We reanalyzed the differentially expressed circRNAs in AML patients with poor or favorable risk status compared to controls by applying the sequence data of cytogenetically normal AML (CN-AML) patients and healthy controls from our previous circRNA microarray platform study dataset (GEO number: GSE94591) [6]. Based on the fold changes and P values of differential expression, the 10 novel circRNAs notated as “AML risk-status related” has been detected, and 3 circRNAs (hsa_circ_0001187, hsa_circ_0011929, and hsa_circ_0000973) satisfied the condition that they had higher expression in cells (Fig. 1A-E, Supplemental Fig. 1A). We further validated their expression in large-scale samples and found that only the expression of Circ_0001187 was significantly decreased in ND AML patients and increased in HCR patients compared with controls, while no significant change was found for the other two circRNAs (Fig. 1F, Supplemental Fig. 1C-D). Moreover, in R/R patients, Circ_0001187 expression was downregulated. To further explore the effect of chemotherapy on Circ_0001187, we followed seven AML patients who received standard chemotherapy until they achieved HCR. We found that the expression level of Circ_0001187 in the HCR stage was significantly increased compared to that in the ND stage (Fig. 1G), indicating that it has potential diagnostic value and antileukemic activity against AML.

Decreased Circ_0001187 in AML correlates with poor prognosis and characteristics of Circ_0001187 in AML cells. A-D Volcano plots of differentially expressed circRNAs between AML patients (favorable risk and poor risk) and healthy controls. E Heatmap of differentially expressed circRNAs between AML patients and healthy controls (blue = down-regulated, red = up-regulated). Fold change was listed above. F The expressions of Circ_0001187 in AML patients and healthy controls. G Circ_0001187 mRNA expression was measured in paired samples from five follow-up AML patients at ND and HCR stages. Data were analyzed using Wilcoxon test. H qRT-PCR analysis of Circ_0001187 expression in AML patients with favorable risk and poor risk. I Kaplan–Meier analysis of the overall survival time of AML patients with different circRNAs expression as indicated by Log-Rank test (n = 40). J-N The results of correlation between the expression level of Circ_0001187 and white blood cell, hemoglobin, platelets, BM% and PB% in ND AML patients. Data were analyzed using Pearson correlation. O Relative expression of Circ_0001187 in age was measured by qRT-PCR. P Diagnostic values of Circ_0001187 in AML patients. AUC: Area under the ROC curve. Q The sequence length of spliced mature Circ_0001187 derived from the linear DOPEY2 mRNA is 301 bp. And the results of Sanger sequencing from the head-to-tail splicing in the RT-PCR product of Circ_0001187. R Expression levels of Circ_0001187 and linear DOPEY2 mRNA in THP-1 AML cells treated with actinomycin D by qRT-PCR. S The qRT-PCR expression results of Circ_0001187 and DOPEY2 using random primer and Oligo dT primer in THP-1 AML cells. T Theagarose gel electrophoresis results of Circ_0001187 PCR products of cDNA or gDNA using convergent and divergent primers in THP-1 and Molm-13 AML cells. U RNA FISH analysis for Circ_0001187 in THP-1 and Molm-13 AML cells. Scale bars = 10 μm. *p