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.