miRNA-383-5p Regulated Migration and Invasion of Tumor Cells by Inhibiting NCKAP1 Expression in Gastric Cancer

Abstract

Gastric cancer (GC) is the second deadliest disease in Asia, so it is crucial to find its promising therapeutic targets. The expression profile data of miR383-5p in the Cancer Genome Atlas (TCGA) were analyzed. The expression levels of miR383-5p in the collected clinical tissue samples and peripheral blood samples were examined by qPCR, and the relationship between its expression and the clinical data of patients was evaluated. MiR383-5p was overexpressed in the AGS cells, and cell biology assays, such as Transwell, were performed to detect the cell proliferation, migration, invasion and other cell biology abilities of miR383-5p. Target prediction and dual luciferase reporter gene assay were performed to find and validate the target genes of miR383-5p. The expression and activity of MMP and related proteins after overexpression of miR383-5p and NCKAP1 were detected by WB and gelatin zymography assay. The expression of miR383-5p was down-regulated in GC tissues, and its low expression was associated with lymph node metastasis. Restoration of miR383-5p expression in GC cells can inhibit the invasion and migration abilities of GC cells. MiR383-5p negatively regulated NCKAP1 through direct interaction with the 3'UTR sequence of NCKAP1. The overexpression of NCKAP1 can improve the migration and invasion abilities of GC cells, whereas overexpression of miR383-5p can inhibit growth of the aforementioned abilities of GC cells induced by NCKAP1 overexpression. The overexpression of NCKAP1 can increase the expression level and activity of MMP2, while the overexpression of miR383-5p can inhibit the increase of MMP2 expression level and activity in GC cells induced by NCKAP1 overexpression. NCKAP1 is a target gene of miR383-5p, and miR383-5p could be a valuable therapeutic target for stomach adenocarcinoma.

Keywords: Gastric cancer; Invasion; Migration; NCKAP1; miR-383-55p.

Fig. 1

miR383-5p expression is down-regulated in STAD A Data in TCGA database show down-regulated miR383-5p expression in STAD; B miR383-5p expression in clinical samples; C miR383-5p expression in peripheral blood

Fig. 2

Cell biological function assay of miR383-5p overexpression cell line. A qPCR verified that miR383-5p overexpression cell line was successfully constructed; B CCK8 results showed that miR383-5p overexpression had no significant effect on cell viability; C EDU results showed that miR383-5p overexpression had no significant effect on cell proliferation amplification, 10 ×; D Representative images of EdU staining; E Representative images of Transwell, amplification, 10 ×; F Transwell results show that miR-383 overexpression inhibited cell invasion; G Wound healing assay results show that miR-383 overexpression inhibits cell migration; H Representative images of Wound healing assay, amplification, 10 ×. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 3

Verification of targeting relationship between miR383-5p and NCKAP1. A miR383-5p had targeting binding sequenced with NCKAP1; B Dual luciferase assay showed that miR-383-5p could bind to NCKAP-Wt; C & D WB assay showed that miR383-5p overexpression inhibited the protein expression of NCKAP1 in GC cells. * p < 0.05; ** p < 0.01

Fig. 4

miR383-5p affected some biological functions of STAD cell lines by inhibiting NAKAP1 expression. A&B Overexpressed miR383-5p inhibited the expression of NAKAP1; C & D Transwell results indicated that overexpression of miR383-5p can inhibit to a certain extent the increase in invasive ability of gastric cancer cells caused by overexpression of NAKAP1, amplification, 10 ×; E & F The wound healing assay results indicated that overexpression of miR383-5p can inhibit to a certain extent the increase in migratory ability of gastric cancer cells caused by overexpression of NAKAP1, amplification, 10 ×. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 5

Activated MMP2 is involved in the process of miR383-5p-NCKAP1 affecting the motility of STAD cells A IHC results showed that the expression level of NCKAP1 in STAD tissues was significantly higher than that in paracancerous tissues, amplification, 10 ×; B & C WB assay showed that miR383-5p could inhibit the increased expression of MMP2, but not MMP9, caused by the overexpression of NCKAP: D Gelatin zymography assay showed that miR383-5p could inhibit the increased activity of MMP2 caused by NCKAP overexpression, but not MMP9. * p < 0.05; ** p < 0.01; *** p < 0.001