Deregulated Expression of Circular RNAs Is Associated with Immune Evasion and Leukemia Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

Background: Circular RNAs (circRNAs) are a novel class of epigenetic regulators that participate in leukemogenesis. However, their roles in leukemia relapse after transplantation remain unclear.

Methods: We defined the circRNAs profile of the bone-marrow-enriched CD34⁺ cells from ten acute myeloid leukemia (AML) patients after transplantation (five relapse [RE] and five continuous complete remission [CR]) and four healthy controls (HCs) by RNA-seq. Differentially expressed circRNAs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) in an independent cohort of six AML patients with pairwise samples at diagnosis and at relapse and six controls.

Results: The bioinformatics analysis revealed a distinct circRNAs profile in relapse patients compared with controls (CR or HCs), while there was no significant difference between CR and HCs. Functional enrichment analysis demonstrated that mRNAs co-expressed with identified circRNAs were primarily involved in immune-related pathways, including the T cell receptor signaling pathway and lymphocyte differentiation. Moreover, we performed a protein-protein interaction network based on the immune-related genes and annotated 20 hub genes. The abnormal expression of hub genes was responsible for impairing T cell co-stimulation and activation, thus contributing to the immune escape of relapse blasts. We further constructed competing endogenous RNAs (ceRNA) regulatory networks based on immune-related genes and identified 10 key circRNAs that are associated with immune evasion. Six candidate circRNAs and their associated miRNA/mRNAs in the ceRNA network were randomly selected to be validated in another set by RT-qPCR.

Conclusions: CircRNAs dysregulation may be involved in the immune evasion of relapse blasts and is associated with AML relapse. Our results identify several promising biomarkers and might provide novel insights into the biology of AML relapse post-transplantation.

Keywords: acute myeloid leukemia; allogeneic hematopoietic stem cell transplantation; circRNA; immune evasion; relapse.

Figure 1

Schematic of RNA-seq analysis of BM-purified CD34⁺ cells from AML patients with relapse (RE), remission (CR) and healthy controls (HC).

Figure 2

CircRNA expression is altered in AML relapse (RE) compared with controls (CR or HC). (A) The Venn diagram (left) and UpSet plot (right) show that numbers of DE circRNAs among the different groups. (B,C) The heatmaps show the DE circRNAs from unsupervised hierarchical clustering present in AML patients with relapse versus remission (B) and relapse versus healthy controls (C–E). Volcano plot graphs for circRNAs expression levels in patients with relapse versus remission (D) and relapse versus healthy controls (E) Red indicates an upregulated expression and blue represents a downregulated expression.

Figure 3

Overview of the DE circRNAs in AML patients with relapse (RE) and controls (CR or HC). (A) Overlapping of identified circRNAs between circBase and our study. (B) Genomic origin of the circRNAs identified in the relapsed samples and controls. (C) The chromosome distributions of exonic circRNAs. (D) The length distributions of exonic circRNAs.

Figure 4

DE circRNAs and mRNAs co-expression networks in the immune-related pathways. (A) Co-expression network of the DE circRNAs and DE mRNAs present in the CD34⁺ cells from patients with AML relapse versus remission. (B) Co-expression network of the DE circRNAs and DE mRNAs present in the CD34⁺ cells from patients with AML relapse versus healthy controls.

Figure 5

Functional enrichment analysis for the DE mRNAs co-expressed with identified DE circRNAs. (A,B) Top10 KEGG pathways enriched in AML relapse compared with remission (A) or healthy controls. (B–D) Top10 biological processes of GO terms enriched in AML relapse compared with remission (C) or healthy controls (D).

Figure 6

The DE circRNAs identified in relapse blasts regulate immune-related mRNAs. (A,B) Heatmaps show that immune-related DE mRNAs co-expressed with DE circRNAs from unsupervised hierarchical clustering present in AML patients with relapse versus remission (A) and relapse versus healthy controls. (B,C) Protein–protein interaction analysis shows the significantly altered expression pattern in the immune-related genes regulated by DE circRNAs in relapse blasts. Edges indicate scores based on the STRING database, and node size indicates shared genes. (D) The construction of a circRNA–miRNA–mRNA ceRNA network based on the selected hub genes related to immune regulation. The ceRNA regulatory network includes 16 mRNAs, 14 miRNAs and 10 circRNAs. The red square nodes represent circRNAs, the purple hexagonal nodes indicate miRNAs and the blue circular nodes denote mRNAs.

Figure 7

Validation of DE circRNAs in AML patients with pairwise diagnosis (DX) and relapse (RE) and controls (CR or HC). The RT-qPCR detected the relative expression levels of DE circRNAs (A,B) and their associated DE miRNAs (C,D) and DE mRNAs (E,F), which were selected randomly from the ceRNA regulatory network in an independent cohort (n = 6 per group). * p < 0.05; ** p < 0.01; *** p < 0.001.