LncRNA FTX介导OCT4调控损伤微环境下牙髓干细胞多能性的机制研究

Regulating the differentiation fate of dental pulp stem cells (DPSCs) under the injury microenvironment is crucial for the dental pulp self-repair. Long non-coding RNAs (LncRNAs), possessing important genetic effects, play essential roles in regulating the proliferation and differentiation of stem cells at the epigenetic level. Our previous study revealed that OCT4 could regulate the injury response of DPSCs and promote their proliferation and differentiation, but the specific mechanism remains unknown. Our preliminary experiments confirmed that OCT4 negatively regulated the expression of LncRNA FTX in hDPSCs, while overexpression of FTX inhibited OCT4 expression. Accordingly, we hypothesize that, FTX and OCT4 may form a feedback loop to regulate the pluripotency of DPSCs and dental pulp repair. This project will firstly construct FTX-overexpressed and interferred dental pulp stem cells to detect the effects of FTX on the proliferation and differentiation of hDPSCs under the injury microenvironment. The interaction between FTX and OCT4 will be revealed by dual-luciferase reporter assays, RNA binding protein immunoprecipitation and RNA pull-down experiments. The downstream regulatory networks of FTX and OCT4 will be uncovered by constructing the gene co-expression spectrum basing on the bioinformatics analysis. Finally, combined with in vivo dentinogenesis experiments, it will demonstrate the capacity of FTX to mediate the ability of OCT4 of inducing injured dental pulp repair and regeneration. This project will reveal the injury response mechanism of dental pulp stem cells from epigenetic and transcriptional regulation perspectives and provide a reference for clinical practice of dental pulp repair and regeneration.

调控损伤微环境中牙髓干细胞的分化命运是牙髓自我修复的关键。LncRNA具有重要遗传学效应，在表观遗传层面精细调控干细胞的增殖分化。课题组前期证实OCT4可调节牙髓干细胞的损伤应答，促进其增殖分化，但机制不明。进一步实验发现OCT4负向调控牙髓干细胞LncRNA FTX的表达，过表达FTX抑制OCT4表达,推测FTX与OCT4可能形成反馈环路调控牙髓干细胞的多能性，介导牙髓损伤修复。本课题拟构建FTX过表达和干扰的牙髓干细胞，检测损伤微环境下FTX对细胞增殖分化的影响；采用双荧光素酶、RIP及RNA pull-down实验明确FTX与OCT4的相互作用关系；利用生物信息学构建基因共表达谱，探索FTX与OCT4下游的分子调节网络；结合体内成牙本质实验验证FTX介导OCT4诱导受损牙髓修复再生的能力，从表观遗传和转录调控角度揭示牙髓干细胞的损伤应答机制，为促进牙髓损伤修复的临床实践提供科学依据

调控牙髓干细胞（Dental Pulp Stem Cells，DPSCs）的增殖分化命运是实现牙髓自我修复与再生的关键。LncRNA在表观遗传、转录调控及转录后调控等多种层面上精细调节多种干细胞的自我更新与分化。课题前期发现多能转录因子OCT4下调DPSCs中LncRNA FTX 表达水平，而FTX则可抑制DPSCs的增殖分化与OCT4表达，但机制不明。MiRNAs可作为LncRNA下游效应因子，在转录后水平调控胚胎干细胞多能性。本课题以LncRNA FTX介导其相关miRNAs调控hDPSCs的多能性为切入点，通过构建LncRNA FTX稳定过表达的hDPSCs，利用小RNA测序筛选调控hDPSCs增殖分化的LncRNA FTX相关miRNA；继而构建miR-122-5p mimic和mimic NC转染hDPSCs模型，明确miR-122-5p可促进hDPSCs增殖与分化；利用双荧光素酶报告基因实验、染色质免疫共沉淀等探究LncRNA FTX下调miR-122-5p具体机制，发现LncRNA FTX下调OCT4进而在转录水平减少miR-122-5p基因转录。并利用生物信息学预测和双荧光素酶报告基因实验筛选LncRNA FTX/miR-122-5p下游靶基因；进而构建FOXO3 siRNA和NC转染hDPSCs模型，明确了FOXO3 siRNA增强hDPSCs增殖与分化能力；最后利用裸鼠皮下植入模型评估LncRNA FTX/miR-122-5p诱导体内异位成牙本质形成效果。研究成果揭示了LncRNA FTX/OCT4/miR-122-5p/FOXO3轴调控hDPSCs增殖和分化的具体分子机制，为寻找调控hDPSCs增殖分化命运的关键基因靶点、促进牙髓损伤修复再生的临床实践提供科学依据。