摘要
目的:探讨miR-520h对缺氧状态下人牙周膜细胞(HPDLCs)MMP-2表达的影响。方法:将HPDLCs置于1%氧浓度的孵箱中培养0、12、24、48和72 h。用实时荧光定量RT-PCR(qRT-PCR)和Western blot检测细胞中MMP-2 mRNA和蛋白的表达。构建野生型和突变型MMP-2表达载体,用TargetScan和miRanda等软件预测作用于MMP-2基因3'-UTR靶向miRNA,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-520h对MMP-2表达的调控。结果:HPDLCs在缺氧培养中MMP-2 mRNA和蛋白的表达量明显下调(P<0.01)。软件预测在MMP-2的3'-UTR 516bp位置有一个与miR-520h的结合位点,HPDLCs被转染pre-miR-520后,其表达量增高(P<0.01),MMP-2载体的荧光素酶活性降低(P<0.05)。miR-520h都抑制了HPDLCs中MMP-2 mRNA和蛋白表达(P<0.05)。结论:miR-520h可抑制缺氧状态下牙周膜细胞MMP-2基因表达。
Objective： To investigate the effect of miR-520h on MMP-2 expression in human periodontal ligament cells （HPDLCs） under hypoxia. Methods： HPDLCs were exposed to 1% oxygen for 0, 12, 24, 48 and 72 h respectively. Real-time fluorescence quantitative PCR（qRT-PCR） and Western blotting were used to detect MMP-2 mRNA and protein expression in the cells. The wild type and mutation type of MMP-2 gene 3＇-UTR luciferase vectors were constructed. TargetScan and miranda software were applied to predict the MMP-2 3＇UTR targeting miRNA. Dual-luciferase reporter gene system was used to screen the predicted miRNA targeting 3＇-untranslat-ed region（3＇-UTR）of MMP-2 gene. The effects of miR-520h regulating MMP-2 mRNA and protein expressions were detected by qRT-PCR and Westem blot. Results： In HPDLCs MMP-2 mRNA and protein were decreased under hypoxia（P 〈0.01 ）. MMP-2 gene con- tained a miR-520h binding site at the 516 bp in 3＇- UTR. Trasfection of pre-miR-520h increased pre-miR-520h expression （ P 〈 0.01 ）, decreased the luciferase activity of wild type MMP-2 3＇-UTR vector in HPDLCs（P 〈0.05）, inhibited MMP-2 mRNA and protein expres-sions（P 〈0.05）. Conclusion： miR-520h can inhibit the expression of MMP-2 in human periodontal ligament cells under hypoxia.
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2014年第3期357-361,共5页
Journal of Practical Stomatology
基金
国家自然科学基金(编号:81170934)
第三军医大学青年创新基金(编号:2011D274)
