MiR-302b-mediated Anti-Osteosarcoma Effect Of Epirubicin And Its Machanisms

Osteosarcoma (OS) is the most common aggressive sarcoma of the bone characterized by producing immature bone or osteoid tissue. Seventy-five percent of patients are diagnosized at15-30years old. Osteosarcoma is highly aggressive and at risk of local relapse or distant metastasis. The prognosis for the OS patients is not good so it is a crippling disease for adolescents and young adults. Thepathogenic genes and pathogenesis of OS are important and key part of the basic research, which could be an important bio-marker for the OS early diagnosis and treatment to improve OS survival.Recently, the investigators find that microRNA could play important role in the target gene regulation. It’s also known that miRNAs and their target genes have the potential to regulate various critical biological processes, including the gene expression, cell signaling, which is highly associated with the inflammation, tumor and endocrine disease. The effects of miRNAs on the gene transcription and protein expression could result in the tumorigenesis.We select the anti-OS effect of epirubicin (EPI) as the research model in the miRNA functions. First, we investigate the inhibitory effect of epirubicin on the OS proliferation and find the differential miRNA expression after the epirubicin exposure in OS cell lines. Based on the bioinfomatic research and the previous literature, we select several interesting miRNAs and validated its differential expression in OS after the epirubicin treatment by Real-time PCR. Those results imply that the differential could play important roles in the inhibitory effect of epirubicin against OS cells. Then we alter the level of the interesting miRNA in OS by the miRNA mimics transfection, so we could detect its functions and target genes in OS. We detect the OS cell proliferation, apoptosis and cell cycle distribution after the miRNA transfection alone or combined with epirubicin treatment. Moreover, we also determine the expression of its target genes which are related with the cell apoptosis and cell cycle arrest. So we could confirm the effective target genes of interesting miRNA in the anti-OS effect of epirubicin.Part I. The differential expression of miRNAs in the anti-OS progress of epirubicinObjective:To study the differential expression of miRNAs after the epirubicin exposure and validate its expression before its function investigation.Methods:We detect the inhibiroty effect of epirubicin on the OS MG-63and SAOS-2c cells by the CCK-8assay and obseve the morphologic features after the epirubicin treatment. Then we determine the miRNA expression in OS by micro-arrays and find out the differential miRNAs after epirubicin treatment. Based on the bio-inofmatics research and previous literature, we select several differential miRNAs as the interesting ones for the future function research. Finally we validate its differential expression in OS cells after the epirubicin exposure by Real-time PCR.Results:We find that1μg/ml epirubicin or more could significantly inhibit the OS cell proliferation in a dose-dependent manner. The cell morphologic features change obviously; the miRNA expressions are also dysregulatedafter1μg/ml epirubicin treatment against OS MG-63and SAOS-2cells. After the comparisons, we find out40differential miRNAs, including16over-expression ones and24down-expression. Of these, miR-302b is significantly up-regulated by the epirubicin, which might play important roles in OS cell apoptosis and cell cycle distribution.Conclusions: Epirubicin could inhibit the OS cell proliferation by the miRNA expression regulation. Forty differential miRNA might contribute to this effect and miR-302b might be one the important ones among those miRNAs.Part II. The miR-302b-mediated effect of epirubicin against osteosarcoma cells and its mechanismsObjective:To investigate the effect of miR-302b and its synergistic role with epirubicin in the anti-OS process by the overexpression of miR-302b.Methods:We validate the relative miR-302b expression in OS MG-63and SAOS-2cells after the miR-302b mimics tranfection with Lipo-2000to confirm the miR-302b restoration. After the exposure in different factors, we detect the OS cell proliferation by CCK-8; investigate the OS cell apoptosis by the Annexin V-PE/7-AAD assays and the cell cycle distribution by the PI staing. To further investigate the machanisms, we detect the Caspase-3activity and the expression levels of several apoptosis and/or cell cycle related proteins.Results:We find that miR-302b mimics transfection could increase the miR-302b expression to attenuate the OS cell proliferation and its effect could be combined with epirubicin. Over-expression of miR-302b could significantly induce OS cell apoptosis and Gl arrest to inhibit OS cell proliferation. In this process, miR-302b could activate Caspase-3, increase the Bim expression, attenuate the expression of AKT、pAKT、Bcl-2、 Cyclin D1、CDK2/4as well to induce OS cell apoptosis and G1arrest.Conclusion:Over-expression of miR-302b could regulate several key apoptosis and cell cycle related protein expression to induce OS cell apoptosis and G1arrest, inhibit cell proliferation. These mechanisms are important to mediate the anti-OS effect of epirubicin.