miR-495-3p对LPS刺激人牙周膜细胞增殖及炎性因子表达的影响

摘要

目的:研究miR-495-3p对脂多糖(LPS)刺激人牙周膜细胞(hPDLC)增殖及炎性因子表达的影响.方法:分离培养原代hPDLC,然后给予10 μg/mL的LPS处理及miR-495-3p模拟物(mimic)转染.实时定量RT-PCR检测LPS处理后miR-495-3p及Toll样受体4(TLR4)的表达;MTT法检测hPDLC细胞增殖;ELISA法检测白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)的含量;双荧光素酶报告基因检测miR-495-3p与TLR4的靶向关系;Western blot法检测miR-495-3p mimic转染后TLR4及磷酸化核因子κB抑制蛋白α(p-IκBα)的表达.结果:LPS处理后,miR-495-3p的表达显著下降,TLR4的表达显著上升.过表达miR-495-3p可显著促进hPDLC细胞的增殖,抑制IL-6及TNF-α的产生;miR-495-3p可靶向结合到TLR4的3'UTR区,并下调TLR4的表达;过表达miR-495-3p可显著抑制p-IκBα的表达.结论:miR-495-3p可通过靶向下调TLR4的表达,抑制核转录因子-κB(NF-κB)通路,进而抑制炎性因子的产生,促进hPDLC的增殖.%Objective: To analyze the effect of miR-495-3p on the proliferation of hPDLC and the production of inflammatory cytokines that induced by LPS.Methods: The primary hPDLC was isolated and was treated with LPS or LPS+miR-495-3p mimic transfection.The expression of miR-495-3p and TLR4 was measured by Real-time PCR.The proliferation of hPDLC was measured by MTT method.The production of IL-6 and TNF-α was measured by ELISA.The target relationship between miR-495-3p and TLR4 was measured by dual luciferase assay.The expression of TLR4 and IκBa was measured by Western blot.Results: LPS significantly down-regulated the expression of miR-495-3p and up-regulated the expression of TLR4.MiR-495-3p mimic dramatically increased the proliferation of hPDLC,decreased the production of IL-6 and TNF-α.MiR-495-3p targeted the 3'UTR region of TLR4.MiR-495-3p mimic significantly decreased the expression of TLR4.Furthermore,miR-495-3p mimic noticeably suppressed the expression of p-IκBα.Conclusions: miR-495-3p increased the proliferation of hPDLC and suppressed the production of inflammatory cytokines through down regulated the expression of TLR4 and inhibited NF-κB pathway.