LncRNA NEAT1靶向miR-495-3p/DKK1轴对LPS诱导的牙周膜干细胞成骨分化的影响OA
Effect of LncRNA NEAT1 on LPS induced osteogenic differentiation of periodontal ligament stem cells by tar-geting miR-495-3p/DKK1 axis
目的:探讨长链非编码RNA核富集转录本 1(long non-coding RNA nuclear-enriched abundant transcript 1,LncRNA NEAT1)靶向微小 RNA-495-3p(microRNA-495-3p,miR-495-3p)/Dick-kopf 相关蛋白 1(Dickkopf-related protein 1,DKK1)对脂多糖(lipopolysaccharide,LPS)诱导的牙周膜干细胞(periodontal ligament stem cells,PDLSCs)成骨分化的影响.方法:将PDLSCs分为对照组(Control组)、LPS组、短发夹阴性对照组(sh-NC组)、短发夹LncRNA NEAT1 慢病毒载体组(sh-NEAT1 组)、sh-NEAT1+拮抗剂阴性对照组(sh-NEAT1+anti-miR-NC组)、sh-NEAT1+miR-495-3p拮抗剂组(sh-NEAT1+anti-miR-495-3p组);除Control组外,其余均建立LPS诱导的细胞模型,qRT-PCR检测各组细胞LncRNA NEAT1、miR-495-3p、DKK1 mRNA表达,ELISA法检测TNF-α、IL-1β、IL-6水平,MTT法与流式细胞仪检测细胞增殖与凋亡,Transwell小室检测细胞迁移,茜素红 S(ARS)与碱性磷酸酶(ALP)染色检测成骨分化,Western blot检测增殖细胞核抗原(PCNA)、Bcl-2 相关 X 蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、DKK1、骨钙素(OCN)、Runt相关转录因子 2(RUNX2)蛋白表达;双荧光素酶报告基因实验验证 LncRNA NEAT1、DKK1 与miR-495-3p之间的靶向关系.结果:LncRNA NEAT1 在LPS诱导的PDLSCs细胞中表达上调,沉默LncRNA NEAT1 表达可显著促进LPS诱导的PDLSCs细胞增殖、迁移与成骨分化,抑制炎症因子水平及细胞凋亡(P<0.05);抑制miR-495-3p表达可逆转沉默LncRNA NEAT1 表达对LPS诱导的PDLSCs细胞增殖、迁移、成骨分化及细胞凋亡的影响(P<0.05);进一步分析验证,LncRNA NEAT1 海绵化miR-495-3p靶向调控DKK1 表达(P<0.05).结论:沉默LncRNA NEAT1 表达可通过调节miR-495-3p/DKK1 轴促进LPS诱导的PDLSCs成骨分化.
Objective:To discuss the effect of long non-coding RNA nuclear-enriched abundant transcript 1(Ln-cRNA NEAT1)on lipopolysaccharide(LPS)-induced osteogenic differentiation of periodontal ligament stem cells(PDLSCs)by targeting microRNA-495-3p(miR-495-3p)/Dickkopf-related protein 1(DKK1).Methods:PDLSCs were separated into Control group,LPS group,the short hairpin negative control group(sh-NC group),the short hairpin LncRNA NEAT1 lentiviral vector group(sh-NEAT1 group),sh-NEAT1+antagonist negative control group(sh-NEAT1+anti-miR-NC group),sh-NEAT1+miR-495-3p antagonist group(sh-NEAT1+anti-miR-495-3p group).Except for the Control group,all others established LPS induced cell models.qRT-PCR was performed to detect the mRNA expression of LncRNA NEAT1,miR-495-3p,and DKK1.ELISA method was performed to measure TNF-α,IL-1β,and IL-6.MTT assay and flow cytometry were performed to measure cell proliferation and apoptosis.Transwell chamber was used to detect cell migration.Alizarin red S(ARS)and alkaline phos-phatase(ALP)staining were used to detect osteogenic differentiation.Western blot was performed to detect proliferating cell nuclear antigen(PCNA),Bcl-2-associated X protein(Bax),Caspase-1,DKK1,osteocalcin(OCN),and Runt-related tran-scription factor 2(RUNX2)proteins.Dual luciferase reporter gene experiment was used to verify the targeting relationship be-tween LncRNA NEAT1,DKK1 with miR-495-3p.Results:The LncRNA NEAT1 expression was upregulated in LPS-induced PDLSCs.Silencing LncRNA NEAT1 clearly promoted LPS-induced proliferation,migration,osteogenic differentiation of PDLSCs,and inhibited inflammatory factors and cell apoptosis(P<0.05).Inhibition of miR-495-3p could reverse the effects of silencing LncRNA NEAT1 on LPS-induced proliferation,migration,osteogenic differentiation,and apoptosis of PDLSCs(P<0.05).Further analysis confirmed that LncRNA NEAT1 targeted the regulation of DKK1 through sponge like miR-495-3p(P<0.05).Conclusion:Silencing LncRNA NEAT1 can promote LPS-induced osteogenic differentiation of PDLSCs by regu-lating miR-495-3p/DKK1 axis.
段颖莹;王景坤;徐强;方圆;李晨曦
唐山市妇幼保健院口腔科 河北 唐山 063000唐山市妇幼保健院口腔科 河北 唐山 063000唐山市妇幼保健院口腔科 河北 唐山 063000唐山市妇幼保健院口腔科 河北 唐山 063000唐山市妇幼保健院口腔科 河北 唐山 063000
生物科学
牙周膜干细胞长链非编码RNA核富集转录本1微小RNA-495-3pDick-kopf相关蛋白1成骨分化
Periodontal ligament stem cellsLong non-coding RNA nuclear-enriched abundant transcript 1MicroRNA-495-3pDickkopf-related protein 1Osteogenic differentiation
《临床口腔医学杂志》 2026 (1)
17-23,7
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