LncRNA和miRNA调控网络在强直性脊柱炎发生和进展中的作用及其分子机制

Previous studies have shown that the expression levels and gene polymorphisms of miRNA in T cells and PBMCs were associated with ankylosing spondylitis (AS), lncRNA may be involved in the abnormal bone differentiation of AS-MSC. There are close relationships between regulatory networks of lncRNA-miRNA and the osteoarthritis, tumors and other diseases. However, direct evidence has not been found between the regulatory mode and AS. We postulated that the abnormal expression of lncRNA in PBMCs combined with miRNA may be involved in the pathogenesis and development in patients with AS. In this project, we first use lncRNA microarray to screen the differentially expressed lncRNA and verified by qRT-PCR. The correlation between candidate lncRNAs and clinical and laboratory indexes will be analyzed. Then bioinformatics database will be used to predict the expression of miRNA and miRNA target gene which related to lncRNA. Finally, the function of the regulatory network will be studied at the cellular level through lentiviral transfection and siRNA interference technology; in addition, the lncRNA was found from the database based on the molecular epidemiology research of our teamwork, and target miRNA as well as target gene SNPs. Furthermore, the susceptibility of these SNPs will be analyzed in a case-control study with large samples. Considering the evidence above, the possible mechanism of lncRNA-miRNA regulatory network in the pathogenesis and progression of AS will be preliminarily revealed.

研究表明miRNA在T细胞以及PBMCs中的表达水平及其基因多态性与AS发病相关，lncRNA可能参与AS-MSC异常的骨分化，lncRNA-miRNA调控网络与骨关节炎、肿瘤等疾病关系密切，但目前缺乏这种模式与AS相关的直接证据。我们假设：AS患者PBMCs中lncRNA表达异常，可与miRNA共同作用参与疾病的发生、发展。本课题先借助lncRNA芯片筛选差异表达的lncRNA并用qRT-PCR验证、分析候选lncRNA与临床特征及实验室指标的相关性；用生物信息学技术预测与lncRNA相关的miRNA及其靶基因；后借助慢病毒转染和siRNA干扰技术等从细胞水平进行功能研究；结合课题组前期研究基础，从数据库中查找lncRNA、靶点miRNA及靶基因SNPs，并在大样本病例对照中对这些SNPs进行易感性分析。综合上述证据，初步揭示lncRNA-miRNA调控网络在AS发病和进展中的分子机制。

研究表明miRNA在T细胞以及PBMCs中的表达水平及其基因多态性与AS发病相关，lncRNA可能参与AS-MSC异常的骨分化，lncRNA-miRNA调控网络与骨关节炎等疾病关系密切，但目前缺乏这种模式与AS相关的直接证据。本研究先借助lncRNA芯片从89459个lncRNA中筛选出154个差异表达的lncRNA，其中已有注释的lncRNA42个，随机挑选4个lncRNA（ZRANB2-AS2，LINC00320，H19和LOC101929023）进行大样本qRT-PCR验证，最终确定H19为本研究的目标lncRNA；利用有关数据库通过生物信息学技术成功的预测到H19的靶点miRNA以及靶点miRNA下游的mRNA，结合参考文献，最终确定H19-miR675-VDR通路为本课题的研究目标分子；然后采用Si-H19和AD-H19来敲低和过表达H19，并用miRNA mimics和miRNA inhibitors来过表达和敲低miRNA的表达； PCR 检测这些lncRNA和miRNAs对细胞通路的影响，Western blot检测mRNA对应的蛋白质表达水平的影响，ELISA检测细胞通路上关键的细胞因子表达水平的改变情况，在HEK293T细胞中进行了双荧光素酶报告基因实验探讨H19与miR22-5p和miR675-5p的调控关系；结果显示H19在AS患者发病过程中起着重要的作用，且其可以充当miR22-5p-VDR-IL-17A/IL-23轴上的ceRNA，或者与miR675-5p-VDR-IL-17A/IL-23通路上的miR675-5p直接相互作用来促进患者炎症的进展，另外本项目还另外本项目还探索lncRNA GAS5、lncRNA MEG3和自噬相关基因LC3、Beclin1在AS发病及进展过程中的作用及意义，研究结果显示AS病例组中lncRNA GAS5的表达水平显著低于对照组；但lncRNA MEG3的表达水平无统计学差异；AS病例组中LC3、Beclin1的表达水平均显著低于对照组。综合上述证据，初步揭示lncRNA-miRNA调控网络在AS发病和进展中的分子机制。