LncRNA NR_120526结合转录因子ILF2/3调控血红蛋白F合成的机制研究

As a harmful inherited blood disorder, there still lack of effective therapies for β thalassemia. Studies suggested that lncRNAs can ameliorate the clinical symptoms of β-thalassemia by upregulating fetal hemoglobin (HbF) expression. However, the exact mechanisms remain poorly understood. Previously, our studies indicate that lncRNA NR_120526 could promote the expression of HbF by binding ILF2/3 and negatively regulating mTORC2 signaling pathway. In addition, our research showed that ARHGAP18 is an activator of RhoA (located on the downstream of the mTORC2 pathway), which could decrease the expression of γ globin gene. This time, our project will enlarge samples of patients with β thalassemia major and normal subjects to test lncRNA NR_120526 expression. The effect and significance of lncRNA NR_120526 will be detected. Whether lncRNA NR_120526 and ILF2/3 directly regulate expression of γ globin gene or not can be confirmed. We further intervene the expression of lncRNA to investigate expression of γ globin gene. Meanwhile, we will explore the changes of key proteins of mTORC2 signaling pathway, downstream RhoA and HbF expression. Thereby, the molecular mechanism for HbF persistence-expressed can be shed new lights and proved theoretical basis for future treament strategies in β thalassemia.

危害严重的遗传性血液病β地贫目前仍缺乏有效的治疗手段。研究表明lncRNA通过促进血红蛋白F（HbF，α2γ2）合成，改善β地贫临床症状，但具体机制仍不明确。我们研究发现lncRNA_NR120526能够结合ILF2/3负调控mTORC2通路，增加HbF合成。此外，还发现ARHGAP18是mTORC2通路下游分子RhoA的激活基因，它可抑制γ珠蛋白基因的表达，使HbF减少。本项目拟进行以下研究：扩大重型β地贫和正常人样本验证lncRNA_NR120526表达的差异，明确其在临床中的意义；检测lncRNA_NR120526结合ILF2/3后是否直接调控γ珠蛋白基因；敲减和过表达lncRNA_NR120526,研究细胞表型和γ珠蛋白基因的变化，同时检测mTORC2信号通路关键蛋白、下游RhoA和HbF表达的改变。从而对HbF持续合成的机制提出新阐释，为将来β地贫的临床靶向治疗提供理论依据。

β-地中海贫血(简称β-地贫)是一种由β-珠蛋白合成障碍引起的遗传性溶血性疾病，严重者可致死。广西是β-地贫的高发区，有证据表明高水平的胎儿血红蛋白（HbF，α2γ2）能缓解重型β-地贫患者的临床症状。重新激活γ珠蛋白基因（hemoglobin subunit gamma 1/2，HBG1/2）的表达可提高HbF水平，改善预后。本课题组前期研究发现长链非编码RNA（Long non-coding RNA，LncRNA） NR_120526在高HbF的β-地贫和HPFH病例组及对照组中存在差异性表达，提示NR_120526可能参与HbF（HBG1/2基因）的表达调控，但具体机制未明。.本项研究扩大了样本量进行qPCR检测，发现lncRNA NR_120526 在重型β-地贫患者与对照组中的表达无差异（P=0.851）。染色质免疫共沉淀-高通量测序（ChIP-Seq）提示NR_120526结合ILF2/ILF3后对HBG1/2的表达可能起间接调控作用。利用CRISPR-Cas9基因编辑技术敲除NR_120526或者敲除ILF3后，K562细胞的增殖减缓、凋亡增加；而过表达NR_120526或者敲除ILF2则对细胞的增殖和凋亡无显著影响。与阴性对照组相比，敲除NR_120526后S6K、mTOR、RhoA和HBG1/2的蛋白表达均显著增高，其中HBG1/2增高1.927倍（p=0.037）；p- mTOR也显著升高，但p-S6K和p-RhoA无明显变化。过表达NR_120526后HBG1/2的蛋白表达无明显变化。敲除ILF2后HBG1/2的蛋白表达增高5.197倍（P=0.001），敲除ILF3后HBG1/2的蛋白表达则增高7.537倍（P＜0.001），但两者对S6K、mTOR和RhoA的蛋白表达影响与敲除NR_120526不一致。提示lncRNA NR_120526负向调控HBG1/2的表达，且可能是通过S6K介导的mTORC2信号通路影响RhoA来实现；ILF2和ILF3也参与负向调控HBG1/2，但不是通过S6K介导的mTORC2信号通路来实现，机制有待进一步研究。.本项研究为LncRNA NR_120526对HbF（HBG1/2）的表达调控提供了机制上的新见解，并为β-地贫患者的精准治疗提供了潜在靶点。

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