LncRNA HOTAIRM1 Regulates Cell Autophagy And Proliferation In NPM1-mutated Leukemia

Objective: Acute myeloid leukemia(AML)with mutated nucleophosmin(NPM1)has been defined as a unique subgroup in the new classification of myeloid neoplasm,which displays a distinct long noncoding RNA(lncRNA)expression profile.However,the biological roles of key lncRNAs in NPM1-mutated AML development are not cleared nowadays.Methods: Based on GEO and TCGA databases,the expression of lncRNA HOX antisense intergenic RNA myeloid 1(HOTAIRM1)in AML cells and its association with clinical prognosis of patients were analyzed with the bioinformatics software.The levels of HOTAIRM1,mi R-152-3p and its target gene ULK3 were detected by q RT-PCR.Chromatin immunoprecipitation(ChIP)assay was used to analyze the enrichment of histone H3 on lysine 27 acetylation(H3K27ac)at the promoter region of HOTAIRM1.The protein levels of histone acetyltransferase CBP,autophagy markers(LC3,p62)and ULK3 were determined by western blot.Immunofluorescence assay was employed to observe LC3 puncta.The effects of HOTAIRM1 knockdown combined with autophagy inducer rapamycin and HOTAIRM1 overexpression combined with autophagy inhibitor 3-MA on the proliferation of leukemia cells were evaluated by CCK-8 and Ed U assays.Nucleocytoplasmic separation and fluorescence in situ hybridization(FISH)assays were used to identify the subcellular localization of HOTAIRM1.Luciferase reporter assay was applied to detect the interaction between HOTAIRM1 and mi R-152-3p,mi R-152-3p and ULK3,respectively.Recovery experiments were performed to clarify the effect of HOTAIRM1-mi R-152-3p-ULK3 axis on the AML cell phenotypes.Results: HOTAIRM1 was highly expressed in NPM1-mutated AML cell lines and clinical samples,and high HOTAIRM1 expression was associated with short overall survival in AML patients.H3K27 ac was enriched at the promoter region of HOTAIRM1.Knockdown of CBP significantly reduced the enrichment of H3K27 ac at the promoter region of HOTAIRM1,and thereby induced a decreased expression of HOTAIRM1.Next,inhibition of HOTAIRM1 downregulated LC3,upregulated p62 protein levels and reduced the accumulation of LC3 puncta numbers,while overexpression of HOTAIRM1 had the opposite effect.In addition,knockdown of HOTAIRM1 depressed cell proliferation,but rapamycin could reverse the inhibitory effect.Overexpression of HOTAIRM1 promoted cell proliferation,while 3-MA could inhibit this effect.Mechanically,cytoplasm HOTAIRM1 could competitively bind with mi R-152-3p to upregulate ULK3 levels.Finally,HOTAIRM1 promoted leukemia cell autophagy and proliferation through the mi R-152-3p/ULK3 axis.Conclusions: HOTAIRM1 was highly expressed in NPM1-mutated AML,which was induced by CBP-mediated H3K27 acetylation on HOTAIRM1 promoter.Importantly,HOTAIRM1 acted as a sponge for mi R-152-3p and thus increased ULK3 expression,leading to enhancement of autophagic activity and cell proliferation.This study elucidates the function of HOTAIRM1 in NPM1-mutated AML,suggesting that HOTAIRM1 may serve as a promising target for the treatment of the distinct leukemia subtype.