Preliminary Study Of Significance And Function Of MiR-17-92Cluster Expression In Osteosarcoma

Background and purposeMicroRNAs (miR) are a new class of multifunctional small non-codingendogenous RNA molecules, which repress translation and cleave mRNA bybase-pairing to the3′untranslated region of the target genes. In our study, ourobjective is to explore the expression of miR-17-92cluster and its six miRNAs inosteosarcoma tissue and osteosarcoma cell lines, and investigate the function ofmiR-19b in osteosarcoma cell lines and the biological behaviors and mechanism ofmiR-19b in osteosarcoma.MethodsThe level of miR-17-92cluster expression in osteosarcoma tissues and matchednormal bone tissues from21patients were detected by RT-PCR and real-time PCR,and evaluated the clinical relevance of miR-17-92cluster. Expression levels of sixmembers of the miR-17-92cluster in MG-63, Saos-2, U2-OS and hFOB1.19cellswere measured by real-time PCR; Select the single miRNA and appropriate cells forfurther experiments; After constructing plasmids (miR-19b Inhibitor), we tranfectedMG-63cells with miR-19b Inhibitor or the Negative Control (NC). The miR-19bexpression in MG-63cells was measured using real-time PCR after transfected. Cellproliferation after transfected was analyzed using the MTS assay in vitro. Cellapoptosis after transfected was analyzed by Tunel assay and Annexin V-FITCanalysis respectively. Cell cycle after transfected was analyzed by Annexin V-FITCanalysis, and invasion assays were performed using Transwell invasion chambers.Using bioinformatics analysis, we predicted Mitofusin1(MFN1) gene as putativetargets of miR-19b and veritfied by Lucifersae reporter assay. Also, the expression ofMFN1in MG-63cells was measured using real-time PCR and Western blot assaysafter transfected to detect the their correlation.Results 1. The relative expression of miR-17-92cluster in osteosarcoma tissues wassignificantly higher than those in adjacent normal tissues. And there was arelationship between miR-17-92cluster upregulation and metastasis of osteosarcomapatients. And there was no significant difference in gender, age, tumor locations andtumor differentiation between two groups;2. Among the six miRNAs, we found that miR-19b was significantly up-regulated inosteosarcoma cells, especially in MG-63. Therefore, we selected the miR-19b andMG-63cells for further experiments;3. Inhibiting miR-19b resulted in promotion of MG-63cell apoptosis, inhibition ofcell proliferation and invasion, and G1phase arrest;4. miR-19b directly and negatively regulate the expression of tumor suppressor geneMFN1. Luciferase reporter assays demonstrates that MFN1is direct targets ofmiR-19b. Western Blots and real-time PCR further confirmed there are significantdifferences in target protein and mRNA expression after transfecting with miR-19bInhibitor. miR-19b represses MFN1protein and mRNA expression though binding to3′-UTR of MFN1.Conclusion1. Upregulated of miR-17-92cluster in osteosarcoma tissues and osteosarcomacancer cells.There exists a positive correlation between the expression of miR-17-92cluster and metastasis in osteosarcoma tissues. However, there was no Significantcorrelation between the expression of miR-17-92cluster and gender, age, tumorlocation and tumor differentiation degree. This indicate that high expression ofmiR-17-92cluster potentially contributes to human osteosarcoma metastasis and actsas one of prognostic factors of osteosarcoma patients;2. miR-19b is a oncomir in Osteosarcoma. Inhibiting expression of miR-19bsignificantly inhibited the function of MG-63cells, such as cell proliferation, invasion,migration and induce cell apoptosis. It suggests that the up-regulation of miR-19bmay play a role in Osteosarcoma cell function.3. miR-19b expression was inversely correlated with tumor suppressor gene MFN1protein and mRNA level in MG-63. Upregulation of miR-19b in osteosarcoma participate in the development process of osteosarcoma through the negatively controldownstream expression of target genes MFN1.Thus miR-19b might become candidatebiomarkers of osteosarcoma diagnosis or treatment in future.