Mex3a promotes oncogenesis through the RAP1/MAPK signaling pathway in colorectal cancer and is inhibited by hsa-miR-6887-3p

Abstract

Background: Although Mex3 RNA-binding family member A (Mex3a) has demonstrated an important role in multiple cancers, its role and regulatory mechanism in CRC is unclear. In this study, we aimed to investigate the role and clinical significance of Mex3a in CRC and to explore its underlying mechanism.

Methods: Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression levels of genes. 5-Ethynyl-2'-deoxyuridine (EDU) and transwell assays were utilized to examine CRC cell proliferation and metastatic ability. The R software was used to do hierarchical clustering analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Overexpression and rescue experiments which included U0126, a specific mitogen activated protein kinase kinase/extracellular regulated protein kinase (MEK/ERK) inhibitor, and PX-478, a hypoxia-inducible factor 1 subunit alpha (HIF-1α) inhibitor, were used to study the molecular mechanisms of Mex3a in CRC cells. Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between two proteins. Bioinformatics analysis including available public database and Starbase software (starbase.sysu.edu.cn) were used to evaluate the expression and prognostic significance of genes. TargetScan (www.targetscan.org) and the miRDB (mirdb.org) website were used to predict the combination site between microRNA and target mRNA. BALB/c nude mice were used to study the function of Mex3a and hsa-miR-6887-3p in vivo.

Results: Clinicopathological and immunohistochemical (IHC) studies of 101 CRC tissues and 79 normal tissues demonstrated that Mex3a was a significant prognostic factor for overall survival (OS) in CRC patients. Mex3a knockdown substantially inhibited the migration, invasion, and proliferation of CRC cells. Transcriptome analysis and mechanism verification showed that Mex3a regulated the RAP1 GTPase activating protein (RAP1GAP)/MEK/ERK/HIF-1α pathway. Furthermore, RAP1GAP was identified to interact with Mex3a in Co-IP experiments. Bioinformatics and dual-luciferase reporter experiments revealed that hsa-miR-6887-3p could bind to the 3'-untranslated regions (3'-UTR) of the Mex3a mRNA. hsa-miR-6887-3p downregulated Mex3a expression and inhibited the tumorigenesis of CRC both in vitro and in vivo.

Conclusions: Our study demonstrated that the hsa-miR-6887-3p/Mex3a/RAP1GAP signaling axis was a key regulator of CRC and Mex3a has the potential to be a new diagnostic marker and treatment target for CRC.

Keywords: MEK/ERK/HIF-1α signaling; Mex3a; RAP1GAP; colorectal cancer; hsa-miR-6887-3p; invasion; microRNA; migration; prognosis; proliferation.