MiR-125b Suppress The Proliferation And Migration Of Osteosarcoma Cells And Its Possible Role In The Signal Pathway

Objective:This project was conducted to investigate the biological role of miR-125b in osteosarcoma by detecting the expression levels of miR125b in osteosarcoma tissues and cells. We used bioinformatics methods to predict the signaling pathways which miR-125b was involved and investigated the role of miR-125b in the molecular mechanisms in occurrence and development of osteosarcoma.Methods:1. QPCR was used for detecting the expression of miR-125b in15cases of osteosarcoma tissues, their paired adjacent normal tissues, and osteosarcoma cell line MG63, Saos-2, U2OS. The human embryonic kidney cells was served as a control.2. Synthetic miR-125b mimics and miR-125b antisense RNA were transfected into osteosarcoma cell MG63and Saos-2, the impact of overexpression and silence of miR-125b on the invasion ability of MG63and Saos-2osteosarcoma cell was detected by Transwell assay。Synthetic miR-125b mimics and miR-125b antisense RNA were transfected into osteosarcoma cell MG63and Saos-2, and then CCK8assay was used to invest the effect of the overexpression and silence of miR-125b on the MG63and Saos-2osteosarcoma cells’proliferation.3. Tumor formation in nude mice was used to observe the impact of overexpression of miR-125b on tumorigenicity of Saos-2cells in mice.4. We used bioinformatics software to predict and analyze the potential target genes of miR-125b involved in the MARK signaling pathway.5. QPCR and Western blot assay were used for detecting the expression levels of MARK signaling pathway upstream gene MKK7mRNA and its specific protein under the state of overexpression and silence of miR-125b in MG-63cells.6. We transfected miR-125b overexpression plasmid, miR-125b silence plasmid, control plasmid and luciferase reporter plasmid, which containing the sequence of MKK73’UTR to MG-63cells, Luciferase activity in different groups was detected to confirm the specific binding between miR-125b and MKK73’UTR region in osteosarcoma cells. Results:1. Compared with the control groups, the expression of miR-125b was downregulated in osteosarcoma tissues and cells.2. overexpression of miR-125b inhibited the ability of migration of MG63and Saos-2while silence of miR-125b promoted the migration of MG-63and Saos-2. Overexpression of miR-125b inhibited the proliferation of the MG-63and Saos-2wihile silence of miR-125b could promote their proliferation.3. The tumorigenicity ability of Saos-2was suppressed by miR-125b in in nude mice.4. Eight genes in upstream in MAPK pathway which miR-125b involved were predicted.3’UTR of MKK7contains highly conserved sequence which is capable of binding to miR-125b specifically.5. Silence of miRNA125bincreased the expression of MEKK7while overexpression of miR-125b decreased the expression of MKK7.6. Compared with the control group, the luciferase activity in overexpressed miR-125b group was significantly decreased, while luciferase activity in silenced miRNA125b group was significantly increased. Conclusions:1. The expression of MiR-125b was downregulated in osteosarcoma tissues and cell lines.2. Overexpression of MiR-125b not only inhibited the proliferation and migration of osteosarcoma cells in vitro, but also inhibited the tumor formation in nude mice.3. MiR-125b can regulate the expression of the target gene MEKK7in osteosarcoma cells, which is the upstream gene of MAPK signal pathway.