The Effects Of MicroRNA-410 On The Biological Behaviour Of Osteosarcoma Cells Via VEGF

Objective To investigate the relationship between miR-410 and VEGF in osteosarcoma cell line and osteosarcoma,and to explore the relationship between miR-410 and VEGF and the molecular mechanism of miR-410 regulating osteosarcoma.To explore the importance of miR-410 in the diagnosis and treatment of osteosarcoma,and to provide a theoretical basis for the development of miR-410 based OS treatment strategy.Methods 1.Identification of potential miRNA that regulate VEGF expression in OS.We first compared the VEGF protein levels in five OS cells.Western blot results show that the VEGF levels in Saos-2 and MG-63 cells were higher than those in other OS cell lines.An online database(http://www.tar getscan.org)was used to predict potential miRNA that regulate VEGF expression.Four miRNA were selected,namely,miR-29 a,miR-579,miR-410,and miR-125 a.To determine if all or some of these miRNA regulate VEGF expression,we constructed specific expression plasmids of each indi-vidual miRNA,and then individually transfected them into Saos-2 and MG-63 cells.Western blot was used to detect the levels of VEGF in each group.Total RNA was extracted from OS frozen tissue samples,miRNA-410 specific primers were used for reverse transcription to detect miR-410 expression,and the expression levels of miR-410 and VEGF were quantified by 2-△△Ct method;Western blot was used to detect VEGF protein expression in OS tissues;To further investigate the targeting relationship between miR-410 and VEGF,luciferase reporter assay was performed.2.Can exogenous miR-410 inhibit OS cell proliferation and invasion,and promote OS cell apoptosis? To assess the function of miR-410 in VEGF-positive OS cells,miR-410 precursor expression plasmids were introduced.MG-63 and Saos-2 cells were transfected with either control plasmid or miR-410 precursor expression plasmids and incubated in a medium containing 10 % FBS.The level of miR-410 was detected by PCR.MTS-based cell proliferation assay detects whether miR-410 inhibits cell growth.To further validate the effect of miR-410 on MG-63 and Saos-2 cells,cell apoptosis,migration and invasive ability were analyzed by flow cytometry and transwell assay.3.Can restoration of VEGF recovery reverse the inhibitory effect of miR-410 on OS cells? To test whether the inhibitory effect of miR-410 in OS cells is mediated by VEGF,VEGF CDS expression plasmid without 3′-UTR was used to resist the negative effects of miR-410 in OS cells.Western blot analysis showed whether transfection of the VEGF expression plasmid altered the protein levels of VEGF and p-AKT(ser473)compared to the control group in miR-410 treated OS cells.Furthermore,Transwell and cytometry analysis showed whether VEGF reversed the effect of miR-410 on cell invasion and apoptosis in OS cells compared to the control group.4.Can miR-410 inhibit tumor growth in OS in vivo? To determine if miR-410 has a similar function in vivo,stable MG-63 and Saos-2 cells with upregulated miR-410,and the control group cells were subcutaneously injected into mice.At 30 d after implantation,the mice were sacrificed and the tumors were removed and photographed.Tumor volume was compared between OS cell group and control group.Six tumors per group were selected for q RT-PCR and Western blot.Results 1.MiR-410 is underexpressed in OS,targeting VEGF and is negatively correlated with its expression.Western blot results showed that VEGF levels in Saos-2 and MG-63 cells were higher than those in other OS cell lines.Four miRNA(i.e.,miR-29 a,miR-579,miR-410,and miR-125a)plasmids were separately transfected into Saos-2 and MG-63 cells,respectively.Transfection of miR-410 resulted in lower levels of VEGF protein than transfection of other miRNA.The results of OS samples showed that miR-410 was negatively correlated with VEGF m RNA(r =-0.7651),and miR-410 was negatively correlated with VEGF protein(r =-0.8342).The dual luciferase reporter system(Promega)measures firefly luciferase and Renilla luciferase activity,and the results indicate that miR-410 directly binds to the 3’-UTR of