摘要
目的 :研究miR-142-3p过表达对脂多糖(LPS)诱导人牙周膜细胞(h PDLCs)分泌炎症因子的影响及其作用机制。方法:组织块法培养原代正常hPDLCs,以不同浓度的LPS处理hPDLCs 6、12、24和48 h;ELISA检测miR-142-3p mimics对2.0μg/mL LPS诱导的hPDLCs作用24 h后炎症因子(TNF-α、IL-6及IL-1β)的表达情况;双荧光素酶报告基因检测miR-142-3p与白介素1受体相关激酶1(interleukin-1receptor-associated-1 kinase1,IRAK1)的靶标关系,qRT-PCR检测miR-142-3p和IRAK1 mRNA的表达水平,Western印迹法检测IRAK1、Toll样受体4(toll like receptor 4,TLR4)及核因子κB(nuclear factor-κB,NF-κB)的蛋白表达水平。采用SPSS 16.0软件包对数据进行统计学分析。结果:2.0μg/mL LPS作用24 h后,hPDLCs表达miR-142-3p最低,存在显著差异(P<0.01);加入miR-142-3p mimics后,miR-142-3p表达量显著升高(P<0.01);miR-142-3p过表达显著降低了炎症因子(TNF-α、IL-6及IL-1β)的表达水平(P<0.05);双荧光素酶报告基因检测结果显示,miR-142-3p与IRAK1存在直接的靶标关系;miR-142-3p过表达可抑制IRAK1蛋白的表达水平(P<0.05),但对IRAK1 mRNA表达水平无显著影响(P>0.05);Western印迹结果显示,miR-142-3p过表达显著下调TLR4蛋白和NF-κB蛋白磷酸化表达水平(P<0.05)。结论:miR-142-3p过表达可减轻LPS诱导的hPDLCs炎症反应,其机制可能通过靶向抑制IRAK1进而抑制TLR4/NF-κB炎症信号通路,从而发挥对牙周组织的保护作用。
PURPOSE： To investigate the effects of miR-142-3p overexpression on secretion of inflammatory factors in human periodontal ligament cells （hPDLCs） induced by lipopolysaecharide （LPS） and its underlying mechanism. METHODS： hPDLCs were cultured by tissue cultivation in vitro and treated with various concentrations of LPS for 6, 12, 24 and 48 h. hPDLCs were treated with miR-142-3p mimics for 24 h in the presence of 2.0 μg/mL LPS and the expression levels of inflammatory factors （TNF-a, IL-6 and IL-1β） were detected by ELISA assay. The target relationship between miR-142-3p and interleukin-1 receptor- associated kinase 1 （IRAK1） was detected by dual-luciferase reporter assay. The miR-142-3p and IRAK1 mRNA expression was measured by qRT-PCR. The protein expression levels of IRAK1, TLR4 and NF-KB were detected by Western blotting. Statistical analysis was performed using SPSS 16.0 software package. RESULTS： The miR-142-3p expression level was decreased significantly at 24 h in 2.0 μg/mL LPS-treated hPDLCs （P〈0.01）. Transfection of miR-142-3p mimics remarkably increased miR-142-3p expression （P〈0.01）. The secretion of inflammatory factors （TNF-a, IL-6 and IL-lβ） in LPS-treated hPDLCs was significantly decreased by miR- 142-3p overexpression （P〈0.05）. Dual-luciferase reporter assay showed a directly targeting relationship between miR- 142-3p and IRAK1. Overexpression of miR-142-3p significantly reduced the IRAK1 protein expression in LPS-treated hPDLCs （P〈0.05）, whereas no obvious effect on IRAK1 mRNA expression was noted （P〉0.05）. Moreover, miR-142-3poverexpression markedly diminished the protein expression of TLR4 and phosphorylated NF-KB in LPS-treated hPDLCs （P〈0.05）. CONCLUSIONS： Overexpression of miR-142-3p can alleviate the inflammatory response induced by LPS in hPDLCs and the underlying mechanism may be associated with targeting and inhibiting IRAK1 expression leading to suppression of TLR4/NF-kB inflaramatory signaling pathway through which miR-142-3p overexpression protects peridentium against inflammation.
出处
《上海口腔医学》
CAS
CSCD
2016年第6期682-687,共6页
Shanghai Journal of Stomatology
