Negative regulation of HBG1/2 expression through S6K by long noncoding RNA NR_120526

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 (HBG1/2 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 HBG1/2. 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 HBG1/2, ribosomal protein S6 kinase B1 (RPS6KB1, S6K), and Ras homologous family member A (RhoA), respectively. RESULTS: We found that NR_120526 interacts with ILF2, ILF3, and S6K. However, ILF2/ILF3 bound to NR_120526 did not interact with HBG1/2, suggesting that NR_120526 may regulate HBG1/2 expression indirectly. The qRT-PCR results showed no statistical difference in the mRNA expression levels of HBG1/2, S6K, and RhoA 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 HBG1/2, S6K, and RhoA in the KO group (P<0.05). It was found that NR_120526 inhibited S6K, thereby downregulating RhoA and leading to decreased HBG1/2 expression. CONCLUSIONS: LncRNA NR_120526 negatively regulates the expression of HBG1/2 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.