【作者】 王明；

【导师】 吕国华；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2022， 博士

【摘要】 研究背景与目的:骨质疏松症是导致骨质疏松性骨折及相关骨科疾病、甚至死亡的主要病因,影响着全球数亿人的健康和生活质量。脂肪间充质干细胞(Adipose-derived Stem Cells,ADSCs)具有多系分化潜能。相对于其他组织来源的间充质干细胞而言,ADSCs具有诸多优越特性,是再生医学领域理想的种子细胞来源。脂肪间充质干细胞成骨分化能力受到诸多因素调节,其中环状RNA(circular RNA,circRNA)是调节ADSCs成骨分化的重要表观遗传调控因素。但对调控ADSCs成骨分化的circRNAs表达谱尚缺乏深入认识,以及对circRNA调节ADSCs成骨分化的具体机制尚不明确。转录因子可以调控circRNAs的表达。本课题旨在通过阐明特定circRNA介导ADSCs成骨分化的具体分子机制,同时阐明转录因子通过调节circRNA表达进而调节ADSCs成骨分化,为ADSCs应用于骨质疏松性骨折及骨科相关疾病提供新的修饰策略。研究方法:获取在人脂肪间充质干细胞(human Adipose-Derived Stem Cells,hADSCs)进行成骨诱导分化,并检测circRNA的表达,筛选差异表达circRNA,最终获取目的circRNA,系统研究关键circRNA分子调节hADSCs的成骨分化机制,采用生物信息学分析获取circ RNNA靶向调节的miRNAs和以及miRNAs的靶基因,采用荧光素酶实验检测circRNA与miRNA,以及miRNA-靶基因的靶向结合关系,同时检测其对hADSCs成骨分化的影响,揭示其调节机制,进一步检测转录因子RUNX3与circRNA的靶向调节关系。研究结果:hADSCs培养14天后,检测发现多个circRNAs差异表达,其中hsa＿circ＿0005752呈现显著上调表达趋势(P<0.001)。我们预测并构建了hsa＿circ＿0005752/miR-496/MDM2-p53调控网络。进一步实验探索hsa＿circ＿0005752在hADSCs成骨分化过程中的功能作用以及与miR-496和MDM2的相互作用关系。采用荧光素酶实验,我们发现hsa＿circ＿0005752和MDM2与miR-496存在靶向结合位点。同时,我们检测过表达miR-496的hADSCs细胞中的MDM2,发现miR-496显著降低了MDM2并影响p53的表达水平(P<0.05),而这一效果却能被hsa＿circ＿0005752过表达挽救。干细胞成骨分化相关实验检测显示,miR-496的过表达抑制了hADSCs的成骨分化,hsa＿circ＿0005752的过表达可以挽救miR-496对hADSCs成骨分化的抑制作用,hsa＿circ＿0005752/miR-496/MDM2-p53形成了ceRNA分子调节网络并影响hADSCs的成骨分化。hsa＿circ＿0005752由溶血磷脂酸受体1(LPAR1)生成,我们进一步揭示了RUNX3可以与溶血磷脂酸受体1(LPAR1)启动子结合,进而调节hsa＿circ＿0005752的表达,并影响hADSCs成骨分化。染色质免疫沉淀实验(Chromatin immunoprecipitation,Ch IP)和荧光素酶报告实验证实RUNX3能够靶向结合LPAR1的启动子。进一步的q PCR结果表明,过表达RUNX3能上调hsa＿circ＿0005752的表达水平,同时可以观察到MDM2和成骨相关因子(RUNX2、Osx、ALP、OCN)表达水平上调,但p53表达水平下调,LPAR1的表达水平无显著变化。研究结论:综上所述,本研究阐明了hsa＿circ＿0005752调控hADSCs成骨分化的分子网络机制,认为hsa＿circ＿0005752和MDM2m RNA共同靶向结合miR-496。hsa＿circ＿0005752通过海绵吸附miR-496形成ceRNA网络靶向调节MDM2-p53影响hADSCs成骨分化。与此同时,hADSCs的hsa＿circ＿0005752表达水平受RUNX3靶向调节,从而影响hADSCs成骨分化。我们的研究可以提升hADSCs成骨分化能力,有望为骨组织工程的发展提供重要的种子细胞基因修饰策略。更多还原

