| Objective Long non-coding RNA is a pivotal modulator in carcinogenesis.Numerious researches have reported that lncRNA H19 plays a crucial role in tumorigenesis,but its expression status and clinical significance in hematologic diseases remain to be elucidated.The study is aimed to detect H19 expression level in acute myeloid leukemia(AML),chronic myeloid leukemia(CML)and myelodysplastic syndrome(MDS)and to explore its clinical significance and potential pathogenic mechanisms.Methods The expression of H19 mRNA in bone marrow mononuclear cells of 36 healthy donors,161 AML patients,78 CML patients and 43 MDS patients was detected by real-time quantitative PCR and the clinical significance was analyzed.Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were carried out to measure the methylation status of H19 in three myeloid malignances.Leukemic cell lines(THP-1 and K562)were treated with demethylation agent to construct a dosing cell model to evaluate whether abnormal expression of H19 resulting from H19 hypermethylation.Then H19 expression in K562 was silenced by siRNA.Cell counting and flow cytometry were applied to detect cell proliferation and apoptosis.Literatures and bioinformatic websites were used to predict downstream target genes.Results The reduced H19 expression was identified in AML,CML and MDS patients as compared with healthy donors.H19 could affect the proliferation and apoptosis of leukemia cells by regulating the downstream target gene ID2.(1)H19 expression in AML patientsThe transcript level of H19 was significantly upregulated in AML patients compared with controls(P=0.003).H19 high patients had significantly older age(P=0.009)and more white blood cells(WBC)(P=0.004).H19 high patients occurred with a lower frequency in favorable karyotype(P=0.013)and a higher frequency in intermediate karyotype(P=0.002).Moreover,H19 high patients had a lower incidence in t(15;17)subtype(P=0.008)and a higher incidence in normal karyotype(P=0.017).In addition,H19 high expression was associated with FLT3-ITD and DNMT3 A mutations(P=0.053 and 0.025).H19 high patients had significantly lower complete remission(CR)rate and shorter overall survival(OS)than H19 low patients(P=0.039 and 0.020,respectively).Moreover,H19 high patients tended to have a shorter leukemia-free survival than H19 low patients in cytogenetically normal AML(P=0.072).Cox regression multivariate analyses demonstrated that H19 overexpression was an independent prognostic factor in non-APL-AML(P=0.063).Bioinformatics analysis using TCGA databases and GEP databases confirmed that H19 high group had shorter OS than H19 low group.Dynamic monitoring of H19 expression in AML patients revealed that H19 expression after CR was lower to the diagnosis time,and increased again after relapse(P<0.001).Furthermore,H19 expression was not associated with H19 methylation in AML patients.However,H19 expression and H19 unmethylation levels were both markedly elevated after treatment with 5-aza-dC in cell line THP-1,indicating that H19 expression in AML might be partially regulated by its methylation.In addition,the expression level of miR-675,encoded by the first exon of H19,was not correlated with H19 expression in AML.(2)H19 expression in CML patientsThe relative abundances of H19 were significantly upregulated in CML patients as compared with controls(P<0.001).Moreover,H19 expression level in blast crisis stage(BC)was higher than in chronic phase stage(CP),acute phase stage(AP)and CP/AP stage(P=0.074,0.115 and 0.061,respectively).Notably,patients with high H19 expression had a tendency of higher WBC and BCR-ABL1 transcript than those with low H19 expression(P=0.088 and 0.086,respectively).Moreover,in five patients with paired samples at diffierent clinical stages,H19 expression levels were significantly up-regulated at AP/BC compared to CP,indicating that H19 expression might be involved in disease progression.Meanwhile,H19 expression was decreased in follow-up patients who achieved complete molecular remission after TKI-based therapy(P<0.001).The analysis of clinical data suggested that H19 expression was associated with H19 methylation in CML patients(R=0.259,P=0.042).Meanwhile,H19 expression and H19 unmethylation levels were significantly increased after demethylating reagent treatment in leukemic cell line K562,indicating that H19 expression in CML might be regulated by its methylation.(3)H19 expression in MDS patientsH19 expression was also significantly up-regulated in MDS patients in comparison to healthy controls(P<0.001).However,H19 expression was not associated with clinical parameters.Kaplan-Meier analysis revealed that H19 high patients had a shorter OS than H19 low patients although there was no statistical difference(P=0.184).Furthermore,multivariate analysis failed to demonstrate the prognostic value of H19 expression in MDS patients(P>0.05).(4)Biological function of H19 in vitroAfter silencing H19 in K562,the proliferation rate of experimental group was slower than that in control group(P=0.002).Total apoptotic rate was significantly higher in experimental group than in control group(P=0.014).In addition,there was no significant difference in the median lethal concentration of demethylated drugs between the two groups(P=0.203).Furthermore,the expression of ID2,a potential target of H19,was downregulated in experimental group,and H19 expression in AML samples was positively correlated with ID2 expression(R=0.262,P=0.002).Conclusions H19 overexpression was a frequent event in myeloid malignancies and the expression level of miR-675 and H19 did not correlate in AML.H19 overexpression was associated with adverse prognosis in AML and might act as a vital biomarker in monitoring AML relapse and CML progression.Silencing H19 could downregulate ID2 expression,reduce cell proliferation and induce apoptosis,suggesting that H19/ID2 abnormalities might be involved in leukomogenesis in myeloid malignancies. |
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