长链非编码基因多态性与煤工尘肺易感性及其分子机制研究

Long non-coding RNAs (lncRNAs) are an important class of genes involved in various biological processes. In recent years, it has gained attention for its complex extensive regulatory functions and genetic variation at the whole genome level. To date, little or nothing has been known about the single nucleotide polymorphisms (SNP) and their functional significance in the lncRNA locus on the susceptibility of coal workers' pneumoconiosis (CWP). Our previous study on a genome-wide association study (GWAS) on silica related pneumoconiosis showed that most SNPs related to CWP do not located in protein coding regions. Therefore, we screened this GWAS database and identified 16 SNPs on 16 lncRNAs which could be significantly related to the susceptibility of CWP. Then, The 600 pair case-control study (of cases and controls) will be used to validate the associations between these SNPs and the risks of CWP. For the significant SNPs, the functional studies will be carried out to explore how the genotype alters the expression and function of lncRNA. To identify the coding gene that regulated by lncRNA, we will construct the cell lines which express high or low lncRNA stably, screen mRNAs that alter in lung tissues of mice treated with silica by microarray and perform the biotin-labeled lncRNA pull-down assay. Additionally, siRNA will be used to interfere the coding gene in cell lines that constructed above, then these cells will be treated with silica. Moreover, a silica-induced pneumoconiosis model that express the target coding gene in mice lung tissues will be established. Finally, whether the occurrence and development of silica-induced lung fibrosis is regulated via coding genes that targeted by lncRNA will be assess comprehensively.

在全基因组水平上对lncRNA遗传变异、功能及调控研究已成为热点，但未见与尘肺病有关的报道。本课题组前期进行的煤工尘肺全基因组关联性分析（GWAS），发现与煤工尘肺病相关的SNPs大多不在编码蛋白质的DNA区域。因此，我们从GWAS数据库中筛选出位于lncRNAs上16个可能与煤工尘肺相关的SNPs，将用600对病例-对照样本进行验证，明确这些SNPs与煤工尘肺的关联性；通过功能学研究明确与煤工尘肺病密切相关SNPs对lncRNA表达及功能的影响；构建稳定高/低表达lncRNA的细胞系，通过芯片筛检差异表达的mRNAs及生物素pull down和RNA免疫共沉淀等确定lncRNA调控的编码基因；用siRNA干涉稳定高/低表达lncRNA细胞系中的编码基因，矽尘处理；同时建立小鼠肺组织高/低表达编码基因的矽肺动物模型。综合评估lncRNA通过编码基因调控矽尘诱导的肺纤维化发生发展。

尘肺病是我国每年新诊断职业病中最多的一类疾病（接近90%），其中煤工尘肺和矽肺占了尘肺病中的90%左右。研究煤工尘肺和矽肺易感性及其机制对预防、干预及靶点治疗均具有重要的意义。.在前期煤工尘肺GWAS数据库中筛选有潜在意义的SNPs，进行人群验证，发现4个功能性煤工尘肺致病位点, 其宿主基因在肺组织中表达显著相关，包括 （rs10797062-ATP1A4，rs1667614-FNBP1P1，rs2540438-ALS2CR12及rs2274554-RBM26）。联合效应分析显示，煤工尘肺的发生风险随着等位基因数量的增加而显著增加，呈等位基因累积的变化趋势。通路富集分析提示ATP1A4, FNBP1P1及 RBM26基因在泛素介导的蛋白水解通路中共表达。.为了鉴定与CWP风险相关的基因，我们首先将来自GTEx数据库中肺组织内的基因表达作为训练数据，将顺式遗传基因归入现有的CWP GWAS数据中。发现NUP35、FAM213B、PSMB9可能是CWP相关基因，用已有的的CWP GWAS和来自GTEx数据库的全血基因表达值进行了另一次TWAS。发现候选基因PSMB9表达与CWP风险相关性最为显着（PTWAS = 0.000211），进一步的eQTL分析显示rs4713600的风险基因型与肺组织和全血中PSMB9的更高表达相关，功能研究结果表明，显示PSMB9基因的表达受rs4713600和与其高联锁的SNPs的转录调节，PSMB9能够调控上皮细胞的凋亡。.在前期GWAS研究的基础上，对发现LAMB1基因上多态性位点rs4320486突变后可降低CWP发生危险性进行了验证，经过动物和细胞实验，初步探究了纤维化过程中可能调控LamB1表达的机制。研究结果提示LamB1可能是肺纤维化的重要生物学标志物。.课题组一直围绕非编码RNA调控矽尘诱导肺纤维化机制研究，力求为干预其发生发展提供科学依据。4年中开展了多条长链非编码RNA和miRNA调控肺纤维化的机制与干预研究，对矽尘诱导肺纤维化的调控网络有了初步的认识，也为尘肺病的干预和治疗研究提供了新的思路和线索，推进了肺纤维化机制研究的进程。.四年中共发表标注本课题资助的文章15篇，其中SCI 13篇，获得专利2件，培养硕博研究生多人，3篇研究生毕业论文获得学校优秀毕业论文奖，获江苏省科技进步2等奖1项。

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