Micro RNA-365-3p as a Tumor Suppressor in Gastric Cancer: Inhibition of NUCKS1-Mediated PI3K/AKT Signaling and Anti-Tumor Effects

Abstract

Increasing evidence supports that microRNAs (miRNAs) are essential regulators of tumor initiation and development in a wide range of human malignancies, including gastric cancer (GC). To date, it has been reported that miR-365-3p is abnormally expressed and influences tumor progression in multiple malignancies. However, the roles of miR-365-3p are yet to be known and are worth investigating in GC. Herein, we screened differentially expressed miRNAs in 3 pairs of matched gastric cancerous and adjacent normal specimens. A series of public datasets and collected clinical GC samples were employed to determine the expression profiles of miR-365-3p and its downstream target protein. Furthermore, the effects of miR-365-3p ectopic expression and depletion on GC cell proliferation, invasion and migration were explored in vitro via cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), clone formation and Transwell assays, and in vivo by applying the nude mice models. Dual-luciferase reporter, western blot and a series of rescue assays were carried out to investigate the potential interactions between miR-365-3p and NUCKS1 (Nuclear, casein kinase and cyclin-dependent kinase substrate 1) and downstream signaling pathways of the miR-365-3p/NUCKS1 axis. According to the analyses of the miRNA microarray and bioinformatics, we first identified miR-365-3p dysregulation in GC. Through miRNA qRT-PCR, we revealed that miR-365-3p was downregulated in GC samples and cell lines. Lower level of miR-365-3p was significantly associated with larger tumor size and advanced lymph node metastasis in GC patients. Downregulation of miR-365-3p accelerated, while ectopic miR-365-3p significantly suppressed GC cell proliferation, migration and invasion in vitro and in vivo. Results also showed that overexpression of miR-365-3p in gastric cancer cells significantly reduced the levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), while miR-365-3p knockdown increased their expression. Moreover, mechanistically, knockdown of miR-365-3p promoted tumor cell aggressiveness by enhancing the expression of NUCKS1 and subsequently resulted in the activation of PI3K/AKT signaling pathway in GC. In addition, either NUCKS1 silencing or the inhibition of PI3K/AKT signaling pathway, at least partially, reversed the promotive effects of miR-365-3p knockdown on GC aggressiveness. Altogether, these findings suggest that miR-365-3p inhibits tumor growth and metastasis by attenuating the expression of NUCKS1 and inactivation of its downstream PI3K/AKT signaling, thus, highlighting the potential of miR-365-3p as a therapeutic target in GC.