H19 overexpression promotes leukemogenesis and predicts unfavorable prognosis in acute myeloid leukemia

Abstract

Background: The long non-coding RNA H19 plays a crucial role in solid tumor initiation and progression. However, the potential role of H19 and its clinical significance in acute myeloid leukemia (AML) remain largely elusive.

Methods: H19 expression was detected by qPCR, and clinical significance in AML patients was further analyzed. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data for AML were used as validation cohorts. The roles of H19 in cell proliferation and apoptosis were determined by cell proliferation assay and flow cytometry analysis.

Results: H19 expression was significantly increased in AML patients but not associated with embedded miR-675 expression. Moreover, H19 overexpression was not dependent on the methylation pattern in H19 differentially methylated region/imprinting control region. Strong association was observed between H19 overexpression and patients' characteristics including sex, higher white blood cells, older age, and intermediate karyotype, FLT3-ITD, and DNMT3A mutations. In addition, H19 overexpression correlated with lower complete remission (CR) rate and shorter overall survival, and further confirmed by multivariate analyses. Importantly, the prognostic effect of H19 expression was validated by TCGA and GEO data. In the follow-up of patients, H19 expression in CR phase was lower than diagnosis time and returned at relapse time. Loss-of-function experiments showed that H19 exhibited anti-proliferative and pro-apoptotic effects in leukemic cell HL60. Furthermore, H19 expression was positively correlated with potential downstream gene ID2 in AML.

Conclusions: Our findings revealed that methylation-independent H19 was a prognostic and predictive biomarker in AML, and H19/ID2 played crucial roles in leukemogenesis with potential therapeutic target value.

Keywords: AML; H19; ID2; Prognosis; Surveillance.

Fig. 1

H19 expression and methylation in AML. a H19 expression level detected by real-time quantitative PCR in controls and AML patients. b H19 unmethylation level detected by real-time quantitative unmethylation-specific PCR in controls and AML patients. c, d H19 methylation density detected by bisulfite sequencing in controls and AML patients, respectively. White cycle, unmethylated CpG dinucleotide; black cycle, methylated CpG dinucleotide. e The correlations between H19 expression and unmethylation. f Relationship between H19 expression and methylation of two different regions using The Cancer Genome Atlas (TCGA) data. Left: H19 expression, value in log2(x + 1) transformation, x is the RSEM value. Middle: H19 methylation in HM27K. Right: H19 methylation in HM450K. Red color: higher expression/methylation. Blue color: lower expression/methylation. White color: intermediate level of H19 expression/methylation. Gray color: no data. Each row represents H19 expression and H19 methylation level of the same patient; each line from right to left in middle and right figures represents the methylation level at different sites

Fig. 2

The impact of H19 expression on survival in AML. a Overall survival (OS) among whole-cohort AML patients. b OS among non-APL-AML patients. c OS among cytogenetically normal AML (CN-AML) patients. d Leukemia-free survival (LFS) among whole-cohort AML patients. e LFS among non-APL-AML patients. f LFS among CN-AML patients

Fig. 3

Prognostic value of H19 expression on overall survival in AML using TCGA and GEO data. a–c The impact of H19 expression on overall survival (OS) in a cohort of 200 AML patients from The Cancer Genome Atlas (TCGA) databases. The patients were classified into H19 low-expressed and high-expressed groups by the median level of ID4 expression. a OS among whole-cohort AML. b OS among non-APL-AML. c OS among cytogenetically normal AML (CN-AML). d–g The impact of H19 expression on OS in two independent cohorts of 78 and 162 CN-AML patients were obtained from Gene Expression Omnibus (GEO) data. Survival analysis was performed through the online web tool Genomicscape. d Probe 224646_at among a cohort of 78 CN-AML patients. e Probe 224997_at among a cohort of 78 CN-AML patients. f Probe 224646_at a cohort of 162 CN-AML patients. g Probe 224997_at among a cohort of 162 CN-AML patients

Fig. 4

H19 expression in the surveillance of AML. a H19 expression in different clinical stages (newly diagnosis, complete remission, and relapse time) of AML patients. b Dynamic change of H19 expression in the follow-up of seven paired AML patients during newly diagnosis, complete remission, to relapse time

Fig. 5

The biological role of H19 on leukemic cell line HL60. a The underlying role of H19 in leukemogenesis determined by Coremine Medical online database (http://www.coremine.com/medical/). b H19 expression in eight common leukemic cell lines. The dotted line indicated the cutoff value to define H19 overexpression. c H19 expression mediated by siRNA-based H19 knockdown. H19 expression was significantly downregulated in siRNA-based H19 knockdown (siH19-1 and siH19-2) and control (siNC) groups. d The effect of H19 knockdown on cell proliferation. The siH19 groups (siH19-1 and siH19-2) showed significantly lower proliferation capacity than the siNC group at 48 and 72 h. e The effect of H19 knockdown on cell apoptosis. The siH19 groups (siH19-1 and siH19-2) showed significantly higher apoptosis rate than the siNC group at 48 h. f–h Flow-type apoptosis figures for siNC, siH19-1, and siH19-2, respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Relationship between H19 and ID2 in AML. a ID2 mRNA expression in after siRNA-based H19 knockdown in HL60 cell line. ID2 mRNA expression was significantly downregulated after siRNA-based H19 knockdown (siH19-1 and siH19-2) and control (siNC) groups. **P < 0.01, ***P < 0.001. b Correlation between H19 and ID2 expressions in AML patients. Correlation analysis was performed by Spearman test