miR-944 inhibits cell migration and invasion by targeting MACC1 in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a common disease in Southern China with high prevalence. miR-944 has been reported to play a vital role in progression of a variety of cancers. The present study aimed to investigate the potential role of miR-944 in NPC cell migration and invasion through elucidating the interaction with its target genes, MACC1. Expression of miR-944 in NPC tissues and cell lines was examined with quantitative RT-PCR. Overexpressed miR-944 and suppressed miR-944 were established with miR-944 mimics and miR-944 inhibitor, respectively. The effect of miR-944 on cell migration and invasion was determined using Transwell cell migration and Matrigel invasion assay. Luciferase assay was used to determine the target of miR-944. Knocked down of MACC1 was done by MACC1 siRNA. Expression of MET related-markers was examined using Western blot analysis. The expression level of miR-944 was downregulated in NPC tissues and cell lines. Overexpression of miR-944 significantly inhibited the cell migration and invasion in NPC 6-10B cells. In contrast, suppression of miR-944 promoted cell migration and invasion in NPC C-6661 cells. MACC1 is a direct target of miR-944. MACC1 expression was repressed in miR-944 mimic transfected cells while it was enhanced in miR-944 inhibitor transfected cells. MACC1 knock down inhibited cell migration and invasion. Either miR-944 restoration or MACC1 knockdown caused enhanced E-cadherin, reduced N-cadherin, and vimentin expression. In conclusion, miR-944 could inhibit MET and metastasis of NPC by targeting MACC1. This study suggests that miR-944 has anti-tumor and anti- metastatic properties and could thus be a novel therapeutic agent for NPC treatment.

Keywords: MACC1; Nasopharyngeal carcinoma; cell migration; invasion; miR-944.

Figure 1

Downregulation of miR-944 in NPC tissues and cell lines. A: Expression of miR-944 in 20 pair of clinical tissue specimens (NPC tissues Kunming 650500, Yunnan Province, China 20, tumor adjacent tissues = 20). B: Expression of miR-944 in human NPC cell lines (CNE2, CNE1, HNE1 and C666-1) and a nasopharyngeal epithelium cell, NP69. ***P < 0.001 vs normal control.

Figure 2

Effects of miR-944 overexpression on 6-10B cell migration and invasion. (A) Relative expression of miR-944 after mimic transfection. (B) Cell migration assessed by wound healing assay. Cell migration (C) and invasion (D) confirmed by Transwell assay in 6-10B cells. ***P < 0.001 vs negative control.

Figure 3

Effects of miR-944 suppression on C666-1 cell migration and invasion. (A) Relative expression of miR-944 after inhibitor transfection. (B) Cell migration assessed by wound healing assay. Cell migration (C) and invasion (D) confirmed by Transwell assay in C666-1 cells. *P < 0.05 vs negative control, ***P < 0.001 vs negative control.

Figure 4

MACC1 is a direct target of miR-944. (A) miR-944 binding site within 3’-UTR of MACC1 mRNA predicted by TargetScan. Dual luciferase activity in wild type and Mut type of 3’-UTR of MACC1 mRNA in cells transfected with miR-944 mimics (B) and miR-944 inhibitor (C). Relative MACC1 expression in cells transfected with miR-944 mimics (D) and miR-944 inhibitor (E) measured by Western blot analysis.

Figure 5

Effects of MACC1 knock down on 6-10B cell migration and invasion. A: Knock down efficiency of siMACC1 measured by Western blot analysis. B: Observation of migratory and invasive cells under light microscope. C: Histogram of number of cells that migrated and invaded after transfected with either siNC or siMACC1. ***P < 0.001 vs negative control.

Figure 6

Expression of ETM related markers measured by Western blot. A: Expression of E-cadherin, N-cadherin, and Vimentin in cells transfected with miR-944 mimics. B: Expression of E-cadherin, N-cadherin, and Vimentin in cells in which siMACC1 is knocked down.