Role of AML1-ETO Related Fusion Circular RNA in the Growth of Leukemia Cells
Authors: Wang Ying , Xu Yingxi , Liu Qian , Xing Haiyan , Tian Zheng , Tang Kejing , Rao Qing , Wang Min , Wang Jianxiang 
Published: October 2019

Abstract
Introduction: The pathogenesis of acute myeloid leukemia (AML) is always associated with chromosomal translocation, such as t(8;21)(q21;q22), which results in the formation of AML1-ETOfusion gene. The AML1-ETO fusion protein leads to hematopoietic differentiation blocked and leukemogenesis. How genomic abnormities such as chromosomal translocation influence the production and biological function of noncoding RNAs is relatively less studied. With the application of high-throughput sequencing and bioinformatics, it is increasing clear that the circular RNAs (circRNA) played important role in multiple biological processes. However, circRNA is rarely studied in hematopoietic malignancies. Here we assessed the roles of AML1-ETO related fusion circRNA (F-CircAE) in AML1-ETO leukemia.
Methods: Total RNA was extracted from leukemia cell lines Kasumi-1 and SKNO-1, then treated with RNaseR or not. PCR assay with divergent primers was performed to identify F-CircAEs. The F-CircAE retroviral expression vector was constructed using Gibson Assembly Cloning Kit. NIH3T3 cells were transduced with retrovirus, the proliferation and foci formation were detected by crystal violet staining. F-CircAE-expressingNIH3T3 cells were injected subcutaneously into nude mice. Tumor growth was monitored at indicated time. To knock down the expression of F-CircAEs, the short hairpin double-stranded oligo targeting the back-splice junction of the F-CircAEs was inserted into the pLKO.1 lentiviral vector. Kasumi-1 cells were transduced with lentivirus, then the proliferation and cell cycle distribution of Kasumi-1 cells was measured by MTS assay and PI staining. Western blot was used to detect the cell cycle associated protein expression. RNA pulldown assay was performed with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the instructions. The bound proteins in the pulldown assay were analyzed by spectrum analysis. RNA-seq were performed using the Illumina platform. Sequencing reads were analyzed by a standard RNA-Seq pipeline. Gene Set Enrichment Analysis (GSEA) were used to analyze the pathway enrichment.
Results: Several F-CircAEswere identified in AML1-ETO leukemia cell lines (Kasumi-1, SKNO-1) and t(8;21) patients' bone marrow mononuclear cells (BMMCs), but not in K562 cells and donors' BMMCs (Figure 1). To further validate F-CircAEs, RNaseR treatment was conducted and amplification products of circular RNAs were still detectable in AML1-ETO positive cells (Figure 1). Subsequently, Sanger sequencing of these amplification circular products revealed the head to tail sites of F-CircAE.To examine the function of F-CircAE, overexprseeion of F-CircAE in NIH3T3 cells resulted in increased proliferation compared with mutated F-CircAE (F-CircAE-mut) overexprssion or control group (Figure 2). Meanwhile, more foci were formed in the overexpressed F-CircAE group. In vivo,F-CircAE overexpression also promoted the growth of NIH3T3 cells in the injection site of nude mice. Furthermore, silencing of two F-CircAEs reduced the growth rates of Kasumi-1 cells (Figure 3)and induced an increase in G1 phase and a decrease in S and G2/M phase compared with scramble group. The protein levels of CDK2 and CyclinD1, which were the cell cycle regulatory components at G1 boundary, were reduced in F-CircAE knockdown groups compared with scramble group. To explore the mechanism by which F-CircAE promoted leukemia cells proliferation, RNA-pulldown assay was performed and more than 70 proteins were found to bind to F-CircAE. Based on the sequencing reads of RNA-seq, GSEA revealed several pathways were enriched following F-CircAE knockdown in Kasumi-1 cells.
Conclusions: F-CircAEs were existed in t (8;21) leukemia cell lines and primary AML leukemia patients' BMMCs. F-CircAE could bind to proteins involved in the growth of leukemia cells. Discovery of F-CircAEs in AML1-ETO leukemia could make an immense progress in understanding their pathogenic mechanism, indicating new marker for diagnosis and therapy target.

Disclosures
No relevant conflicts of interest to declare.