MiR-134 Inhibits Osteosarcoma Cell Invasion And Metastasis Through Targeting Of MMP1 And MMP3 Expression In Vitro And In Vivo

Objective:To explore the role of miR-134 in mechanism of invasion and metastasis of osteosarcoma,and provide a theoretical basis for the new therapeutic mechanism and target in clinical treatment of osteosarcoma,as well as the novel insight for the treatment of osteosarcoma.Methods:(1)qRT-PCR was used to detect the expression of miR-134,MMP1 and MMP3 in human osteosarcoma cell line Saos-2,MG-63 and human chondrocyte cell line CHON-001,and the differences were compared.Immunohistochemical staining was used to detect the differential expression of MMP1 and MMP3 between human osteosarcoma samples and normal bone tissues.(2)Transfected human osteosarcoma Saos-2 cells with lentiviral-miR-134 to overexpressed miR-134;the cells transfected with miR-134-Control lentivirus were used as control group;verified the transfection efficiency by qRT-PCR.Scratch test was used to detect the effect of miR-134 on the migration ability of osteosarcoma cells.The plasmid was transfected into Saos-2 cells to overexpress miR-134,and the effect of miR-134 on the invasion ability of osteosarcoma cells was detected by Transwell assay.(3)The nude mice were randomized and implanted subcutaneously with Saos-2-lentiviral-miR-134 cells or Saos-2-lentiviral-miR-Control cells mixed with Matrigel,to establish an osteosarcoma transplanted xenograft model,and detect the effect of miR-134 on osteosarcoma growth in vivo.Lung tissue HE staining was used to observe the effect of miR-134 on metastasis in nude mice.At the same time,qRT-PCR,immunohistochemistry,ELISA,and Western blot were used to detect the effect of miR-134 on the expression of MMP1 and MMP3 in vivo.(4)Dual-luciferase reporter assay was used to verified the direct targeting of miR-134 to MMP1 and MMP3.Results:(1)qRT-PCR results showed that miR-134 expression was under expressed in Saos-2(p<0.001),MG-63 cells(p<0.01);But MMP1 and MMP3 were overexpressed in human osteosarcoma cell lines(p<0.001).Immunohistochemistry results also showed that MMP1 and MMP3 overexpressed in human osteosarcoma tissues(p<0.001).(2)Lentivirus was transfected into human osteosarcoma Saos-2 cells to upregulate miR-134 expression,and the transfection efficiency was over 95%,and qRT-PCR results confirmed its high expression(p<0.05)).Wound healing assay showed that the cells in the miR-134 group had weaker migration ability than that in control group(p<0.001);Transwell assay showed that the invasive ability of Saos-2 cells in the miR-134 group was weaker than that in the Control group(p<0.001).(3)In vivo experiments showed that miR-134 inhibited MMP1 and MMP3 mRNA expression(p < 0.001);meanwhile,ELISA(p < 0.05,p < 0.001),immunohistochemistry(p<0.001),Western blot(p<0.001)and FMT(p<0.05)results showed miR-134 inhibits the expression of MMP1 and MMP3 proteins.At the same time,MMP1 and MMP3 were highly expressed in the osteosarcoma model compared to Saos-2 cells(p<0.001),while miR-134 was low expressed in the osteosarcoma model(p<0.001).(4)HE staining of lung tissue of nude mice showed that the lung metastasis of miR-134 group was significantly lower than that of Control group(p<0.001).Conclusions:(1)miR-134 was negatively correlated with MMP1 and MMP3 expression in osteosarcoma cell lines Saos-2,MG-63 and human osteosarcoma tissues.(2)miR-134 inhibits the migration and invasion of osteosarcoma cells in vitro.(3)miR-134 inhibits the growth,invasion and lung metastasis of osteosarcoma in vivo;and also suppresses the expression of MMP1 and MMP3.(4)miR-134 inhibits the invasion and metastasis of osteosarcoma in vivo and in vitro,as well as suppresses the expression of MMP1 and MMP3,which attributed to the direct target of miR-134 by MMP1 and MMP3.