MiR-496 Inhibits Invasion And Metastasis Of Osteosarcoma Cells By Targeting The Wnt/β-catenin Pathway

Objective:To study the expression levels of miR-496 and β-catenin in osteoscarcoma, analyze whether there is binding of miR-496 and β-catenin, and explore the molecular mechanism by which miR-496 inhibits invasion and metastasis of ostesarcoma cells by targeting the Wnt/β-catenin pathway.Methods:Firstly, by using bioinformatics software-miRwalk and miRanda, this study looked for imRNAs that could bind with the target gene, β-catenin(CTNNB1), finding that 20 miRNAs might be potential target miRNAs of β-catenin; by further analysis, it found that the binding between miR-496 and β-catenin showed the best specificity and the strongest stability. In osteosarcoma tissues and adjacent fibrosic tissues, expression of miR-496 was detected by qRT-PCR, western blot was applied to detect the expression of β-catenin, and the relationship between expressions of miR-496 and β-catenin was analyzed; in osteosarcoma cell lines with different metastatic potentials, expression of miR-496 was detected by qRT-PCR, western blot was applied to detect expressions of β-catenin, CyclinD1 and MMP-7, and the relationships between expression of miR-496 and those of β-catenin, CyclinD1 and MMP-7 were analyzed; MG63 osteosarcoma cells were transfected with synthetic miR-496 mimics, sequences with point mutation(PM) and Scramble, respectively; expressions of miR-496 in different cell lines were detected by qRT-PCR, western blot was applied to detect expressions of β-catenin, CyclinDl and MMP-7, and the relationships between expression of miR-496 and those of β-catenin, CyclinD1 and MMP-7 were analyzed;Results:(1) There were 20 target miRNAs that could bind with CTNNB1, as predicted by bioinformatics methods; among all the bindings, the one between miR-496 and β-catenin showed the best specificity and the strongest stability.(2) miR-496 was lowly expressed in osteosarcoma tissues, while it was highly expressed in adjacent fibrosic tissues, with significant difference in expression level of miR-496 between them (P<0.01); β-catenin was highly expressed in osteosarcoma tissues but lowly expressed in adjacent fibrosic tissues, with significant difference in expression level of β-catenin between them (P<0.01). Expressions of miR-496 and β-catenin were negatively correlated and expression of β-catenin might be inhibited by miR-496 (P<0.01)(3) miR-496 was lowly expressed in high metastatic cell Line SOSP-9607, while β-catenin, CyclinDl and MMP-7 were all highly expressed; miR-496 was highly expressed in low metastatic cell line U-2OS, while β-catenin, CyclinD1 and MMP-7 were all lowly expressed; there was significant difference between expression level of miR-496 and those of the three proteins in different metastatic cell lines (P<0.01) Expression level of β-catenin could be regulated by miR-496, while expression level of β-catenin was positively correlated to CyclinD1 and MMP-7, which might associated with metastasis of osteosarcoma.(4) Expression of miR-496 was increased in cell line MG63-miR-496 mimics, while expression of β-catenin was down-regulated; however, there was no change in expression level of miR-496 in MG63-miR-Scramble, MG63-miR-496-PM and MG63 cell lines, and there was no significant change in expression level of β-catenin as well. It indicated that increased expression of miR-496 could down regulate the expression of β-catenin; however, after the occurrence of point mutation in miR-496, there was no change in expression level of β-catenin, because miR-496 couldn’t bind with β-catenin for degradation reaction, confirming that miR-496 could bind with β-catenin.(5) Count of invasive cells in cell line MG63-miR-496 mimics with high expression of miR-496 was significantly lower than those in the other three cell lines (P<0.01), and there was no significant difference among counts of invasive cells in MG63, MG63-miR-Scramble and MG63-miR-496-PM cell lines (P>0.05). High expression of miR-496 could inhibit the expression of β-catenin, and thus to inhibit the invasion and metastasis of osteosarcoma cells.Conclusions:(1) For β-catenin, miRNA-496 might be the target miRNA with the best binding stability and specificity, as found by bioinformatics methods.(2) Expressions of miRNA-496 and β-catenin were negatively correlated in osteosarcoma tissues.(3) Expressions of miRNA-496 and β-catenin were negatively correlated in osteosarcoma cells, and were associated with metastasis of osteosarcoma cells.(4) By targeting β-catenin and down-regulating the expressions of CyclinD1 and MMP-7, miRNA-496 could inhibit the invasion and metastasis of osteosarcoma cells.