The Expression,cell Function And Mechanism Of Circ＿0093335 In Acute Myeloid Leukemia

Objective:Circular RNA(circular RNA,circ RNA)plays an important role in tumorigenesis and development.BMI1 is widely used in tumor research.BMI1 has been confirmed to be overexpressed in AML and is an independent risk factor affecting the overall survival time of patients with acute myeloid leukemia(AML).The clinical significance of circular RNA BMI1(circ-BMI1,circ 0093335)in AML is unclear.In this paper,the expression level of circ0093335 in bone marrow mononuclear cells(BMMNCs)of AML patients and controls was detected,differentially expressed circ 0093335 was used for cell function and animal experiments,and preliminary mechanism studies were conducted to explore the clinical significance and potential causes of circ 0093335.Methods:1.The expression levels of circ 0093335 in bone marrow mononuclear cells of 130 cases of newly diagnosed AML patients and 22 controls were detected by real-time quantitative PCR(q PCR),and clinical significance was analyzed.2.Overexpressing circ 0093335 lentiviral vector and mock vector were constructed to infect AML cell lines Thp1,HEL,and the effect of circ 0093335 differential expression on AML cell line proliferation,apoptosis,and chemotherapeutic drug sensitivity experiments were analyzed.3.circ＿0093335-Thp1 cells,NC-Thp1 cells or PBS were respectively used to inject NCG mice into the tail vein to construct a mouse model of leukemia to explore the effects of circ＿0093335 on the occurrence and development of AML in animals.4.The circ＿0093335/miR-338-5p/ID4 signaling pathway was predicted by bioinformatics analysis.Luciferase experiments and RNA immunoprecipitation(RNA immunoprecipitation,RIP)experiments were performed to verify the circ＿0093335/miR-338-5p/ID4 signaling pathway.5.Western blot technology was used to detect the protein expression of ID4 in circ0093335-Thp1 and NC-Thp1 cells.Results:1.Compared with the control group,circ 0093335 was significantly down-regulated in AML(P = 0.0070).The Receiver Operating Characteristic(ROC)curve showed that the expression level of circ＿0093335 could be used as a potential marker for distinguishing patients with total AML,Non-APL-AML and CN-AML subtypes from the control group,which had auxiliary diagnostic significance.2.The expression level of circ 0093335 in circ 0093335-AML cells detected by q PCR was significantly higher(P < 0.01).The growth rate of circ 0093335-AML cells decreased significantly(P < 0.05).The sensitivity of Thp1 cells to daunorubicin and homoharringtonine was enhanced by circ＿0093335(P < 0.05).The apoptotic rate of circ 0093335-AML and NC-AML cells was not statistically different(P > 0.05).3.circ＿0093335-Thp1 and NC-Thp1 caused leukemia symptoms such as hemorrhage,lower limb paralysis,lymph node and hepatosplenomegaly,and ovarian enlargement in mice.Thp1 blasts could be seen in peripheral blood and bone marrow smears,suggesting that the leukemia mouse model was successfully constructed.There was no significant difference between NC-Thp1 group and circ＿0093335-Thp1 group in body weight and bone marrow phenotype flow cytometry(P > 0.05).Compared with NC-Thp1 group,the blast cells in the bone marrow smear of mice in the circ＿0093335-Thp1 group had a decreasing trend.Ovarian volume of mice in the circ＿0093335-Thp1 group had a decreasing trend(P = 0.1386).Proportion of ID4 positive cells in the liver,ovary and spleen tissues of mice in the circ＿0093335-Thp1 group was significantly higher than that in NC-Thp1 group(P < 0.05).Peripheral blood white blood cell count of mice in the circ＿0093335-Thp1 group had a downward trend(P = 0.0814).4.The luciferase test reports showed that circ＿0093335 had the same binding site as miR-338-5p,and miR-338-5p had the same binding site as ID4(P < 0.01).RIP result showed that miR-338-5p could bind to ID4(P < 0.01).5.Western blot detection of ID4 protein expression in circ 0093335-Thp1 cells was significantly higher than that in NC-Thp1 group(P < 0.05).Conclusions:1.The expression level of circ＿0093335 decreased in AML patients.The expression level of circ＿0093335 could be used as a potential marker to distinguish AML patients from healthy individuals.2.Overexpression of circ＿0093335 inhibited the proliferation and survival of AML cells,and increaseed the sensitivity of AML cells to daunorubicin and homoharringtonine.3.circ＿0093335 might inhibit the growth of AML cells in leukemia mice.4.circ＿0093335 might regulate the development of AML through circ＿0093335/miR-338-5p/ID4 signaling pathway.