The Expression And Biological Function Of Circ₀093335 In Acute Myeloid Leukemia

Objective:Acute myeloid leukemia?AML?is a clinical and molecular heterogeneous disease,which is mainly treated with high-dose chemotherapy.Cytogenetic abnormalities and chemotherapy resistance lead to the high recurrence rates.Therefore,it is urgent to determine new targets to reduce recurrence and improve patient survival.This study was to detect the transcript level of circ₀093335 in the bone marrow of AML patients,and to conduct cell function experiments to explore the biological function of circ₀093335 in the occurrence and development of AML.Methods:1.The transcript levels of circ₀093335 in bone marrow samples from 125 patients with initial AML and 17 healthy controls were detected by real-time quantitative PCR?RQ-PCR?,and clinical relevance was analyzed.2.HL60 cell line transfected with circ₀093335 was sorted by flow cytometry to construct stably transfected NC-HL60 and 93335-HL60 cells,and the transfection efficiency was detected by RQ-PCR.3.Cell proliferation,apoptosis and chemotherapeutic drug sensitivity were carried out by CCK8 and flow cytometry to delineate the function of circ₀093335 in HL60 cells.4.The bioinformatics website predicts targeted miRNAs for circ₀093335 and further searches for downstream target genes.5.The levels of miR-338-5p,miR-153-5p and ID4 transcripts in circ₀093335 transfected cells were detected by RQ-PCR.The protein expression of ID4 in circ₀093335 transfected cells was detected by western blot.6.Analysis of the correlation between circ₀093335 and ID4 in AML clinical samples.Result:1.Compared with the control,circ₀093335 was down-regulated in AML patients?P=0.001?.Receiver operating characteristic?ROC?curves show that circ₀093335 transcript levels are able to distinguish between AML and healthy individuals?AUC = 0.736?.In the Non-APL AML patients,the low expression group had a higher white blood cell count compare with high expression group?median 36.2 × 109 / L and 17.35 × 109 / L,P = 0.024?.In total AML andNon-APL AML,the low expression rate of circ₀093335 was significantly higher in male patients than in female patients?P = 0.003 and P = 0.013?.In addition,the low expression group had lower platelet counts than the high expression group?P = 0.007 and P = 0.007?.2.The stably transfected 93335-HL60 cells were detected by RQ-PCR,and the expression of circ₀093335 was significantly higher than that of NC-HL60?P<0.01?.3.The growth rate of 93335-HL60 cells was significantly slower than that of NC-HL60,and the amplification ability was also slower than that of NC-HL60 in serum-free environment.The inhibition rate of daunorubicin and homoharringtonine on HL60 cells with circ₀093335overexpression was increased.The apoptosis rates of NC-HL60 and 93335-HL60 cells were not significantly different between the two groups,but the apoptosis rates of 93335-HL60 cells were increased after the treatment with homoharringtonine?P<0.05?.4.The bioinformatics website predicts that the targeted miRNAs of circ₀093335 are miR-338-5p and miR-153-5p,and further search for miR-338-5p and miR-153-5p downstream target genes,and consult the literature to find out gene associated with AML: ID4.5.The expression of miR-338-5p and miR-153-5p was down-regulated in HL60 cells overexpressing circ₀093335?P<0.05?,and the expression of ID4 was up-regulated?P<0.05?.Western blot results showed that the protein level of ID4 was also up-regulated.6.In the AML clinical samples,the expression of circ₀093335 and ID4 was positively correlated?P<0.055,r=0.32?.Conclusions:Circ₀093335 low expression is a common molecular event in AML,and the transcript level of circ₀093335 can be used as a potential marker to distinguish between total AML and Non-APL AML subtypes and healthy controls,which has the significance of auxiliary diagnostic markers.Circ₀093335 inhibits the proliferation of HL60 cells,increaseing the sensitivity of HL60 cells to daunorubicin and homoharringtonine,and promotes the apoptosis induced by homoharringtonine.Circ₀093335 may act as spongs for miR-338-5p and miR-153-5p,and then promote the expression of ID4 to regulate the function of AML cells and the development of AML.