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. 2013 Oct;114(10):2375-83.
doi: 10.1002/jcb.24586.

HOX antisense lincRNA HOXA-AS2 is an apoptosis repressor in all trans retinoic acid treated NB4 promyelocytic leukemia cells

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HOX antisense lincRNA HOXA-AS2 is an apoptosis repressor in all trans retinoic acid treated NB4 promyelocytic leukemia cells

Hang Zhao et al. J Cell Biochem. 2013 Oct.

Abstract

HOXA cluster antisense RNA 2 (HOXA-AS2) is a long non-coding RNA located between the HOXA3 and HOXA4 genes in the HOXA cluster. Its transcript is expressed in NB4 promyelocytic leukemia cells and human peripheral blood neutrophils, and expression is increased in NB4 cells treated with all trans retinoic acid (ATRA). Knockdown of HOXA-AS2 expression by transduced shRNA decreases the number of viable cells and increases the proportion of apoptotic cells, measured by annexin V binding and by activity and cleavage of caspases-3, -8, and -9. The increase in death of HOXA-AS2 knockdown cells was accompanied by an elevated TNF-related apoptosis-inducing ligand (TRAIL) levels, but ATRA-induced NB4 cells treated with TRAIL did show an increase in HOXA-AS2 expression. These results demonstrate that ATRA induction of HOXA-AS2 suppresses ATRA-induced apoptosis, possibly through a TRAIL-mediated pathway. HOXA-AS2-mediated negative regulation thus contributes to the fine-tuning of apoptosis during ATRA-induced myeloid differentiation in NB4 cells.

Keywords: APOPTOSIS; Hox GENE CLUSTER; LONG INTERGENIC NON-CODING RNA (lincRNA); MYELOID; NON-CODING RNA.

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Figures

Figure 1
Figure 1
ATRA induction of HOXA-AS2 expression in NB4 cells. (A) lower panel: UCSC Genome Browser map of expressed sequences in the region between HOXA3 and HOXA4 in the HOXA cluster on chromosome 7. Upper panel: RNA-Seq signal from this region in RNA from NB4 cells with and without ATRA induction (1 μM for 5 days) and normal peripheral blood polymorphonuclear leukocytes (PMN). (B) HOXA-AS2 transcripts measured by qRT-PCR in NB4, NB4R2 (ATRA-resistant) or K562 cells were incubated with ATRA (1 μM) for the indicated number of days. Point and error bars represent the mean ± SD of four independent experiments.
Figure 2
Figure 2
Effect of IFNγ and TNFα on HOXA-AS2 gene expression in PMN. Relative expression of (A) HOXA-AS2 and (B) TRAIL in normal PMN incubated with or without IFN-γ (1000U/ml), TNF-α (1000U/ml), or IFN-γ plus TNF-α for 30 min or 2 hours. Bars and error bars represent the mean ± SD of three measurements in a representative experiment, of three independent experiments with PMN from two donors. Horizontal bars with asterisks indicate comparisons that reached statistical significance at p<0.05.
Figure 3
Figure 3
Effect of HOXA-AS2 knockdown on NB4 cell proliferation. (A) HOXA-AS2 gene expression, measured by qRT-PCR, in NB4 cells transfected with three HOXA-AS2 shRNAs. (B,C) NB4 cells transfected with the indicated constructs, with or without ATRA induction, as represented by the colors and symbols in the insert, were seeded at 2 × 105/mL and cell number (B) and viability (C) determined at the indicated numbers of days in culture. Points and error bars represent the mean ± SD of three independent experiments.
Figure 4
Figure 4
Effect of HOXA-AS2 knockdown on NB4 cell apoptosis. Annexin V staining of NB4 cells transfected with scramble shRNA or the indicated HOXA-AS2 targeting shRNAs, incubated with or without ATRA (1 μM for 3 days) as indicated. (A) Scatter plots; (B) Bar plot, with bars and error brackets representing mean ± SD of four independent experiments; p<0.001 for differences between scramble and knockdown clones A and C. Caspase-3 activity was measured (C) colorimetrically with substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide, bars and error brackets representing mean ± SD of four independent experiments, p<0.001 for differences between scramble and knockdown clones A and C; and (D) by western blot detection of cleaved caspase-3 protein; the blot is representative of three experiments.
Figure 5
Figure 5
Effect of HOXA-AS2 on ATRA-induced extrinsic apoptosis pathways. (A) Caspase-8 and (B) caspase-9 activity, measured respectively as acetyl-Ile–Glu–Thr–Asp–p-nitroanilide-cleaving and acetyl-Leu-Glu-His-Asp-pnitroanilide-cleaving activity in NB4 cells with or without treatment with ATRA (1 μM for 4 days). Bars and error brackets represent mean ± SD of four independent experiments. Horizontal bars with asterisks indicate comparisons that reached statistical significance at p<0.05. (C) Western blot of cleaved caspases-8 and -9, Bax, and β-actin in the indicated cell lysates; the blot is representative of three experiments.
Figure 6
Figure 6
Effect of HOXA-AS2 on TRAIL expression and function. NB4 cells transfected with the indicated knockdown constructs were cultured with or without ATRA (1 μM for 3 days). (A) Western blot of TRAIL and β-actin in the indicated cell lysates; blot representative of three experiments. (B) TRAIL transcripts measured by qRT-PCR. Bars and error brackets represent mean ± SD of three independent experiments; p<0.01 for difference between scramble and knockdown clone A, and p<0.01 for difference between scramble and knockdown clone C. (C) Western blot of cleaved caspases-8 and 9, and β-actin, in NB4 cell lysates with or without ATRA (1 μM for 3 days) and with anti-TRAIL or isotype control antibodies as indicated; the blot is representative of three experiments.
Figure 7
Figure 7
Effect of TRAIL on HOXA-AS2 expression. NB4 cells were incubated with ATRA (1 μM) or with the indicated concentrations of recombinant human TRAIL for 2 days. (A) HOXA-AS2 expression was analyzed by qRT-PCR. Bars and error brackets represent mean ± SD of three independent experiments. (B) Western blot analysis of cleaved caspase-8 and β-actin protein; the blot is representative of three experiments.

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