长链非编码RNA NR_120526通过S6K对表达的负调控。

Negative regulation of expression through S6K by long noncoding RNA NR_120526.

作者信息

Jia Wenguang, Wu Xiaojing, Chen Zhaohui, Lin Weixiong, He Yunyan

机构信息

Department of Pediatrics, The First Affiliated Hospital of Guangxi Medical University, Nanning, China.

NHC Key Laboratory of Thalassemia Medicine, Chinese Academy of Medical Sciences, Nanning, China.

出版信息

Transl Pediatr. 2023 May 30;12(5):907-917. doi: 10.21037/tp-23-174. Epub 2023 May 24.

DOI:10.21037/tp-23-174

PMID:37305725

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10248940/

Abstract

BACKGROUND

High levels of fetal hemoglobin (HbF) may alleviate clinical symptoms in patients with β-thalassemia. A previous study showed that the long noncoding RNA NR_120526 (lncRNA NR_120526) might be involved in regulating HbF levels (/ gene expression). However, the function and mechanism by which NR_120526 regulates HbF expression remains unknown. Here, we investigated the effect of NR_120526 on HbF and its mechanism so as to provide an experimental basis for treating patients with β-thalassemia.

METHODS

Chromatin isolation by RNA purification-mass spectrometry (ChIRP-MS) assay, database query, and bioinformatics analysis were performed to explore the proteins that specifically bind to NR_120526 and their interactions. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) were used to determine whether NR_120526 directly regulates the expression of /. The NR_120526 gene was knocked out (KO) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in K562 cells. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the messenger RNA (mRNA) and protein expressions of /, ribosomal protein S6 kinase B1 (, ), and Ras homologous family member A (), respectively.

RESULTS

We found that NR_120526 interacts with ILF2, ILF3, and S6K. However, ILF2/ILF3 bound to NR_120526 did not interact with , suggesting that NR_120526 may regulate expression indirectly. The qRT-PCR results showed no statistical difference in the mRNA expression levels of /, , and between the NR_120526-KO group and negative control (NC) group (P>0.05). However, Western blot results showed a significant increase in the protein levels of /, , and in the KO group (P<0.05). It was found that NR_120526 inhibited S6K, thereby downregulating RhoA and leading to decreased / expression.

CONCLUSIONS

LncRNA NR_120526 negatively regulates the expression of / through S6K. These new findings provide mechanistic insights into the regulation of HbF and offer potential therapeutic targets for precision medicine in patients with β-thalassemia.

摘要

背景

高水平的胎儿血红蛋白（HbF）可缓解β地中海贫血患者的临床症状。先前的一项研究表明，长链非编码RNA NR_120526（lncRNA NR_120526）可能参与调节HbF水平（/基因表达）。然而，NR_120526调节HbF表达的功能和机制仍不清楚。在此，我们研究了NR_120526对HbF的影响及其机制，以便为治疗β地中海贫血患者提供实验依据。

方法

采用RNA纯化-质谱染色质分离分析（ChIRP-MS）、数据库查询和生物信息学分析，探索与NR_120526特异性结合的蛋白质及其相互作用。采用染色质免疫沉淀结合高通量DNA测序（ChIP-seq）来确定NR_120526是否直接调节/的表达。利用成簇规律间隔短回文重复序列（CRISPR）/Cas9技术在K562细胞中敲除（KO）NR_120526基因。最后，采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分别检测/、核糖体蛋白S6激酶B1（，）和Ras同源家族成员A（）的信使核糖核酸（mRNA）和蛋白质表达。

结果

我们发现NR_120526与ILF2、ILF3和S6K相互作用。然而，与NR_120526结合的ILF2/ILF3并不与相互作用，这表明NR_120526可能间接调节表达。qRT-PCR结果显示，NR_120526基因敲除组与阴性对照组之间/、和的mRNA表达水平无统计学差异（P>0.05）。然而，蛋白质免疫印迹结果显示，敲除组中/、和的蛋白质水平显著升高（P<0.05）。研究发现，NR_120526抑制S6K，从而下调RhoA并导致/表达降低。