LncRNA HOTAIRM1 is involved in the progression of acute myeloid leukemia through targeting miR-148b

Abstract

LncRNAs have been shown to be involved in the biological and pathological processes of acute myeloid leukemia (AML). Hox antisense intergenic RNA myeloid 1 (HOTAIRM1) was reported to be highly expressed in AML. However, the detailed role and molecular mechanism of HOTAIRM1 in AML pathogenesis remain undefined. In the present study, HOTAIRM1 and miR-148b expressions in AML patients and healthy controls were detected by qRT-PCR. Cell proliferation and apoptosis were evaluated by CCK-8 and flow cytometry assays, respectively. The regulatory interaction between HOTAIRM1 and miR-148b was explored by bioinformatics analysis using starBase v3.0 software and The Cancer Genome Atlas (TCGA) AML dataset. We found that the miR-148/miR-152 family members including miR-148a, miR-148b, and miR-152 were predicted to be potential targets of HOTAIRM1. HOTAIRM1 expression was negatively correlated with miR-148b expression but had no correlation with miR-148a/miR-152 expressions in AML samples from the TCGA dataset. HOTAIRM1 expression was higher while miR-148b expression was lower in AML patients than in healthy controls. A negative correlation between HOTAIRM1 and miR-148b in AML patients was observed. HOTAIRM1 silencing and miR-148b overexpression both suppressed cell proliferation and induced apoptosis in AML cells. miR-148b was identified as a target of HOTAIRM1 in AML cells. Moreover, HOTAIRM1 knockdown inhibited proliferation and induced apoptosis in AML cells by negatively regulating miR-148b. In summary, HOTAIRM1 was involved in the progression of AML through targeting miR-148b, shedding light on the biological function and molecular mechanism of HOTAIRM1 in AML.

Fig. 1. The expressions of HOTAIRM1 and miR-148b in AML. (A) The predicted complementary binding sites of the miR-148/miR-152 family members to HOTAIRM1 by bioinformatics analysis using starBase v3.0 software. (B–D) The correlation between HOTAIRM1 and the miR-148/miR-152 family member expressions in a cohort of AML patients from TCGA dataset. qRT-PCR analysis of HOTAIRM1 (E) and miR-148b (F) in the peripheral blood samples from 29 AML patients and 17 age-matched healthy individuals. (G) Pearson's correlation analysis of the correlation between HOTAIRM1 and miR-148b expression in AML patients.

Fig. 2. HOTAIRM1 silencing suppressed cell proliferation and induced apoptosis in AML cells. (A–C) qRT-PCR was applied to examine HOTAIRM1 expression in HL60, THP-1, and NB4 cells after transfection with si-HOTAIRM1 or si-con for 48 h. (D–F) Cell proliferation was evaluated by CCK-8 assay after HL60, THP-1, and NB4 cells were treated with si-HOTAIRM1 or si-con for 24, 48, and 72 h. (G) Apoptosis of si-HOTAIRM1 or si-con-transfected HL60 and THP-1 cells was analyzed by flow cytometry analysis. *P < 0.05 vs. si-con.

Fig. 3. miR-148b overexpression suppressed cell proliferation and induced apoptosis in AML cells. (A–C) miR-148b expression was detected by qRT-PCR in HL60, THP-1, and NB4 cells transfected with miR-148b or miR-con for 48 h. (D–F) Cell proliferation was assessed by CCK-8 assay in HL60, THP-1, and NB4 cells transfected with miR-148b or miR-con for 24, 48, and 72 h. (G) Apoptosis of miR-148b- or miR-con-transfected HL60 and THP-1 cells was analyzed by flow cytometry analysis. *P < 0.05 vs. si-con.

Fig. 4. The interaction between HOTAIRM1 and miR-148b in AML cells. (A) The WT or MUT fragments of HOTAIRM1 carrying the predicted miR-148b binding sites. (B) HL60 cells were cotransfected with WT-HOTAIRM1 or MUT-HOTAIRM1 and miR-148b or miR-con for 48 h, and luciferase activity was then measured by luciferase reporter assay. (C) qRT-PCR analysis was performed to detect miR-148b expression in HL60 and THP-1 cells transfected with si-HOTAIRM1 or si-con for 48 h. *P < 0.05.

Fig. 5. Effects of HOTAIRM1 or along with miR-148b on AML cell proliferation and apoptosis. (A) qRT-PCR analysis of miR-148b expression in HL60 and THP-1 cells transfected with anti-miR-con or anti-miR-148b for 48 h. (B) qRT-PCR analysis of HOTAIRM1 expression in HL60 and THP-1 cells transfected with anti-miR-con or anti-miR-148b for 48 h. (C and D) CCK-8 assay was performed to detect cell viability in HL60 and THP-1 cells transfected with si-HOTAIRM1 + anti-miR-con, si-con + anti-miR-con, or si-HOTAIRM1 + anti-miR-148b for 48 h. (E and F) Flow cytometry analysis was performed to evaluate apoptosis of HL60 and THP-1 cells after transfection with si-HOTAIRM1 + anti-miR-con, si-con + anti-miR-con, or si-HOTAIRM1 + anti-miR-148b for 48 h. *P < 0.05.