【Abstract】 Background and purpose:Osteoporosis is the crucial cause of osteoporotic fractures,related orthopedic diseases,and even death,affecting the health and quality of life of hundreds of millions of people worldwide.Adipose-derived mesenchymal stem cells(ADSCs)are multilineage potential.Compared with other tissue-derived mesenchymal stem cells,ADSCs have many superior properties and are an ideal source of seed cells in regenerative medicine.Multiple factors regulate the osteogenic differentiation of ADSCs,osteogenesis-related signaling pathways,and epigenetic modification.Circ RNAs are essential epigenetic regulators for osteogenic differentiation of ADSCs.However,the circRNA expression profile of ADSCs osteogenic differentiation is unexplored.And the specific mechanism of how circRNAs regulate the osteogenic differentiation of ADSCs is still unclear.Transcription factors can regulate the expression of circRNAs.This project aims to clarify the specific molecular mechanism of circr Na-mediated osteogenic differentiation of ADSCs,and to clarify that transcription factors regulate the expression of circRNA and then regulate the osteogenic differentiation of ADSCs,so as to provide a new modification strategy for the application of ADSCs in osteoporotic fractures and orthopedic diseasesMethod: Human adipose mesenchymal stem cells(h ADSCs)were obtained for osteogenic induction differentiation,and the expression of circRNA was detected,the differentially expressed circRNA was screened,and the target circRNA was finally obtained.The mechanism of circRNA regulating h ADSCs osteogenic differentiation was systematically studied,and the circRNA was obtained by bioinformatics analysis.The targeted binding relationship between circRNA and miRNA,and miRNA-target genes was detected by luciferase assay.Meanwhile,its effect on h ADSCs osteogenic differentiation was detected to reveal its regulatory mechanism,and transcription factor RUNX3 and circRNA were further detected.Resutls: To analyze circRNAs that regulate osteogenic differentiation,we used the h ADSCs after osteogenic differentiation for14 days.And then,we found that multiple circRNAs were differentially expressed,among which hsa＿circ＿0005752 showed a significantly up-regulated expression trend.We then explored the regulatory role of hsa＿circ＿0005752 in osteogenic differentiation.According to the ceRNA theory,circRNAs usually share the same miRNA as m RNA.Therefore,we predicted and constructed the hsa＿circ＿0005752/miR-496/MDM2 network.Then experiments were performed to explore the function of hsa＿circ＿0005752 in osteogenic differentiation and its interaction with miR-496 and MDM2.Through multiple experiments,we found that miR-496 could target hsa＿circ＿0005752 and MDM2.Meanwhile,we detected MDM2 in h ADSCs overexpressing miR-496 and found that miR-496 significantly reduced MDM2 expression but could be rescued by hsa＿circ＿0005752 overexpression.We also found that overexpression of miR-496 inhibited the osteogenic differentiation of h ADSCs,and overexpression of hsa＿circ＿0005752 could rescue the miR-496-mediated inhibition of osteogenic differentiation of h ADSCs.In further experiments,we explored the mechanism responsible for the increased expression of hsa＿circ＿0005752.We revealed the mechanism by which RUNX3 binds to the LPAR1 promoter and regulates the expression of hsa＿circ＿0005752,thereby affecting the osteogenic differentiation of h ADSCs.Chromatin immunoprecipitation(Ch IP)and luciferase reporter assays confirmed that RUNX3 could target the LPAR1-binding promoter.Further q PCR results showed that overexpression of RUNX3 could up-regulate the level of hsa＿circ＿0005752.At the same time,we observed that the levels of MDM2 and osteogenesis-related factors(RUNX2,Osx,ALP,OCN)were up-regulated.In contrast,the level of p53 was down-regulated,while the level of LPAR1 had no significant change.Conclusion: In conclusion,our research elucidates the molecular mechanism of hsa＿circ＿0005752 regulating the osteogenic differentiation of h ADSCs.We believe that hsa＿circ＿0005752 and MDM2 m RNA jointly targeting bind to miR-496 and regulate the osteogenic differentiation of h ADSCs through the hsa＿circ＿0005752/miR-496/MDM2-p53 ceRNA network.At the same time,the expression of hsa＿circ＿0005752 is targeted by RUNX3,thereby affecting the osteogenic differentiation of h ADSCs.Our findings provide a gene modification strategy to enhance osteogenic differentiation of h ADSCs and offer an essential cell resource for the development of bone tissue engineering.更多还原