微小RNA-218-5p靶向TLR2基因对LPS所致人牙周膜干细胞炎症反应的影响

Effect of MicroRNA-218-5p on Inflammation of Human Periodontal Ligament Stem Cells Induced by Lipopolysaccharide through Targeting TLR2 Gene

目的:研究微小RNA(microRNA,miR)-218-5p对脂多糖(LPS)诱导的牙周膜干细胞(PDLSCs)增殖、凋亡和炎症的影响及作用机制。方法:分离培养PDLSCs,用LPS处理PDLSCs细胞建立炎症模型,MTT法和流式细胞术分别检测细胞存活率和凋亡率,qRT-PCR和Western blot分别检测miR-218-5p和Toll样受体2(TLR2)蛋白的表达,检测细胞中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-ɑ(TNF-ɑ)的含量,双荧光素酶报告系统检测miR-218-5p和TLR2的靶向关系。结果:LPS处理人PDLSCs 0、12、24和48 h,PDLSCs细胞中miR-218-5p表达下调,TLR2表达上调,细胞存活率下降,凋亡率升高,炎症因子IL-6、IL-1β和TNF-α含量升高,呈时间依赖趋势,差异均有统计学意义(P<0.05);miR-218-5p靶向调控TLR2表达;过表达miR-218-5p或沉默TLR2均可提高细胞存活率,降低细胞凋亡率和炎症因子IL-6、IL-1β、TNF-α的含量;过表达TLR2可逆转miR-218-5p过表达对LPS诱导的PDLSCs细胞炎症和细胞增殖的作用。结论:miR-218-5p通过靶向TLR2调控LPS诱导的PDLSCs细胞增殖、凋亡和炎症反应。

Objective:To investigate the effects and mechanism of miR-218-5p on proliferation,apoptosis,and inflammation of periodontal ligament stem cells(PDLSCs)induced by lipopolysaccharide(LPS).Methods:PDLSCs were isolated and cultured.PDLSCs cells were treated with LPS to establish an inflammatory model.MTT assay and flow cytometry were applied to detect the cell survival rate and apoptosis rate,respectively.qRT-PCR and Western blot were performed to detect the expression levels of miR-218-5p and TLR2 protein,respectively.The kits was used to detect the contents of interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)in PDLSCs.The dual luciferase reporting system was used to detect the targeting relationship between miR-218-5p and TLR2.Results:Human PDLSs were treated by LPS for 0h,12h,24h,and 48h.miR-218-5p in PDLSs cells was down-regulated,TLR2 was up-regulated,cell survival rate was down-regulated,apoptosis rate was up-regulated,and the contents of inflammatory factors IL-6,IL-1β,and TNF-αwere up-regulated,showing a time-dependent trend with statistical significance(P<0.05).MiR-218-5p targeted and regulated the expression of TLR2.Over-expression of miR-218-5p or silencing of TLR2 both improved cell survival rate and reduced cell apoptosis rate and levels of inflammatory factors IL-6,IL-1β,and TNF-α.Over-expression of TLR2 reversed the effect of miR-218-5p over-expression on inflammation and proliferation of PDLSCs induced by LPS.Conclusion:MiR-218-5p regulates the proliferation,apoptosis,and inflammatory response of PDLSCs cells induced by LPS through targeting TLR2.