MiRNA Expression Profiles And Functional Identification In Osteosarcoma

MicroRNAs(miRNAs)play important roles in the development,differentiation,and function of different cell types and in the pathogenesis of various human diseases.MiRNAs are differentially expressed in normal and cancer cells.Osteosarcoma is the most common primary malignant bone tumour,predominantly affecting children and adolescents.The mechanisms of formation and development of osteosarcoma remains unknown.Recently,Several studies have indicated that abnormal expression of miRNAs occurs frequently in human osteosarcoma.MiRNA in osteosarcoma research is still in its infancy.The investigation of miRNA expression in osteosarcoma is crucial for future clinical trials.These paper includes two parts,aiming at screening osteosarcoma miRNA expression profiling and the biological analysis and Subsequent miRNA functional identification,to explore the occurrence of osteosarcoma and transfer mechanisms provide a theoretical basis.Part 1 MiRNA expression profiles in osteosarcomaObjective Screening the different miRNA expression in osteosarcoma,and analysis the tumor related genes and signal pathway to provide basic data for further study;Method Gathering formalin-fixed,paraffin-embedded tissue samples to to extraction total RNA,including 50 cases ofosteoblastoma,50 cases of osteosarcoma and 20 cases of metastatic osteosarcoma;Performing miRNA microarray analysis on 8 samples,including 3 cases of osteoblastoma,5 cases of osteosarcoma and 2 cases of metastatic osteosarcoma;And confirming the results of the microarray analysis by using reverse transcription polymerase chain reaction;Then analysing the tumor related genes and signal pathway of miRNA profiles through biological information analysis.Result 1.The standard set of differentially expressed is p<0.05,log2>1,signal values above 1000.Using miRNA microarray to screen out of 10 differentially expressed miRNAs.Among the 10 miRNAs,3 miRNAs(miRNA494?miRNA4267?miRNA6133)were down-regulated in osteosarcoma;2 miRNA(miRNA4632-5p.miRNA 1227-5p)were up-regulated,whereas 5 miRNAs(miRNA3141?miRNA1908?miRNA3185?miRNA4467?miRNA6722-3p)were down-regulated in metastatic osteosarcoma,.2.By qPCR validation of the expanded sample about 10 differentially expressed miRNA,we got 5 significant different miRNAs.3 miRNAs(miRNA494?miRNA4267?miRNA6133)were down-regulated in osteosarcoma.Comparing with miRNA microarray,Results were consistent?whereas 2 miRNAs(miRNA3141?miRNA1908)were down-regulated in metastatic osteosarcoma.Comparing with miRNA microarray,partial results were consistent.3.Selecting four differentially expressed miRNAs(miRNA494?miRNA4267,miRNA 1908?miRNA3141)for bioinformatic analysis,we got 3780 related genetic information,These genes located in three categories including the cellular component,molecular function,biological process information;They play roles in 27 signaling pathways;They had 1467 target genes.Conclusion 1.MiRNA expression profiles of osteosarcoma were successfully screened by miRNA microarray and qPCR validation.2.Through bioinformatics analysis,more signaling pathways and a lot of target genes were obtained,they provided the basis for subsequent experiments.Part 2 MiRNA function identificationObjective:Based on the miRNA expression profiles of first part,choosing 2 down-regulated miRNA(miRNA1908?miRNA3141)in metastatic osteosarcoma,to verify miRNA functions in the high potential metastatic osteosarcoma cell.Method:MG63 cell transfected miRNAmimics(miRNA1908mimics,miRNA3141 mimics),comparing with the blank group and negative control group was transfected.Detecting the cell cycle and apoptosis rate of transfected MG63 cells by flow cytometry;Finding transfer efficiency of MG63 cells by transwell cell migration assay.Result:1.After transfected miRNA 1908mimics in MG63 cells,detected by flow cytometry,MG63 cells apoptosis increased(p<0.05),but no significant changes in cell proliferation;By transwell cell migration assay,it was not obvious that miRNA 1908mimics inhibited cell metastasis.2.After transfected miRNA3141 mimics in MG63 cells,detected by flow cytometry,MG63 cells proliferation decreased,apoptosis increased;By transwell cell migration assay,it was obvious that miRNA3141mimics inhibited cell metastasis.(p<0.05)Conclusion1.miRNA1908 acts as a tumor suppressor gene to promote apoptosis in osteosarcoma,but not obvious in inhibiting cell proliferation and metastasis.2.miRNA 3141 acts as a tumor suppressor gene to inhibit cell proliferation and metastasis,to promote apoptosis in osteosarcoma.