The Role And Molecular Mechanism Of MiR-409-3P In Colorcctal Cancer Mctastasis

Colorectal cancer (CRC) is one of the most common cancers and the third leading cause of cancer death in men and women worldwide. Its metastasis accounts for the majority of CRC-related death. However, the molecular mechanisms underlying this process are not well characterized. MiRNA is proven to play important role in CRC progression and metastasis. Therefore, identification of key miRNAs in CRC metastasis and exploration of their mechanisms of action will contribute to the understanding of CRC metastasis. Our previous and other reported studies have shown that miR-409-3p is a pivotal miRNA that could be involved in various tumor develoment and progression, but its role in CRC is still unclear. In this study, we aim to explore the role and mechanism of action of miR-409-3p in CRC metastasis.To establish the association of miR-409-3p and CRC, we first assessed the miR-409-3p expression levels in82paired samples of CRC and their adjacent tissues. The results showed that miR-409-3p expression was significantly down-regulated in CRC tissue compared to adjacent non-tumor tissue, and reduced miR-409-3p expression was correlated with CRC metastasis. We further detected the expression of miR-409-3p in CRC cells. The results revealed that its expression in cells isolated from CRC metastatic sites was lower than that in cells from the CRC primary sites. These data suggest that miR-409-3p is a CRC metastasis-related miRNA. The in vitro and in vivo function studies revealed that miR-409-3p negatively regulated CRC metastatic capacities, including suppressing cancer cell migration, invasion, and metastasis, but without affecting CRC cell growth. These data indicate that miR-409-3p acts as a metastatic suppressor of CRC.To explore the mechanism of action of miR-409-3p, we adopted a pathway and pathophysiological event-based target screening and validation approach, and found9known metastasis-related genes as potential targets. The3’UTR binding assays between the candidates and miR-409-3p suggested that only GABl, NR4A2, and LM04were directly regulated by the miRNA. Considering that:1) The different microenviroment of293T/17comparing to CRC cells may lead to the misjudgement of potential target;2) Besides binding to the3’UTR of a target gene, miRNA could also regulate the expression of its target though interacting with the5’ UTR or a coding region. We therefore conducted western blot assay to directly measure the protein levels of all9predicted and the reported miR-409-3p targets in CRC cells. However, due to the absence of appropriate antibody, endogenous expression analysis revealed that only GABl was modulated by miR-409-3p in CRC cells at protein levels. Furthermore, in vitro evidence showed that restoration of GAB1rescued the impaired cell migration and invasion to～80%of the WT level. Finally, we confirmed the decreased expression of miR-409-3p and increased GAB1expression in eight paired fresh CRC tissues.Taken together, our data demonstrate that miR-409-3p is a metastatic suppressor, and post-transcriptional inhibition of the oncoprotein GAB1is one of the mechanisms of action of this miRNA. Our finding suggests miR-409-3p might be a novel target for CRC metastasis early diagnosis and/or treatment.