lncRNA-miRNA-mRNA双重调控BMSCs异常分化预防激素性股骨头坏死的实验研究

The steroid-induced osteonecrosis of femoral head(ONFH) is a common disease that can lead to physical disability in high rate. The effective preventive means are poor at present. The important pathogenesis is that steroid can induce a large number of adipogenic differentiation of BMSCs and inhibit osteogenic differentiation of them. Novel lncRNA-miRNA-mRNA network regulate and control mode is better than previous gene therapy for disease prevention. We made a preliminary experiment with microarray technology and bioinformatics analysis, screened and obtainted one low expression lncRNA （TCONS_00041960）and two high expression miRNAs (miR-125a-3p and miR-204-5p) in the rat BMSCs treated with steroid. This project intends to use the network control mode of dual regulating BMSCs abnormal differentiation. We can constructe the recombinant lentivirus tageting lncRNA, transfect it into rat BMSCs in vitro, and inject into animal femoral head of steroid-induced ONFH model in vivo respectively. And then，the high expression of lncRNA can cause downregulation of miRNAs，upregulation of GILZ (PPAR gamma inhibitor) expression and suppress PPAR gamma expression, and promote the expression of Runx2 at the same time. The results can inhibit cell adipogenic and actively promote the osteogenic differentiation simultaneously. To regulate the abnormal differentiation of BMSCs through advantages of the network regulation can higher efficiently prevent the onset of steroid-induced ONFH from the key aspects of the pathogenesis. This innovative idea has a more important scientific significance and application prospects.

激素性股骨头坏死致残率高，尚无有效预防方法。重要发病机制是激素诱导BMSCs大量成脂分化并抑制成骨分化。新颖的lncRNA-miRNA-mRNA网络调控方式预防疾病优于以往基因疗法，我们前期通过基因芯片及生物信息学分析筛选出激素诱导的大鼠BMSCs中低表达目标lncRNA(TCONS_00041960)和高表达的miRNAs(miR-125a-3p和miR-204-5p)。本项目拟采用此网络调控方式双重调控BMSCs，构建目标lncRNA重组慢病毒，分别转染大鼠体外BMSCs及注入激素性股骨头坏死动物模型股骨头内，使lncRNA高表达，下调效应miRNAs，上调靶基因GILZ(PPARγ抑制剂)表达、阻断PPARγ，同时上调Runx2表达，抑制细胞成脂并促进成骨分化。从发病机制关键环节通过网络调控优势调节BMSCs异常分化预防激素性股骨头坏死发生。这一创新思路更具重要的科学意义和应用前景。

激素性股骨头坏死发病机制的源头环节是激素诱导BMSCs大量成脂并抑制成骨分化。为探索预防该病的新策略，本项目构建lncRNA TCONS_00041960重组慢病毒载体，转染大鼠BMSCs，并注入ONFH模型大鼠股骨头内，探讨预防效果。.实验结果显示: (1)基因芯片筛选出激素与正常组大鼠BMSCs差异表达lncRNAs和miRNAs,并证明目标lncRNA(TCONS_00041960)与目标miRNAs(miR-204-5p和miR-125a-3p)之间存在结合位点及相互作用，Runx2是miR-204-5p的靶基因、GILZ是miR-125a-3p的靶基因。(2)细胞学:成功构建目标lncRNA重组慢病毒载体，转染激素诱导的BMSCs，lncRNA重组慢病毒组(Ex-Lnc+Dex)与Dex、Ex-NC+Dex组比较，PPARγ mRNA表达降低(P<0.05)，GILZ、Runx2 mRNA表达升高(P<0.05)。各组中蛋白表达与mRNA趋势一致。与Dex、Ex-NC+Dex组比较，Ex-Lnc+Dex组中TG含量降低及ALP活性升高(均P<0.05)，且细胞内脂滴聚集明显减少。上调目标lncRNA与下调目标miRNAs对激素诱导BMSCs异常分化作用一致；上调目标lncRNA的表达抑制了激素诱导的BMSCs成脂分化且促进其成骨分化。(3)动物学:将目标lncRNA重组慢病毒注入ONFH模型大鼠股骨头内，与Dex、Ex-NC+Dex组比较，Ex-Lnc+Dex组股骨头细胞内PPARγ mRNA表达明显降低(P<0.01)，Runx2、GILZ mRNA表达显著升高(P<0.01)。各组蛋白表达与mRNA表达一致。Ex-Lnc+Dex组大鼠股骨头组织结构接近正常组，无脂肪聚积。体内外实验证实:上调lncRNA TCONS_00041960可通过双重调控miR-204-5p和miR-125a-3p表达，靶向阻断体外BMSCs及ONFH模型活体股骨头细胞内PPARγ表达，阻止成脂分化；同时上调细胞中Runx2表达，促进成骨分化，增强股骨头骨组织修复，获得遏制成脂且促进成骨的双重效果。.本研究证明了lncRNA-miRNA-mRNA双重调控BMSCs异常分化的优势，能够从发病机制源头环节有效预防激素性ONFH，为阐明发病机制和有效防治提供了重要的科学依据。