The Role Of MiR-195in Metastasis Of Osteosarcoma And Its Mechanism

Osteosarcoma is the most common malignant tumor of bone, and about40%of cases develop lung metastasis with much lower survival rate. Therefore,it is extremely urgent to find effective early diagnostic tools and treatment inosteosarcoma.miRNA, a class of non-coding small RNA molecules, is involved inpost-transcriptional regulation that have been identified in various plants andanimals. Studies have demonstrated that miRNAs are associated with a varietyof tumor and play a key role in controlling the metastasis of tumor. So, it couldbe regarded as biomarkers and therapeutic targets for tumor metastasis.Our laboratory has carried out basic research in bone tumors for a long time.miRNA expression in the different metastatic potential osteosarcoma cell line F4and F5M2was investigated using miRNA microarrays.8miRNAs that weredifferentially expressed were identified. These differentially expressed miRNA may become metastasis-related biomarkers in osteosarcoma, but these requirefurther verification and research.Objective:To study the function and mechanism of miR-195in the metastasis ofosteosarcoma; to clarify target genes of miR-195in the metastasis ofosteosarcoma; to explore the possibility of miR-195as metastasis-relatedbiomarkers and therapeutic targets.Methods:1. To verify the differential expression of miR-195in the differentmetastatic potential osteosarcoma cell lines F4and F5M2by using the TaqManprobe real-time quantitative PCR analysis2. To construct the expression plasmid of miR-195with pSilencer4.1-CMV vector, and transfect it into F5M2cells, and obtain G418-selectedstable cell lines.3. Gain-of-function research. In vitro, to evaluate the effects of miR-195on proliferation, apoptosis, migration and invasion of osteosarcoma; In vivo, toobserve the effects of miR-195on the capacities of tumor formation andpulmonary metastasis of osteosarcoma in nude mice.4. To predict miR-195target genes by using bioinformatics software, incombining with47genes differentially expressed between the differentmetastatic potential of osteosarcoma cell line F4and F5M2. To verify thepotential target gene in gene and protein levels.5. To detect the expression of miR-195and FZD10in clinical osteosarcomasamples using real-time quantitative PCR and immunohistochemical staining, andanalyze the associativity of their expression trends and osteosarcoma metastasis. Results:1. The Realtime RT-PCR results showed that the expression of miR-195was lower expressed in F5M2cells as compared with F4cells.2. The pSilencer-miR-195was successfully constructed, and the stableosteosarcoma cell line F5M2+miR-195, which can up-regulate the expressionof miR-195, and its control group cell lines F5M2+NC, were also successfullyconstructed.3. In vitro, miR-195significantly inhibited the invasion and migration ofosteosarcoma cells F5M2, and also had some pro-apoptotic effect; Invivo,miR-195could significantly inhibit the capacities of tumor formation andpulmonary metastasis.4. FZD10was predicted to be a potential target gene of miR-195. Theimmunohistochemical staining and west blotting showed that expression ofFZD10was reduced in the F5M2+miR-195, and the results of luciferasereporter detection system showed that miR-195could bind the direct binding ofthe3’UTR of FZD10.5. In clinical osteosarcoma samples which were followed up to metastasis,the relative expression level of miR-195was significantly reduced, whileFZD10protein expression was significantly increased.Conclusion:1. The expression of miR-195in osteosarcoma cells was closelycorrelated with the capacity of metastasis.2. F5M2+miR-195cells provided the experimental basis for furtherstudy of miR-195function and mechanism in osteosarcoma.3. miR-195could significantly inhibit the capacity of metastasis ofosteosarcoma cells F5M2in vitro and in vivo. 4. FZD10, a target gene of miR-195, provided the theoretical basis for thestudy of miR-195in the regulatory mechanism in the osteosarcoma metastasis.5. miR-195and FZD10could be regarded as biomarkers and therapeutictargets for osteosarcoma metastasis.