Study On The Mechanism Of CircMTOR In Imatinib Resistance Of Chronic Myeloid Leukemia

BackgroundChronic Myelogenous Leukemia(CML)is a clonal proliferative disease of bone marrow caused by T(9;22)(q34;Q11).The BCR-ABL1 fusion gene produced by translocation has high tyrosine kinase activity,which hinders the abnormal proliferation and apoptosis of tumor cells?The application of tyrosine kinase inhibitors(TKI)represented by imatinib(IM)has greatly improved the long-term survival of Chronic Myeloid Leukemia(CML),but some patients develop TKI resistance during the treatment of IM,which is still an important reason for the failure of TKI treatment.It has recently been discovered that circRNAs may be involved in CML resistance to imatinib by regulating the expression of oncogenes.In this study,high-throughput sequencing technology was used to screen the expression profiles of differentially expressed circRNAs between the imatinib-sensitive and imatinib-resistant groups,and to explore the specific molecular regulation mechanism of circMTOR in CML drug resistance.MethodsA total of five imatinib-sensitive CML patients and five imatinib-resistant CML patients without ABL mutation were recruited in this study.The differentially circRNA expression was identified in peripheral blood mononuclear cell(PBMC)by using RNA-seq and validated by RT-PCR.Bioinformatic analysis was furtherly conducted including circRNA-miRNA network,Gene Ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.Dual luciferase was used to confirm target relationship of the identified miRNA and circRNA.The function studies of identified cicrRNA were performed in CML cell line(K562)and imatinib resistance CML cell line(K562/IM)by using siRNA,MTS assay,Real-Time PCR and flow cytometry.ResultRNA-seq analysis revealed that 1852 circRNAs were differentially expressed in imatinib-resistant CML patients(800 upregulated and 1052 downregulated)compared to drug-sensitive CML patients.Quantitative reverse transcription PCR showed that hsa_circ₀1 10442(circMTOR)was the most significantly upregulated in both patients PBMC and K562R cells.hsa_circ₀110442 derived from Exon20 and Exon23 of mTOR gene,termed here as circMTOR.We constructed a competitive endogenous RNA(ceRNA)regulatory network mediated by circMTOR through bioinformatics.KEGG analysis revealed that it is associated with PD-L1,ErbB and VEGF cancer-related pathways,and GO analysis indicated that most of its target genes are involved in biological processes.Bioinformatics analysis predicted circMTOR acts as a binding target of miR-34a and c-Myc may be the target gene of miR-34a.Silencing of circMTOR significantly inhibited the proliferation and arrested cell cycle of k562 cells by targeting miR-34a and c-Myc may be the target gene of miR-34a.By silencing circMTOR with siRNA,miR-34a expression level was up-regulated,apoptosis rate of K562R cells was increased,and susceptibility to imatinib was enhanced.Conclusions1)Using high-throughput sequencing technology,1852 differentially expressed circRNAs were identified in PBMC cells between imatinib sensitive and resistance patients.Bioinformatics analysis showed that circRNAs may be involved in various signaling pathways to regulate imatinib resistance in CML.2)qRT-PCR verified that hsa_circ₀110442(circMTOR)was the most significantly up-regulated circRNA in K562R cells;Knocking down of circMTOR by siRNA could significantly inhibit proliferation and induce apoptosis of K562R cells,thus facilitate the sensitivity of CML cells to imatinib.3)Dual luciferase reporter gene assay and function rescue assay were perforemd to demonstrate that circMTOR acted as a ceRNA by sponging miR-34a,and regulated the sensitivity of K562R cells to imatinib;4)Western Blot assay showed that miR-34a affected the proliferation and apoptosis of K562R cells by regulating the expression of target gene c-myc.5)Abnormal activation of the circRNAmTOR/miR-34a/c-Myc axis may be one of the mechanisms of imatinib resistance independent of BCR-ABL1.