The Role Of MiR-34a In Metastasis Of Osteosarcoma And Its Mechanism

miRNA (microRNA) is a class of endogenous non-protein-coding single-stranded small RNA, which is about 20-25 nt in length. Its coding genes account for approximately 1% of the entire genome. miRNA plays an important role in the post-transcriptional regulation of gene expression. Studies showed that miRNA expression is prevalent in a variety of eukaryotic cells and involved in the regulation of growth and development of organisms, signal transduction, tissue differentiation, disease occurrence and so on. Meanwhile, studies also showed that miRNA plays an important role in the occurrence and development of a variety of tumors.miR-34a, which was initially found in C. elegans (Caenorhabditis elegans), is a member of an evolutionarily conserved miRNA family, miR-34s. miR-34a, whose encoding gene is on chromosome 1p36, is correlated with tumor occurrence and frequently missed in human neuroblastoma. miR-34a is directly regulated by p53 tumor suppressor. There is a highly conserved p53 binding site which is approximately 30kb above the miR-34a encoding gene. miR-34a plays the tumor inhibitory role by down-regulating its targets such as CDK4, CDK6, E2F3, E2F5, c-Met and so on. miR-34a also plays an important role in the p53-induced G1 phase arrest of cell cycle, cell senescence, apoptosis and other biological behavior. The inactivation and absence of miR-34a may be related to the pathogenesis of a variety of tumors. However, the relationship between osteosarcoma and miR-34a has been reported rarely. Therefore, it is of great significance to study the fuction and mechanism and to further explore the clinical application of miR-34a in osteosarcoma.Previously, we have evaluated the expression of miR-34a in osteosarcoma with different metastatic capacities by using microRNA microarray assay. The results show that the expression of miR-34a was down-regulated in high-metastatic osteosarcoma cell lines E10. Therefore, in this study, we will further study the function and mechanism of miR-34a in osteosarcoma.Objective:To verify the differential expression of miR-34a in the osteosarcoma cell lines with different metastatic capacities (E10, H9); To study the function and mechanism of miR-34a in osteosarcoma SOSP-9607 cells in order to further promote the clinical application of miR-34a in osteosarcoma prognosis and treatment.Methods:1. To verify the differential expression of miR-34a in the osteosarcoma cell lines with different metastatic capacities (E10, H9) by using Northern blotting and Realtime RT-PCR.2. To construct a recombinant eukaryotic expression plasmid of miR-34a, pcDNA-miR34a. To transfect it into osteosarcoma SOSP-9607 cells, and obtain G418-selected stable cell lines. 3. To evaluate the effects of miR-34a on proliferation, invasion and migration of osteosarcoma in vitro. To construct an osteosarcoma in situ tumor formation and pulmonary metastasis model, and then evaluate the effects of miR-34a on the capacities of in situ tumor formation and pulmonary metastasis.4. To predict miR-34a target genes by using bioinformatics calculation methods, such as miRanda, TargetScan and PicTar in combining with the fuctions of target genes. Ultimately, to selected the potential target genes that will be further verified.Results:1. We evaluated the differential expression of miR-34a in the osteosarcoma cell lines with different Metastatic capacities (E10, H9) by using Northern blotting and Realtime RT-PCR. The results showed that miR-34a was lower expressed in E10 cells as compared with H9 cells, which were consistent with the previous results from microRNA microarray assay.2. The human miR-34a eukaryotic expression vector pcDNA-miR34a was successfully constructed. pcDNA-miR34a can up-regulate the expression of miR-34a in osteosarcoma SOSP-9607 cells. Finally, we obtained stable osteosarcoma cells which expressed a high level of miR-34a.3. In vitro, miR-34a significantly inhibits the proliferation, invasion and migration of SOSP-9607 cells. And in vivo, miR-34a also remarkably inhibits the capacities of in situ tumor formation and pulmonary metastasis.4. We predicted miR-34a target genes by using bioinformatics methods, such as miRanda, TargetScan and PicTar in combining with the fuctions of target genes. Ultimately, we selected potential target genes that would be further verify. They are c-Met, Bcl2, Notch1 and Notch2.Conclusion: 1. The expression of miR-34a in osteosarcoma cells, which is different in E10 and H9 cells, is closely correlated with the capacity of metastasis. Thus, it is of great significance to to further study the fuction and mechanism of miR-34a in osteosarcoma.2. The eukaryotic expression plasmid of miR-34a was successfully constructed and effectively expressed in osteosarcoma SOSP-9607 cells. This facilitated the further investigations of function and mechanism of miR-34a in osteosarcoma.3. Functioning as a tumor suppressor, miR-34a can be used as a potential target for the treatment of osteosarcoma.4. We predicted miR-34a target genes by using bioinformatics methods, and initially chosed three target genes that may correlate to the fuction of miR-34a. This facilitated the further verification of miR-34a target genes.