Screening for and identification of targets of let-7a microRNA in A375 melanoma cells by using iTRAQ technology
10.3760/cma.j.issn.0412-4030.2016.05.011
- VernacularTitle:iTRAQ技术筛选人黑素瘤A375细胞系let-7a靶标蛋白
- Author:
Yan WANG
;
Qian ZHANG
;
Xiaowei ZHOU
;
Zhenying WANG
;
Fang FANG
;
Jianfang SUN
- Publication Type:Journal Article
- Keywords:
Melanoma;
Cell line,tumor;
MicroRNAs;
Proteomics;
iTRAQ
- From:
Chinese Journal of Dermatology
2016;49(5):342-347
- CountryChina
- Language:Chinese
-
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-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-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.
