miRNA-194在骨肉瘤中靶基因的预测和验证

The Prediction and Verification of Target Genes of miRNA-194 in Osteosarcoma

目的:对miRNA-194在骨肉瘤中的作用机制进行探索和研究,为研究miRNA-194在骨肉瘤中的作用及可能的生物治疗提供理论依据和研究基础。方法:使用生物信息学软件对miRNA-194在骨肉瘤中可能的靶基因进行预测,并在基因水平和蛋白水平进行相应的验证。此外,利用荧光素酶基因报告实验对其靶基因(CDH2)进行DNA水平的直接验证。结果:使用生物信息学软件预测出了多个可能的潜在靶基因(367个,6014个和212个);荧光素酶基因报告实验显示野生PTEN-3'-UTR在miRNA-194上调组明显的低表达,同时下调组明显的高表达(P<0.05);同时,western实验显示在蛋白水平miRNA-194直接靶向作用于CDH2(P<0.05),而在m RNA水平则无明显作用(P>0.05)。结论:经预测和验证,CDH2是miRNA-194在骨肉瘤中的直接靶基因,为接下来关于miRNA-194在骨肉瘤中的各项研究奠定了良好的实验基础。

Objective： To forecast and verify the target genes of miRNA-194 in human osteosarcoma and establish some theoretical basis for osteosarcoma treatment and element experience of miRNA-194 for further research. Methods： Potential target gene of miRNA-194 was predicted using bioinformatics and were verified in gene and protein levels. Moreover, luciferase reporter assay was used to confirm whether CDH2 was direct targets of miRNA-194. Results： Potential target genes of miRNA-194 were predicted by using bioinformatics. Moreover, luciferase activity of the wild-type PTEN-3＇-UTR reporter was significantly suppressed in miRNA-194up-regulation group compared with other groups in SOSP-9607 cells（P〈0.01） while down-regulation group could significantly increase it（P〈0.01）. At the same time, western blot analysis revealed that miRNA-194 expression could affect the expression of CDH2 protein while there was no significant difference on m RNA level（P〉0.05）. Conclusion： CDH2, one target gene of miRNA-194, was predicted and verified, which provided the theoretical basis for the next study of miRNA-194 in the osteosarcoma.