MicroRNA-338-5p suppresses metastasis of esophageal squamous cell carcinoma by targeting EGFR and cMET

Abstract

Esophageal squamous cell carcinoma (ESCC) is a subtype of esophageal cancer of which the incidence is relatively high in Eastern Asia. Despite recent advances in therapy methods against esophageal cancer, the outcome of ESCC patients is still dismal, with a 5-year survival rate of only 20%-30%. Its poor prognosis is partly due to the high rate of local invasion and distant metastasis. The involvement and roles of microRNAs (miRNAs) in metastasis have become one of the hot topics of research in recent years. MicroRNAs (miRNAs) are a group of small non-coding RNAs which functionally suppress gene translation or promote degradation of target mRNA by binding to the 3’-untranslated region (UTR). Frequent dysregulation of miRNAs has been observed in many tumor types, including ESCC. Increasing evidence supports the importance of miRNAs as regulators of tumorigenesis in a tumor-suppressive or oncogenic capacity. Previous studies performed by others and our group found that miR-338-5p is frequently downregulated in primary ESCC, and can modulate chemo- and radioresistance in ESCC by targeting Survivin and Id-1 in a suppressive way. However, the functions of miR-338-5p in ESCC are still far from being fully understood, especially in modulating cancer invasion and metastasis. Therefore, this study focuses on finding novel targets of miR-338-5p, and investigating its role and mechanism in regulating ESCC metastasis. Gain- and loss-of-function experiments confirmed that miR-338-5p had inhibitory effect on ESCC cell migration and invasion in vitro. Furthermore, stable overexpression of miR-338-5p downregulated matrix metalloproteinase 2 (MMP2) expression, but did not affect cell proliferation and epithelial-mesenchymal transition (EMT). Several predicted targets of miR-338-5p including epidermal growth factor receptor (EGFR), receptor tyrosine kinase MET (cMET), p21-activated kinase 4 (PAK4) and proliferating cell nuclear antigen (PCNA) were evaluated. Data from dual-luciferase reporter assay showed that miR-338-5p interacted with the 3’UTR of EGFR and inhibited its expression by promoting degradation of EGFR mRNA. Functionally, the miR-338-5p/EGFR targeting axis inhibited EGFR downstream signaling cascade and blocked EGF-induced cancer cell invasion. Moreover, cMET was found to be another novel target of miR-338-5p in ESCC cells, and introducing miR-338-5p into ESCC cells resulted in cMET signaling blockade. Re-introducing EGFR and cMET rescued the metastatic phenotype abolished by miR-338-5p overexpression in vitro and in vivo, indicating that EGFR and cMET mediated the inhibitory function of miR-338-5p on ESCC cell motility and metastasis. Moreover, systemically delivered miR-338-5p mimicking oligonucleotides inhibited metastasis of ESCC cells in vivo without obvious adverse effect on the wellbeing of animals, which suggests that miR-338-5p-based therapy might be an effective strategy in preventing ESCC metastasis. In summary, this study showed that the miR-338-5p exerts metastasis-suppressive function in ESCC through repressing EGFR and cMET protein expression and downstream signaling. The results provide a better understanding of how miR-338-5p dysregulation contributes to ESCC tumor invasion and metastasis, which may have further impact on understanding miRNA function in human cancer. Importantly, systemic delivery of miR-338-5p was found to effectively suppress metastatic burden, which highlights the therapeutic potential of miR-338-5p replacement therapy in improving the disease outcome of patients with advanced ESCC.