MiR-362-5p targets DKK1 and promotes the proliferation and invasion in tongue squamous cell carcinoma

Abstract

Objective To investigate the expression level of miR-362-5p in tongue squamous cell carcinoma (TSCC) tissues and cell lines, and to explore the effect of miR-362-5p on TSCC cells proliferation and invasion, and their related mechanisms. Methods Real-time quantitative PCR was performed to detect the relative expression of miR-362-5p in TSCC tissues and cell lines. SCC-9 and UM1 cells were transfected with miRNA mimic or antagomiR, cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation, and transwell assay was carried out to assess cell invasion. TOP/FOP luciferase ratio assay was used to test the Wnt/β-catenin pathway activity. Western blotting was carried out to measure the accumulation of β-catenin in nuclear. Bioinformatics, Western blotting and dual luciferase assay were performed to investigate the target genes of miR-362-5p. Statistical differences were determined by the two-tailed Student′s t-test between two groups of data; a P value<0.05 was considered statistically significant. Results miR-362-5p was highly expressed both in TSCC samples and cell lines, P values were less than 0.001. SCC-9 and UM1 cells transfected with antagomiR-362-5p or miR-362-5p mimic significantly inhibited (tSCC-9= 26.53, PSCC-9= 0.0083; tUM1= 24.45, PUM1= 0.0094) or upregulated (tSCC-9=-257.87, PSCC-9= 0.0008; tUM1=-270.44, PUM1= 0.0007) miR-362-5p expression levels. MiR-362-5p inhibition markedly decreased SCC-9 and UM1 cells proliferation (tSCC-9=32.44, PSCC-9= 0.0009; tUM1= 22.32, PUM1= 0.0013) and invasion (tSCC-9= 39.55, PSCC-9= 0.0008; tUM1= 23.78, PUM1= 0.0092) , while the cell proliferation (tSCC-9=-23.35, PSCC-9= 0.0084; tUM1=-17.47, PUM1=0.0138) and invasion (tSCC-9=-26.23, PSCC-9= 0.0072; tUM1=-18.69, PUM1= 0.0145) was apparently enhanced by ectopic expression of miR-362-5p. Compared with that in control cells, miR-362-5p overexpression enhanced the Wnt/β-catenin signaling pathway by down-regulating DKK1 expression. Conclusion miR-362-5p activated Wnt/β-catenin signaling by directly targeting DKK1, which enhanced TSCC cells proliferation and invasion. Key words: Carcinoma, squamous cell; Tongue; Neoplasm invasiveness; MiR-362-5p; DKK1; Wnt/β-catenin signaling pathway