[microRNA-16-5p targeted tetraspanin 15 gene to inhibit the proliferation, migration and invasion of osteosarcoma cell through phospoinositide 3-kinase/protein kinase B signaling pathway].

ABSTRACT

Objective: To study the effects of miR-16-5p on proliferation, migration and invasion of osteosarcoma cells and its mechanism. Methods: Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression of miR-16-5p and TSPAN15 in human normal osteoblasts hFOB 1.19 and osteosarcoma cells MG63, Saos2 and HOS. The miR-16-5p or si-TSPAN15 was transfected into MG63 cells to observe its role in cell proliferation, migration and invasion. Cell proliferation was measured with MTT assay, cell migration and invasion were examined by Transwell, and the protein expression of CyclinD1, matrix metalloproteinase 2 (MMP-2), MMP-9, tetraspanin 15 (TSPAN15), phospha-tidylinositol3-kinase(p-PI3K) and phospha-protein kinase B(p-AKT) were determined by using Western blotting. The starbase website prediction combined with dual luciferase gene reporter assay was performed to analyze the targeting relationship between miR-16-5p and TSPAN15. miR-16-5p and pcDNA-TSPAN1 were co-transfected to assess the effect of high expression of TSPAN15 on overexpression of miR-16-5p-induced proliferation, migration and invasion of MG63 cells. Data comparison between the two groups was performed by using t test. Results: Compared with hFOB 1.19 cells (1.00±0.12), the expression of miR-16-5p was significantly decreased in MG63, Saos2 and HOS cells (0.32±0.05, 0.40±0.04, 0.45±0.06, respectively)(F=156.204, P<0.05), and TSPAN15 mRNA and protein levels were greatly increased (F=71.718, 110.350, both P<0.05). Overexpression of miR-16-5p obviously reduced the expression of CyclinD1, MMP-2, MMP-9 protein, cell viability, cell migration and invasion (F=150.136,117.228, 154.971, 89.479, 98.373, 130.880, all P<0.05) in MG63 cells. Knockdown of TSPAN15 greatly reduced CyclinD1, MMP-2, MMP-9 protein levels, cell survival rate, cell migration, and invasion number (F=93.206, 107.030, 109.326, 115.625, 146.113, 139.300, all P<0.05). Overexpression of miR-16-5p markedly decreased the expression of p-PI3K and p-AKT protein in MG63 cells (F=156.755, 181.419, both P<0.05). miR-16-5p targeted to regulate the expression of TSPAN15. High expression of TSPAN15 partially reversed the inhibitory effect of miR-16-5p on TSPAN15, CyclinD1, MMP-2, MMP-9, p-PI3K, p-AKT protein expression, cell viability, cell migration number and invasion number in MG63 cells. Conclusion: miR-16-5p inhibits the proliferation, migration and invasion of osteosarcoma cells by targeting the TSPAN15 gene and regulating the PI3K/AKT signaling pathway.