAS 蛋白质组学 ITRAQ 

分 类 号：R739.5[医药卫生—肿瘤]