The Research On The Mechanism Of MicroRNA-27a Regulating The Characteristics Of Osteosarcoma Biological Behaviors

Objectives:The present study aimed to elucidate miRNA expression profiles and expression differences in OS cell lines. Verification of miRNA expression provided a basic understanding of research and direction for ongoing work of underlying mechanism in OS.Methods:To characterize and compare the different expression of miRNA in four OS cell lines, including U-2OS, Saos-2, MNNG/HOS, MG63with human osteoblast cell line hFOB, total RNA was extracted from cells. For establishing a novel microRNA expression profile of OS cell lines, microRNA microarray chips were performed to detect the expression of global microRNA profiling.Results:For screening of differential expression miRNAs, the microRNA microarray chips was applied to compare the miRNAs profiles in four OS cell lines, including U-2OS, Saos-2, MNNG/HOS, MG63with human osteoblast cell line hFOB. The miRNA-profiling based screening assay for the investigation indicated that23miRNAs were significantly up-regulated while4miRNAs were down-regulated more than2.0-fold in four OS cell lines, including U-2OS, Saos-2, MNNG/HOS, MG63compared with human osteoblast cell line hFOB. The list of miRNAs which were up-regulated more than2.0-fold:let-7e-5p, miR-20b-5p, miR-92a-3p, miR-125a-5p, miR-196a-5p, miR-214-3p, miR-20a-5p, miR-181a-5p, miR-29b-3p, miR-10b-5p, miR-301a-3p, miR-181d, miR-21-5p, miR-206, miR-7-5p, miR-29a-3p, miR-17-5p, miR-210, miR-25-3p, miR-18a-5p, miR-181c-5p, miR-32-5p, miR-27a-3p, while the down-regulated miRNA were miR-142-5p, miR-133b, miR-16, miR-144-3p. Conclusion:The results of miRNA microarray chips study of four OS cell lines (U-2OS, Saos-2, MNNG/HOS, MG63) in comparison with human osteoblast cell line hFOB revealed27differentially expressed miRNAs. These differential expression results of miRNA provided novel insights and research qualifications for further miRNA functional study in OS. Objectives:The aim of the present study was to determine the changes in miR-27a expression levels affect the proliferation、apoptosis and invasion of OS and investigate the underlying mechanism.Methods:MiR-27a inhibitor、miR-27a negative control、miR-27a mimics and miR-27a mimics control was transfected into OS cell lines using Lipofectamine2000transfection reagent respectively. Caspase-3/8/9activities were assayed by Caspase-3/8/9Activity Assay Kit. The flow cytometry was used for investigation of apoptotic cells. The cell viability was measured by MTT assay. Migration and invasion assays were performed using transwell chambers. Colony formation assay were performed to evaluate cells colony formation. The expression of PUMA、BAK and BAX was determined by quantitative real-time PCR (qRT-PCR) and Western blot. The mice were observed for tumor formation, tumor volumes and tumor weights。Tumor sizes were measured to draw a tumor growth curve.Results:There was an increase activity of caspase-3/9/8and apoptosis in miR-27a inhibitor group compared with other groups Transfection of miR-27a remarkably suppressed proliferation, motility and colony formation of OS cells (P<0.01) Expression of PUMA was negatively correlated with miR-27a levels in OS cells, and PUMA was confirmed to be potential targets of miR-27a using luciferase assays. These results indicated that miR-27a regulated PUMA expression by directly binding to its3’-UTR target site. The tumor size, volume and tumor growth speed were significantly reduced in miR-27a-inhibitor group compared to mice in miR-27a inhibitor negative control group Conclusion:Activation of caspase-3、8and9activity and apoptosis in OS cells by miR-27a inhibitor, an antagomir of miR-27a, is resulted from overexpression of PUMA. Furthermore, inhibition of miR-27a expression suppresses OS cell proliferation, migration, invasion and tumor growth in nude mice...