中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (5): 342-347.

• 论著 • 上一篇    下一篇

iTRAQ技术筛选人黑素瘤A375细胞系let-7a靶标蛋白

王焱1,张倩2,周晓伟3,王震英4,方方1,孙建方1   

  1. 1. 南京 中国医学科学院北京协和医学院皮肤病研究所
    2. 中国医学科学院皮肤病医院
    3. 中国医学科学院北京协和医学院皮肤病研究所
    4. 山东省立医院皮肤科
  • 收稿日期:2015-07-16 修回日期:2015-11-30 出版日期:2016-05-15 发布日期:2016-05-04
  • 通讯作者: 孙建方 E-mail:fangmin5758@aliyun.com

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

  • Received:2015-07-16 Revised:2015-11-30 Online:2016-05-15 Published:2016-05-04

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 100 nmol/L let-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。

Abstract:

Wang Yan, Zhang Qian, Zhou Xiaowei, Wang Zhenying, Fang Fang, Sun Jianfang Department of Dermatologic Surgery, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College; Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China (Wang Y, Zhang Q, Zhou XW, Fang F); Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China (Sun JF); Department of Dermatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China (Wang ZY) Corresponding authors: Sun Jianfang, Email: fangmin5758@aliyun.com; Fang Fang, Email: fangfangjh@126.com 【Abstract】 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-1et-7a mimics (hsa-1et-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-1et-7a 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-1et-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.