VEGF m RNA.2.Exogenous miR-410 inhibits OS cell proliferation,invasion,migration,and promotes OS cell apoptosis.MG-63 and Saos-2 cells were transfected with control plasmid or miR410 precursor plasmid.PCR results showed that miR-410 levels increased after miR-410 precursor transfection,and the difference between the two groups was statistically significant(P<0.05).MTS-based cell proliferation assay showed that miR-410 strongly inhibited cell growth compared to cells transfected with control plasmid,and the difference between the groups was statistically significant(P<0.05).The cell invasion ability was analyzed by Transwell assay.The results showed that miR-410 overexpression inhibited the invasion of OS cells,and the difference between the two groups was statistically significant(P<0.05).The cell migration ability was analyzed by Transwell assay.The results showed that miR-410 overexpression inhibited the migration of OS cells,and the difference between the groups was statistically significant(P<0.05).Apoptosis was analyzed by Annexin V-PE/7-AAD double staining.The results showed that the apoptotic rate of the transfected miR-410 group was significantly higher than that of the control group,and the difference between the groups was statistically significant(P< 0.05).3.Restoration of VEGF reversed the inhibitory effect of miR-410 on OS cells.The VEGF CDS expression plasmid without 3’-UTR was used to counter the negative effects of miR-410 in OS cells and Western blot detection was performed.The protein levels of VEGF and p-AKT(Ser473)in Saos-2 transfected miR-410+VEGF group were significantly higher than those in Saos-2 transfected miR-410 group,and Saos-2 transfected miR-410+ pcDNA3.1 group.The difference was statistically significant(P<0.05).The protein levels of VEGF and p-AKT(ser473)in MG-63 transfected miR-410+VEGF group were significantly higher than those in MG-63 transfected miR-410 group,and MG-63 transfected miR-410+ pcDNA3.1 group.The difference was statistically significant(P<0.05).To further verify that VEGF restoration reversed the inhibitory effect of miR-410 on OS cells,cell invasion ability was analyzed by Transwell assay.The results showed that the invasive ability of Saos-2 and MG-63 transfected miR-410+VEGF group was significantly higher than that of transfected miR-410 group and transfected with miR-410+ pcDNA3.1 group,which was statistically significant(P<0.05).To further verify that VEGF restoration reversed the inhibitory effect of miR-410 on OS cells,apoptosis was analyzed by Annexin V-PE/7-AAD double staining.The results showed that the apoptosis rate of Saos-2 and MG-63 transfected miR-410+VEGF group was significantly lower than that of transfected miR-410 group and transfected miR-410+ pcDNA3.1 group,which was statistically significant(P<0.05)..4.MiR-410 inhibits tumor growth of OS in vivo.The tumor volume of Saos-2 transfected miR-410 group was significantly smaller than that of Saos-2 transfected pcDNA3.1 group(P<0.05).The tumor volume of MG-63 transfected miR-410 group was significantly lower than that of MG-63 transfected pcDNA3.1 group(P<0.05).Quantificational Real-time PCR was performed for each group of six tumors.The results showed that the expression of miR-410 was significantly up-regulated in the Saos-2 transfected miR-410 group and the MG-63 transfected miR-410 group,while the VEGF expression was in the miR-410 up-regulated group.It was significantly down-regulated,and the difference was statistically significant(P<0.05).Western Blot was performed in each group of six tumors.The results showed that the levels of VEGF protein in Saos-2 transfected miR-410 group and MG-63 transfected miR-410 group were significantly lower than those in the control group.Conclusions 1.MiR-410 is lowly expressed and negatively correlated with VEGF expression in OS samples.MiR-410 regulates the biological behavior of OS cells by targeting VEGF 2.Up-regulation of miR-410 inhibits OS cell proliferation,invasion,migration,and promotes OS cell apoptosis.3.Restoration of VEGF reversed the inhibitory effect of miR-410 on OS cells.4.MiR-410 inhibits OS tumor growth in vivo.