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

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 dualluciferase 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 upregulated proteins with iTRAQ ratio > 1.2 and 176 downregulated proteins with iTRAQ ratio < 0.8. Of 176 down-regulated proteins, 47 were predicted as miRNA targets by the miRWalk software. The dualluciferase 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. Key words: Melanoma; Cell line, tumor; MicroRNAs; Proteomics; iTRAQ