MicroRNA-145 inhibits tumour growth and metastasis in OS by targeting cyclin-dependent kinase, CDK6

Osteosarcoma (OS) is reported to be one of the most familiar forms of the primary malignant tumor to adolescents and adults, which is usually found in their long bone1,2. OS is closely related to the abnormality in differentiation induced by genetic and epigenetic changes. Current treatments include chemotherapy, surgery, and sometimes radiotherapy all together, but the prognosis remains very poor, the 5-year survival rate of patients with recurrent or metastatic osteosarcomas is only 50%-60%3. Despite researchers having identified many carcinogens and tumor suppressor genes, which are believed to account for the growth of OS, the possible mechanism of migration and invasion of OS remains mysterious in many aspects. MicroRNAs (miRNAs) are a kind of RNA with the length of 22-25 nucleotide that down-regulate the expression of the gene in plants and animals4,5. MicroRNAs are believed to play vital roles in oncogenesis, cellular differentiation programs and developmental biology6. Decades of studies have found that the proliferation, apoptosis, metastasis, and invasion are tightly associated with abnormal expression of miRNAs in human cancer cells, including osteosarcoma. Several miRNAs were reported to be associated with OS growth and metastasis, which includes miR-327, miR-142-3p8, miR-1949, miR20210, miR-21711, and let-7a12. A decrease in the level of miR-145 has been detected in OS. Decreased leAbstract. – OBJECTIVE: Osteosarcoma (OS) is reported to be one of the most familiar forms of the primary malignant tumor to adolescents and adults, which is usually found in their long bones. Evidence and data have shown that abnormality in micro-RNA expression is closely related to tumorigenesis. The aim of this study is to investigate the expression, function and underlying mechanism of miR-145 in OS. MATERIALS AND METHODS: MiR-145 expression in tumor tissues of patients and cell lines were detected by SYBR green qPCR. Kaplan-Meier method and log-rank test were applied to analyze the overall survival of patients with different miR-145 expression. MG-63 and U2OS cells were transfected with miR-145 or miR-NC, after which we recorded and analyzed the proliferation, migration and invasion respectively. Potential binding sites of miR-145 in CDK6 was identified using TargetScan 6.2. Meanwhile, OS cells were transfected with miR-NC, miR-145, or transfected with miR-145 and CDK6 vector together to find out whether the effect of miR145 inhibit growth and mobility of OS cells were via targeting CDK6. Moreover, the expression of miR-145 was mediated by a constructed lentivirus vector to inoculate nude mice to observe the inhibiting effect of miR-145 in OS in vivo. RESULTS: MiR-145 downregulation was remarkably related with the occurrence of a phenotype of OS with more aggressiveness. The univariate analysis was applied which showed that with a lower miR-145 level in OS patients led to a more unfavorable prognosis, or, a lower overall survival rate. In OS cells, cell proliferation, motility and invasion were inhibited by ectopic overexpression of miR-145 in vitro. Further studies into the mechanism indicated that its expression probably has a close interrelationship with the 3’untranslated region (3’-UTR) of CDK6 mRNA, whose mRNA and expression levels were inhibited. And it is found that CDK6 expression was also reversely related to miR-145 expression in clinical specimens. In in vivo experiment, the miR-145 inhibits the growth of OS tumor. CONCLUSIONS: miR-145 plays a crucial role in inhibiting invasive and metastatic of OS, European Review for Medical and Pharmacological Sciences 2016; 20: 5117-5125