Long non-coding RNA HOXB-AS3 promotes myeloid cell proliferation and its higher expression is an adverse prognostic marker in patients with acute myeloid leukemia and myelodysplastic syndrome

Abstract

Background: Long non-coding RNAs (lncRNAs) represent the majority of cellular transcripts and play pivotal roles in hematopoiesis. However, their clinical relevance in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains largely unknown. Here, we investigated the functions of HOXB-AS3, a lncRNA located at human HOXB cluster, in the myeloid cells, and analyzed the prognostic significances in patients with AML and MDS.

Methods: shRNAs were used to downregulate HOXB-AS3 in the cell lines and the effect was evaluated by quantitative polymerase chain reaction. The proliferation of the cell lines was illustrated by proliferation and BrdU flow assays. Further, we retrospectively analyzed the HOXB-AS3 expression in 193 patients with AML and 157 with MDS by microarray analysis, and evaluated its clinical importance.

Results: Downregulation of HOXB-AS3 suppressed cell proliferation. Mechanistically, HOXB-AS3 potentiated the expressions of several key factors in cell cycle progression and DNA replication without affecting the expressions of HOX genes. In AML, patients with higher HOXB-AS3 expression had shorter survival than those with lower HOXB-AS3 expression (median overall survival (OS), 17.7 months versus not reached, P < 0.0001; median relapse-free survival, 12.9 months versus not reached, P = 0.0070). In MDS, patients with higher HOXB-AS3 expression also had adverse prognosis compared with those with lower HOXB-AS3 expression (median OS, 14.6 months versus 42.4 months, P = 0.0018). The prognostic significance of HOXB-AS3 expression was validated in the TCGA AML cohort and another MDS cohort from our institute. The subgroup analyses in MDS patients showed that higher HOXB-AS3 expressions could predict poor prognosis only in lower-risk (median OS, 29.2 months versus 77.3 months, P = 0.0194), but not higher-risk group.

Conclusions: This study uncovers a promoting role of HOXB-AS3 in myeloid malignancies and identifies the prognostic value of HOXB-AS3 expression in AML and MDS patients, particularly in the lower-risk group.

Keywords: Acute myeloid leukemia; HOXB-AS3; Myelodysplastic syndrome.

Fig. 1

Identification of HOXB-AS3 as a prognostic biomarker and overview of its locus in human HOXB. a The numbers of genes influenced overall survival in TCGA AML cohort and NTUH AML cohort. b The neighborhood of HOXB-AS3 locus in human HOXB cluster. The genomic data from ENCODE project was taken from UCSC genome browser. The exon localizations of eight HOXB-AS3 isoforms and HOXB5, HOXB6, and HOXB7 genes are shown. Arrows indicate the transcription direction of these genes. The NCBI Reference Sequence (RefSeq) numbers were labeled in the end of each variant. c PhyloCSF analysis for predicting the noncoding nature of HOXB-AS3. Exon 1, exon 4, exon 5 and exon 6 are zoom-in to see the details of PhyloCSF scores of the specific exons. All PhyloCSF scores are negative in the exons of HOXB-AS3

Fig. 2

HOXB-AS3 promotes cell proliferation in myeloid cells. a Proliferation assay of OCI/AML3 cells infected with lentivirus carrying pLKO-vector (control), shHOXB-AS3#1, or shHOXB-AS3#2. The infected cells were sorted by the expression of GFP. The sorted GFP-positive cells were seeded with a number of 1 × 10⁶ in 5 mL culture medium on Day 0. Cell number was counted for 6 days. P values were calculated by Kruskal-Wallis test. b Quantitative PCR analysis of HOXB-AS3 relative to β2 microglobulin expression in OCI/AML3 cells stably carrying pLKO-vector (control), shHOXB-AS3#1, or shHOXB-AS3#2 by lentivirus infection. c The percentage of OCI/AML3 cells in the S phase of cell cycle. The result was derived from triple repeats. P values were calculated by Kruskal-Wallis test. d Quantitative PCR analysis of HOXB-AS3 relative to β2 microglobulin expression in TF-1 cells stably carrying pLKO-vector (control) or shHOXB-AS3#1 by lentivirus infection. e The percentage of TF-1 cells in the S phase of cell cycle. The result was derived from triple repeats. P values were calculated by Student t test. f Quantitative PCR analysis of HOXB-AS3 relative to β2 microglobulin expression in TF-1 cells infected with lentivirus carrying pLAS5w.Pbsd vector (control) or pLAS5w.Pbsd-HOXB-AS3 (HOXB-AS3 overexpression). g The percentage of cells in the S phase of cell cycle. The result was derived from the triple repeats. P values were calculated by Student t test. (* meant that P value was less than 0.05)

Fig. 3

HOXB-AS3 regulates genes involving in cell cycle progression and DNA replication instead of HOX clusters. a The expressions of HOX genes were not influenced by HOXB-AS3 expression. RNA was purified from OCI/AML3 infected by lentivirus carrying pLKO-shLacZ (control#1), pLKO-vector (control#2), pLKO-shHOXB-AS3#1 or pLKO-shHOXB-AS3#2. b Differentially expressed genes between the control cells and HOXB-AS3 knockdown cells were revealed by microarray analysis. c GSEA analysis of the differentially expressed genes in HOXB-AS3 knockdown cells compared to the control cells. d Quantitative PCR analysis of the expressions of indicated genes in OCI/AML3 cells infected with lentivirus carrying pLKO-vector (control), shHOXB-AS3#1, or shHOXB-AS3#2. Data were derived from the triple repeats of experiments. P values were calculated by Kruskal-Wallis test. e Quantitative real time PCR analysis of RNA18S5 RNA (18 s rRNA), HOXB-AS3, MALAT1, NEAT1 and GAPDH in the nuclear and cytoplasmic fractions of OCI/AML3 cells. (* meant that P value was less than 0.05)

Fig. 4

Survival analysis of de novo AML patients stratified by the expressions of HOXB-AS3. a Overall survival (OS) in the NTUH AML cohort. b Relapse free survival in the NTUH AML cohort. c OS in the AML patients with intermediate-risk cytogenetic changes. d OS in the TCGA AML cohort. The patients in the NTUH AML cohort were stratified by the expressions of transcript cluster: TC17002858.hg.1 on Affymetrix GeneChip® HTA 2.0 arrays

Fig. 5

Overall survival of MDS patients stratified by the expressions of HOXB-AS3. a OS in the NTUH MDS training cohort stratified into 4 groups: HOXB-AS3 expression highest, intermediate high, intermediate low and lowest groups. b OS in the NTUH MDS training cohort stratified into 2 groups: HOXB-AS3 expression higher group (HOXB-AS3 expression highest group in Fig. 5a) and lower group (HOXB-AS3 expression lowest, intermediate low, and intermediate high groups in Fig. 5a). c OS in the NTUH MDS validation cohort stratified into 2 groups as Fig. 5b. The patients were stratified by the expressions of transcript cluster: TC17002858.hg.1 on Affymetrix GeneChip® HTA 2.0 arrays

Fig. 6

OS of MDS patients stratified by the expressions of HOXB-AS3 in different IPSS risk groups. a OS in the patients with IPSS low or intermediate-1 risk, stratified by HOXB-AS3 expressions. b OS in the patients with IPSS intermediate-2 or high risk, stratified by HOXB-AS3 expressions. The patients were stratified by the expressions of transcript cluster: TC17002858.hg.1 on Affymetrix GeneChip® HTA 2.0 arrays into 2 groups as Fig. 5b