MicroRNA对β-地中海贫血表型的修饰作用及其分子机制

A study on effects of modifier genes in modulating phenotypes of Mendelian disorders is becoming a new project for the current understanding of genotype- phenotype correlation. Regulators of fetal-to-adult hemoglobin switching have been identified as major genetic factors associated with considerable phenotypic variation of β-thalassemia but microRNAs and their target genes involved in modulating mechanism is still unclear. In the preliminary study, we have identified a few groups of microRNAs and their target genes with mutations occurred within 3’UTR, which are responsible for regulating fetal hemoglobin levels. In the present proposal, using deep sequencing，CRISPR-Cas9 editing and RNA ChIP-seq technologies, etc, we plan to investigate these microRNAs and their target genes focused on the four aspects of the mechanisms as follows: （1）Demonstration of the molecular mechanism under β-thalassemia stress erythropoiesis by analyzing the variance of microRNA expression and interplay of microRNA and their target genes; (2) Exploration of the fine regulation of fetal-to-adult hemoglobin switching by discovering gene regulatory networks composed of transcription factors and microRNA;（3）Study on the contribution of variations in the microRNA binding sites of human genes to β-thalassemia phenotypic severity and elucidation of association between clinical phenotypes and modifier gene genotypes; （4）Clarification of the molecular mechanism of silencing or enhancing of HbF due to modifier gene mutations for explanation of phenotypic severity in β-thalassemia. The results obtained from this study will expand related knowledge about the regulation of hemoglobin switching. Our revealed epigenetic mechanism of γ-globin regulation will be applicated in precision diagnosis/prediction of this disease and provide potential targets for treatment of β-hemoglobinopathy patients.

遗传修饰基因对孟德尔遗传病表型的修饰作用是遗传病表型-基因型研究面临的新课题，胎儿血红蛋白（fetal hemoglobin, Hb F）表达水平差异大是目前解释β地中海贫血（β地贫）患者表型严重性的主要遗传因素，但miRNA及其相互作用基因对疾病表型的修饰作用机制目前尚不清楚。本课题拟以前期在β地贫患者中鉴定得到的有Hb F调节功能的miRNA及其靶基因3’UTR结合位点变异为对象，采用深度测序，CRISPR-Cas9和ChIP-seq等技术，研究在β地贫突变红系压力下，miRNA的表达变化规律及其与靶基因的相互作用的分子病理学机制，探索Hb F表达精细调控的miRNA联系网络；同时系统分析这类新的靶基因遗传变异对β地贫临床表型的贡献，并揭示解释β地贫表型严重性的遗传突变作用机制。研究结果可扩充和延伸Hb F基因调控的相关知识，并可为中国人群临床精确诊治和预测β-地贫患者表型严重性提供新靶标。

遗传修饰基因对孟德尔遗传病表型的修饰作用是遗传病表型-基因型研究面临的新课题，胎儿血红蛋白（Hb F）表达水平差异大是目前解释β地中海贫血（β地贫）患者表型严重性的主要遗传因素，但miRNA及其相互作用基因对疾病表型的修饰作用机制目前尚不清楚。本课题基于过于对β-地贫患者大样本队列的表型-基因型数据积累，完成了影响HbF表达水平的表观遗传学探索，核心的方向是鉴定出一批直接或间接参与HbF表达调控的microRNA（miRNA）。 本研究表明，miRNA是影响HbF表达的重要因素之一，它的作用机制是通过结合在关键的HbF调控基因的3’UTR区域，介导靶基因的降解或低表达，最终实现对HbF水平的调节。.本研究共鉴定出miR-326是关键红系转录因子KLF1的重要上游调控因子，并通过体内外实验发现并证实了miR-326在KLF1的3’UTR端直接结合的序列 (Exp Hematol. 2018 Jul; 63:33-40.e2)。 同时，本课题亦从KLF1的5’UTR端出发，系统研究了KLF1启动子区甲基化与β-地贫患者表型多样性的关系 (Blood Cells Mol Dis. 2018 Jul;71:16-22)； 最后，我们发现了一个可直接影响红系转录因子KLF6表达的miR-2355-5p，并且探索了KLF6受miR-2355-5p转录后调控的作用机制 (Br J Haematol. 2020 Dec 24. Doi: 10.1111/bjh.17134)。..综上，本研究鉴定出了可显著影响β-地贫患者体内HbF表达的miRNA，并且通过系统功能学实验验证其作用机制，该研究为β-地贫表型精确诊断及分子治疗提供了有效的靶点。