Comprehensive analysis of hsa-miR-654-5p’s tumor-suppressing functions

Abstract

BackgroundLung cancer is one of the most malignant types of cancer worldwide. Recently, the pivotal role of miRNAs in carcinogenesis and tumor metastasis have been reported for their direct regulation of specific target genes. However, the precise roles of miR-654-5p in lung adenocarcinoma have been poorly investigated. In this study, we designed a series of closed-loop integrated bioinformatic analyses and in vitro experimental validation to explore the main function and regulation pattern of miR-654-5p, and elucidate its roles in lung adenocarcinoma metastasis and prognosis both in silico and in vitro.MethodData from The Cancer Genome Atlas (TCGA) were used to analyze the pan-cancer prognostic value of miR-654-5p. miRanda, TargetScan, RNA22 and miRWalks were utilized to screen the targets of miR-654-5p. Metascape and miRWalks were used to perform gene ontology analyses and multiple enrichment analyses. STRING database and Cytoscape were used to construct protein-protein network and identify hub genes. GEPIA 2.0 were used to perform pan-cancer expression and survival analyses of hub genes. The regulation between miRNAs and predicted genes were verified by qPCR, western blotting and dual-luciferase system. In addition, EMT hallmarks detection, cell proliferation assay and wound healing assay were used to demonstrate the predicted functions of miR-654-5p experimentally.ResultsWe identified 275 potential targets of miR-654-5p and validated the direct regulatory effects of miR-654-5p on RNF8 in vitro as a proof of concept. Furthermore, we revealed the pivotal functions and disease association of miR-654-5p and experimentally validated the tumor suppressor roles of miR-645-5p that inhibited lung cancer cell epithelial-mesenchymal transition process, cell proliferation, and migration capacity. Among these 275 targets, 11 highly interconnected hub genes in lung adenocarcinoma were selected and studied through pan-cancer expression and pan-cancer survival analysis, in order to assess these targets’ clinical value.Conclusionsour research not only identified 275 hub target genes of miR-654-5p in silico and performed experimental validation but also revealed the tumor-suppressive roles of miR-654-5p in lung adenocarcinoma metastasis in vitro for the first time.