The Role And Mechanism Of MiR-200b-5p In Proliferation,migration,invasion And Apoptosis Of Human Osteosarcoma

Objective:Explore the effect of mi R-200b-5p on proliferation,migration,invasion and apoptosis of osteosarcoma and its underlying mechanism.Methods:(1)The RNA expression of mi R-200b-5p,c-Myc and c-Met in human chondrocytic cell line(C28),osteosarcoma cell line(Saos2,U2OS)and the collected osteosarcoma tissue and normal bone tissues were detected via q RT-PCR,and the protein expression of c-Myc and c-Met in C28,Saos2 and U2 OS cells was verified by Western Blot.(2)Saos2cells were transfected with mi R-200b-5p mimic lentivirus respectively,mi R-200b-5p overexpression cells(mi R-200b-5p-mimic)were constructed as experimental group.Saos2 cells also transfected with negative virus which does not carry mi R-135a-5p,and these corresponding negative cells(Control)were constructed as control group.The transfection efficiency and the changes of downstream target gene m RNA were detected by q RT-PCR.CCK-8 method and colony formation test were used to detect the effect of mi R-200b-5p on the proliferation of osteosarcoma cells.The effects of mi R-200b-5p on the invasion and migration ability of osteosarcoma cells were verified by Transwell assay and wound healing assay.Flow cytometry was used to test the effect of mi R-200b-5p on the anti-apoptosis function of osteosarcoma cells.The effects of mi R-200b-5p on target genes c-Myc,c-Met,angiogenesis related proteins VEGF,MMP2,MMP9 and PI3K-AKT-m TOR,NF-κB pathway proteins were analyzed by Western Blot.(3)The xenograft tumor models of nude mice were established by injecting mi R-200b-5p-mimic and Control Saos2 cells respectively.According to the change of tumor size with time,the effect of mi R-200b-5p on the growth of osteosarcoma in vivo was studied.The lung tissue of nude mice was stained with HE to observe the lung metastasis of xenograft tumor,the effect of mi R-200b-5p on metastasis was studied.In xenograft tumors,the effect of mi R-200b-5p on the expression of angiogenesis related indexes VEGF,MMP2 and MMP9 was verified by immunohistochemistry.Results:(1)The result of q RT-PCR suggested that compared to the human chondrosarcoma cell C28,the expression of mi R-200b-5p in human osteosarcoma cell lines(Saos2,U2OS)decreased,while the expression of c-Myc and c-Met increased,and compared to the normal bone tissues,the expression of mi R-200b-5p in clinical osteosarcoma tissue decreased,while the expression of c-Myc and c-Met increased.West Blot results showed that the c-Myc and c-Met protein level in human osteosarcoma cell lines(Saos2,U2OS)was higher than that in human chondrocyte line C28.(2)As lentivirus mi R-200b-5p-mimic and corresponding control negative virus were transfected into Saos2 and U2 OS cells respectively,more than 90% of the cells were fluorescent observed by fluorescence microscope.Verified by q RT-PCR,the expression of mi R-200b-5p in mi R-200b-5p-mimic group was significantly higher than that in Control group,and the expression of c-Myc,c-Met and angiogenesis related indexes VEGF,MMP2 and MMP9 decreased.Verified by CCK-8 assay,the proliferation curve of cells in mi R-200b-5p-mimic group was lower than Control group.The colony formation assay showed that the colony number and colony size of cells in mi R-200b-5p-mimic group were lower than those in Control group.Both CCK-8 and colony formation assay all indicate that overexpress mi R-200b-5p will decrease the proliferation ability of OS cells.Verified by wound healing assay,that compared to the control group,the migration distance of cells in mi R-200b-5p-mimic group was less,indicating that the migration ability of OS cells became weaker after high expression of mi R-200b-5p.Verified by Transwell assay,the number of cells reach the lower surface of the Transwell membrane was much lower in the mi R-200b-5p-mimic group than control group,suggesting that overexpress mi R-200b-5p decreased the invasive ability of OS cells.The apoptosis detected by flow cytometry showed that the percentage of apoptotic in mi R-200b-5p-mimic group was significantly higher than that in control group,meaning that overexpress mi R-200b-5p will significantly weak the anti-apoptotic ability of OS cells.The Western Blot test showed that compared to the control group the expression of c-Myc,c-Met,PI3K-AKT-m TOR and NF-κB pathway in mi R-200b-5p-mimic group was lower.(3)The results of in vivo experiment suggested that the growth of xenograft in mi R-200b-5p-mimic group was significantly inhibited than that in Control group.Observed the HE staining of lung tissues which from the nude mice with xenograft,the amount of lung metastases in mi R-200b-5p-mimic group was less than Control group.The immunohistochemical results of xenograft in nude mice showed that the expression of angiogenesis related indexes VEGF,MMP2 and MMP9 decreased in mi R-200b-5p-mimic group.Conclusion:(1)The expression of mi R-200b-5p decreased and abnormal high expression of c-Myc and c-Met in osteosarcoma tissues of clinical osteosarcoma patients and osteosarcoma cell lines.(2)Overexpression of mi R-200b-5p weaken the proliferation of Saos2 cells in vitro,negatively regulate their migration,invasion and anti-apoptosis ability,reduce the expression of angiogenesis related indexes such as VEGF,MMP2,MMP9 and inhibit the angiogenesis of HUVEC cells.Overexpression of mi R-200b-5p down regulates the expression of c-Myc and c-Met in osteosarcoma,and also acts on the PI3K-AKT-m TOR and NF-κB pathways.(3)Overexpression of mi R-200b-5p can inhibit xenograft tumor growth and reduce the expression of VEGF,MMP2 and MMP9 in xenograft,suggesting that mi R-200b-5p may target VEGF,MMP2 and MMP9 in osteosarcoma and affect angiogenesis of osteosarcoma.