Effects On Invasion And Migration Of Human Osteosarcoma MG63 Cells By Targeting Regulation Of MicroRNA-192 On Matrix Metallo Proteinases-11

Background and PurposeOsteosarcoma is a malignant tumor originated from primitive mesenchymal stem cells. It is the most important primary bone tumor in children and adolescents. Surgery and multiple drug chemotherapy is the standard treatment for the disease, new developments in the treatment of osteosarcoma has made the prognosis of the patients had been improved significantly, but there are still about 40% of the tumor metastasis, suggesting that the tumor cell resistance to chemotherapy. Therefore, it is urgent to discover new biomarkers to predict the prognosis of these patients and to provide treatment methods. Micro RNA(miRNA) is a small non coding single stranded RNA, with a length of 19 to 25 nucleotides, which are present in the genomes of plants and animals. The normal function of miRNA including the regulation of cell differentiation pathways, cell cycle and apoptosis. The expression of miRNAs dysregulation may lead to tumor development. Recently, miR-192 expression was down regulated in lung cancer, bladder cancer, colorectal cancer, breast cancer and other cancer cells, and the invasion and migration of tumor cells were often inhibited. But so far no expression of miR-192 in osteosarcoma and its effect on osteosarcoma were found. Matrix metalloproteinases(MMPs) are a family of zinc dependent metalloproteinase. The organ of MMP-11 in embryo implantation and development, plays the role of organizing degeneration and repair process, but also the impact of many diseases, such as atherosclerosis, rheumatoid arthritis and cancer. The bad prognosis of primary MMP-11 patients with high level of tumor is closely related to the.This experiment aims to study of miR-192 target to regulatethe influence of MMP-11 to osteosarcoma invasion and migration of, to understand the relationship between miR-192, MMP-11 and osteosarcoma and to osteosarcoma diagnosis and gene target to provide the basis for treatment. Methods1. Clinically, 20 patients with osteosarcoma were collected, and the adjacent tissues and adjacent tissues were collected. m RNA expression of miR-192 and MMP-11 gene was detected by q RT-PCR method. In addition, Western blot was used to detect the expression level of MMP-11 protein in the tissues.2. The synthesis of miR-192 mimics and negative control was transferred into osteosarcoma MG63 cells by liposome. The cells were divided into three groups according to the different transfection materials. Mi R-192 group: transfection of miR-192 mimics and liposome; NC group: miR-192 negative control and liposome transfection;Blank group: liposome only. The transfected cells were cultured in a carbon dioxide incubator for subsequent experiments.3. The effect of miR-192 on the migration of MG63 cells was observed by scratch test on three groups of cells.4. Transwell assay was used to evaluate the cell invasion effect of miR-192 on MG63 cells.5. Bioinformatics software were used to predicte the targets of miR-192. Constructed wild type and mutant type recombinant vectors thatcontained MMP-11 3′UTR region. Respectively, co-transfected them with miR-192 mimics or Scramble into MG63. We use western blotting assays and luciferase reporter to confirm the major targets of miR-192. Results1. The mir-192 expression level in osteosarcoma was significantly lower than that in paracancerous tissues(P<0.05). The m RNA expression level and protein expression levelof MMP-11 in osteosarcoma was significantly higher than that of cancer adjacent tissues(P<0.05).2. After transfection, miR-192 expression level in miR-192 group was significantly higher than that in NC group and Blank group(P<0.05), which indicated that miR-192 successfully expressed in transfected cells.3. The scratch test results showed that compared with NC group and Blank group, miR-192 group migration of osteosarcoma MG63 cells decreased significantly.4. Transwell assay showed that compared with the Blank and NC groups, the mean number of osteosarcoma MG63 cells penetrating the membrane for miR-192 group were significantly decreased(P<0.05).5. Western Blot and dual luciferase reporter assay proved that MMP-11 was the target of miR-192. Mi R-192 targeted to the MMP-11 3’UTR, negative regulating its expression. Conclusions1. High expression of miR-192 can effectively inhibit the migration and invasion of the osteosarcoma cells.2. miR-192 negative regulates the expression of MMP-11 by targeting to the 3’UTR of MMP-11.