靶向VDR miRNAs和VDR多态性在结核病中的作用及机制研究

Augmentation of immune responses to Mycobacterium tuberculosis could shorten the course of antimicrobial treatment in drug-sensitive tuberculosis or improve outcome in drug-resistant disease. Calcitriol,the active metabolite of vitamin D by binding vitamin D receptors expressed by antigen-presenting cells and activated lymphocytes induces antimycobacterial activity in vitro.This metabolite modulates the host response to mycobacterial infection by induction of reactive nitrogen and oxygen intermediates,suppression of matrix metalloproteinase enzymes implicated in the pathogenesis of pulmonary cavitation, and induction of the antimicrobial peptide cathelicidin,which induces autophagy. Vitamin D(Vit D) is often used as adjuvant therapy of TB treatment because of its immunoregulation and anti-tuberculosis by VDR. But the machemican and effect of VDR works is not clear.our previous study proved that gene polymorphism of VDR was associated with TB infection and treatment, and many miRNAs SNPs such as miR-125a/b can target VDR gene, so we hypothesized that miRNAs and SNPs may had some kind of combined effects in TB and influence the effect of Vit D in TB treatment. Therefore, based on the results of previous study, we will design case-control study to research the associations of VDR related miRNAs and VDR, and related factors and TB , and interaction by generalized multifactor dimensionalityreduction（GMDR）and haplo.stats in this study.we also prove the impact of miRNAs and SNPs on the effection of Vit D on anti-TB by cell model, and explore the mechanism. The results of this study would assist to understand TB, and utilize Vit D and targeted therapy in medicine drug discovery.

当前抗结核药物研发困难和耐药结核病流行，维生素D（VitD）由于其可通过其受体（VDR）发挥重要免疫调节和抗结核作用受到临床重视，而目前相关调控机制远未阐明限制了临床应用。本课题组前期研究证实VDR基因多态性与结核病发生和治疗效果均存在关联，同时发现miR-125a/b等四个具有SNPs的miRNAs可靶向结合VDR基因，推测这些miRNAs及SNPs可调控VDR基因，且效应存在差异，从而影响结核病的发生及疗效。因此，本研究将采用小样本及大样本病例对照研究分析VDRs与靶向VDR的相关miRNA的SNPs与结核病发生的关联及其交互作用；同时采用细胞模型对相关的miRNAs及其SNPs对VitD抗结核作用的影响进行生物功能学验证和机制研究。该研究结果对于进一步了解结核病发病机制、充分利用VitD抗结核作用和开展个体靶向性治疗药物研发有重要意义。

抗结核药物研发困难和耐药结核病流行，因维生素D3（VitD3）其可通过维生素D受体(VDR)发挥重要免疫调节和抗结核作用受到临床重视。miRNAs可通过调控VDR活性影响结核病的发生及治疗，但相关miRNAs尚未阐明。课题组采用3个生物信息预测平台同时进行靶向VDR的 miRNAs筛选，结合结核病病例和健康对照的差异表达miRNAs谱，筛选出可能靶向VDR，且在结核病患者中高表达的miRNAs；进一步采用病例对照研究（N=360），校正年龄、性别等混杂因素，明确这些miRNAs与结核病的相关性，进而通过细胞模型证实其能靶向VDR，且具有生物学效应。该研究结果对于进一步了解结核病发病机制，充分利用VitD3抗结核作用和开展个体靶向性治疗药物研发有重要意义。. 通过TargetScan、Starbase、MiRDB三个数据库分别进行靶向VDR的miRNAs预测结果取交集后，得到了最有可能靶向VDR的hsa-miR-10a-5p、hsa-miR-27a-3p和hsa-miR-30b-5p等33条miRNAs。. 对5例结核病人和5例健康对照miRNAs高通量测序，发现23条差异表达显著的miRNAs。在结核病例中，hsa-miR-432-5p等15条miRNAs表达下调，hsa-miR-1291、hsa-miR-27a-3p和hsa-miR-30b-5p等8条miRNAs表达上调。. 以181例结核病例和179例对照者进行病例对照研究，校正年龄、性别等混杂因素后，hsa-miR-27a-3p（OR=1.321,95%CI1.093-1.598）和hsa-miR-30b-5p（OR=1.233,95%CI1.012-1.502）与结核病呈显著相关性，进一步证实这2个miRNAs与结核病的相关性。. 在单核-巨噬细胞系模型中，进一步证实了hsa-miR-27a-3p和hsa-miR-30b-5p能够靶向并调控VDR，使VDR表达量降低，VitD3免疫调节功能减弱, 且NF-kB信号通路激活，促进IL-1β等炎症因子释放，进而影响结核病的易感性及发展进程。. 通过以上研究，筛选出并证实了hsa-miR-27a-3p和hsa-miR-30b-5p能靶向结合VDR，且能产生影响结核杆菌生长繁殖的生物学效应，为其抗结核机制研究奠定基础。