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目的探讨miR-519家族对人牙周膜细胞内MMP-2表达的影响。方法构建MMP-2双荧光素酶野生型和突变型表达载体,应用TargetScan和miRanda等软件预测作用于MMP-2 3’-UTR靶向miRNA,利用双荧光素酶报告基因检测系统验证靶向MMP-2 3’-非翻译区(3’-UTR)的miRNA。用qRT-PCR和Western blot技术检测miR-519家族对MMP-2表达的调控。结果软件预测在MMP-2的3’-UTR位置有一个与miR-519家族的结合位点;mimics-miR-519实验组与转染control miR组和突变型载体组相比,能明显降低MMP-2载体的荧光素酶活性(P<0.05)。qRT-PCR结果显示实验组的miR-519较对照组表达显著增高(P<0.01)。qRT-PCR、Western blot结果表明miR-519抑制MMP-2mRNA和蛋白水平表达(P<0.05)。结论 miR-519家族可负性调控人牙周膜细胞内MMP-2基因的表达。
Objective To investigate the effect of miR-519 family on the expression of MMP-2 in human periodontal ligament cells. Methods The wild-type and mutant expression vectors of MMP-2 dual luciferase were constructed. The target miRNAs were predicted by TargetScan and miRanda software to predict the target miRNAs in the 3’-UTR of MMP-2. The dual-luciferase reporter assay -2 3’-untranslated region (3’-UTR). The regulation of MMP-2 expression by miR-519 family was detected by qRT-PCR and Western blot. Results The software predicted that there was a binding site to the miR-519 family in the 3’-UTR of MMP-2. Compared with the control miR group and the control group, the mimics-miR-519 experimental group could significantly reduce the MMP -2 vector luciferase activity (P <0.05). qRT-PCR results showed that the expression of miR-519 in experimental group was significantly higher than that in control group (P <0.01). qRT-PCR and Western blot showed that miR-519 inhibited the expression of MMP-2 mRNA and protein (P <0.05). Conclusion The miR-519 family negatively regulates the expression of MMP-2 gene in human periodontal ligament cells.