MicroRNA-27a Promotes Proliferation,Migration And Invasion By Targeting MAP2K4in Human Osteosarcoma Cells

Osteosarcoma is a high-grade malignant bone neoplasm that occurs primarily in children and adolescents. The clinical treatment for osteosarcoma is very difficult. Despite incorporation of chemotherapy into initial treatment, which significantly increases the cure rate, the5-year event-free survival and overall survival rates are around50-60%, and patients treated with amputation alone often died of pulmonary metastasis within one year. Molecular pathways contributing to osteosarcoma development and progression have recently been discovered, and various studies have been carried out to investigate the genes that are involved in metastasis of osteosarcoma. However, the highly complex molecular mechanism of metastasis is still poorly understood. Recently, microRNAs have become a new research "hot topic" for molecular pathways. MicroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs of19-25nucleotides in length, which can regulate gene expression at the post-transcriptional level by inhibiting the translation of a protein at the mRNA level or by promoting mRNA degradation. However, their biological function remains largely unknown and only a few mRNAs that are directly regulated by miRNAs in animals have been verified empirically. Comparison between human cancer and their normal tissue counterparts have revealed distinct miRNAs expression profiles. In addition, miRNAs may function as either oncogenes or tumor suppressors by specifically regulating the expression of their target genes. miR-27a, a member of an evolutionarily conserved miRNA family, is abnormally increased in several types of cancers and has been identified as being increased in osteosarcoma and has a pro-metastatic role in osteosarcoma cell lines. However, the effects of miR-27a on osteosarcoma have not been totally elucidated. Therefore, it is of great significance to further study the mechanism of miR-27a in osteosarcoma.Methods:In this study, Three Biological softwares were conducted to predict miR-27a target in MAP2K4. a specific miR-27a inhibitor was used to inhibit endogenous miR-27a activity in human osteosarcoma cell line MG63. Cell proliferation assay, colony formation assay, migration and invasion assay were performed to assess the effect of miR-27a on proliferation, metastasis and invasion of MG63. The expression levels of several proteins evolved in the JNK/p38signaling passage was detected by Western blotting.Results:The luciferase activity of the wild-type pGL3-MAP2K43’UTR vector was significantly inhibited after the transfection of miR-27a precursor or the control precursor into MG63cells, but not by the mutant-type pGL3-MAP2K43’UTR vector. Meanwhile, inhibition of miR-27a increased the luciferase activity after the transfection of miR-27a inhibitor or the control inhibitor into MG63cells with the wild-type pGL3-MAP2K43’UTR vector. These results demonstrated that MAP2K4is a potential target of miR-27a and can be directly regulated by miR-27a. Inhibition of miR-27a significantly suppressed cell proliferation after72hours compared with the negative control group, and statistical analysis indicated that inhibition of miR-27a suppressed the colony formation of MG63cells by39.6%. Transwell migration and invasion assays demonstrated that the number of migratory and invasive cells transfected with the miR-27a inhibitor was reduced by63.5%and69.1%, respectively. After transfection with miR-27a inhibitor in MG63cells, the level of phosphor-JNK1and phosphor-p38raised (by25%and29%, respectively) along with the up-regulation of MAP2K4protein.In summary, this is the first study to propose that miR-27a functions as an oncogene by targeting MAP2K4in osteosarcoma MG63cell lines. Inhibition of miR-27a increases MAP2K4, which in turn inhibits cell proliferation and migration through the JNK/p38signaling pathway in MG63cells. These findings may help us to understand the molecular mechanism of miR-27a in tumorigenesis of osteosarcoma and provide new diagnostic and therapeutic options for treatment of this neoplasia.