BMP2/Gm26778/β-catenin信号通路参与调控老龄化BM-MSCs成骨分化的机制研究

Osteoporosis is closely linked with aging. The prognosis of osteoporotic fracture healing has great influence on the physical and mental health of the elderly, and the responsiveness of aging BM-MSCs to BMP signals is weakened, which is closely related to the reduction of endochondral osteogenesis and the delay of callus formation. .In the previous study, employing RNA sequencing, we identified 26 long non-coding RNA (lncRNA) genes that are highly selectively expressed in BMP-non-responsive imef cell with n19 library, which display inhibited osteoblastic differentiation and ectopic bone formation, compared with parental iMef induced by BMP signal. Among these lncRNA genes we detected that Gm26778 is signficantly upregulated by BMP signal induced MSCs but repressed in BMP-non-responsive iMef cells. And Gm26778 roles in regulating BMP-2-induced osteogenic differentiation of BM-MSCs. Bioinformatics analysis found that Gm26778 may regulate osteogenic differentiation through binding with β-catenin dependent classical Wnt signaL pathway and then effect on its downstream osteogenic transcription factor. .In this project, we first analyzed the relationship between Gm26778 and age-related factors influencing bone regeneration induced by BMP2. After siRNA and overexpression of Gm26778, the effect of BMP2-induced osteogenic differentiation of BM-MSCs was analyzed in vitro and in vivo, further analysis of β-caitenin and phosphorylation level of several sites on it and nuclear transfer, using RNA pull down and RIP to verify the binding of Gm26778 and β-caitenin . Finally, we examined whether BMP2 synergize with the classical β-catenin signal pathway via Gm26778, then function in the osteogenic differentiation of aged BM-MSCs.

骨质疏松性骨折的预后对老年人身心健康影响巨大，而老龄化BM-MSCs对BMP信号的响应性减弱，与软骨内成骨作用的减少和骨痂形成的延迟密切相关。前期研究中通过RNAi文库筛选到BMP信号抵抗细胞株中异常下调的LncRNA-Gm26778，参与介导BMP-2诱导BM-MSCs成骨分化，生物信息学分析Gm26778可能结合经典β-catenin信号通路作用于下游成骨转录因子。本项目首先分析Gm26778与年龄因素影响BMP2诱导骨再生的相关性，再采用siRNA干扰和过表达Gm26778后，体内外实验分析其影响BMP2诱导BM-MSCs 的成骨分化的效应，进一步分析β-caitenin 及其不同位点磷酸化水平和核转移的变化，采用RNA pull down和RIP验证两者相互结合。最终明确BMP2可通过Gm26778与经典β-caitenin 信号通路发生交互对话，从而影响老龄化MSCs 成骨分化。

骨质疏松性骨折的预后对老年人身心健康影响巨大，而老龄化BM-MSCs对BMP信号的响应性减弱，与软骨内成骨作用的减少和骨痂形成的延迟密切相关。前期研究中通过RNAi文库筛选到BMP信号抵抗细胞株中异常下调的LncRNA-Gm26778，本项目通过体内外实验明确敲低 Gm26778 明显抑制了 BMP2 诱导 BM-MSCs 的成骨分化，而提高了其成脂分化能力，但对β-catenin的表达没有明显影响。其次比较青年组和老年组小鼠BM-MSCs发现，老年组的BM-MSCs干性下降和细胞衰老的发生，经 BMP2 诱导后成骨分化能力亦明显下降，但Gm26778 在老年组BM-MSCs 经 BMP2 处理后表达水平未有明显变化，其意义有待于进一步阐明。最后经RNA sequencing 检测和Ven分析比较两组，明确青年组BM-MSCs经BMP2诱导后有455个差异mRNA和25个差异LncRNA，老年组BM-MSCs经BMP2诱导后有349个差异mRNA和176个差异LncRNA，两组间有共同的193个差异mRNA和8个差异LncRNA，其中筛选到ITGAV（Integrin alpha V，CD51），Ankrd1，Calcr1，CXC15，Prrg1和Zbtb26 6个表达模式相悖的基因。