CircPAN3 Regulates The Proliferation And Apoptosis Of Acute Myeloid Leukemia Cells Through The MiR-370-3p/TSPAN3

Leukemia is a malignant hematological disease originating from hematopoietic stem and progenitor cells.The uncontrollable proliferation of leukemia cells is its typical pathological feature.On the one hand,the proliferation of leukemia cells can cause the reduction of multiple lines of normal blood cells by interfering with normal hematopoiesis.On the other hand,they can infiltrate various tissues and orga ns and affect their physiological functions.Acute myeloid leukemia(AML)is one of the most common acute leukemias in adults.It has a rapid onset and can die within a few weeks if not treated in time.It is currently believed that the pathological causes of AML are mainly hindered differentiation and maturation of hematopoietic cells,excessive proliferation,and abnormal differentiation.It is a hematological malignancy with high heterogeneity.At present,the treatment of AML is based on traditional high-dose combined chemotherapy,and hematopoietic stem cell transplantation is feasible for middle and high risk groups,but recurrence of leukemia is still the main cause of death in this type of disease.The occurrence of leukemia is affected by many factors such as heredity,environment,genes,etc.The study of the mechanism of leukemia is of great significance to its treatment.Although there are many causes or causes of leukemia,these factors need to change the biological state of normal hematopoietic cells to obtain uncontrollable prolif eration and eventually become malignant.Therefore,studying the key molecular biological events that occur or participate in the malignant transformation of leukemia cells is the key to uncovering the causes of leukemia and finding precise therapeutic targets.circRNA was discovered when studying RNA viruses in the 1970 s,and the ubiquity of circRNA was also found in eukaryotes.circRNA is mostly composed of exons,and its side chain is an intron containing the reversed ALU sequence.circRNA is different f rom linear RNA,and it is not easily degraded by exonuclease,so circRNA is more stable in cells.Participate in protein binding,miRNA binding,protein translation and other processes.In recent years,studies have fou nd that circRNA can affect tumor progression and metastasis by regulating tumor-related miRNAs.circPAN3 is a kind of circRNA found to be related to tumors.Studies in leukemia indicate that it may be involved in the progression of AML disease and mediate drug resistance.In order to further clarify the biological process of circPAN3 regulating the proliferation and apoptosis of AML cells,we designed and implemented this research topic,hoping to provide more understanding and molecular targets for the precise treatment of AML.This topic includes the following three parts:Part 1: The clinical significance of circPAN3 in the treatment of acute myeloid leukemia;Part 2: The regulatory mechanism of circPAN3 on miR-370-3p/TSPAN3;Part 3: circPAN3 passes miR-370-The 3p/TSPAN3/Wnt/β-Catenin axis regulates the functional verification of AML.Part One The clinical significance of circPAN3 in the treatment of acute myeloid leukemia Methods:1.Use qRT-PCR to detect the change of circPAN3 expression in 40 AML patients.2.Use qRT-PCR to detect the change of circPAN3 expression in AML cells and normal bone marrow stromal cells.3.Actinomycin D and RNase R detect circPAN3 stability.Results:1.The expression level of circPAN3 was not related to the age and gender of the patient,white blood cell counts and platelet counts at the initial diagnosis,but related to the FAB typing and cytogenetics of the patient.2.The expression level of circPAN3 in normal bone marrow stromal cells was significantly lower than that in AML cells.3.circPAN3 was a stable circular RNA in AML cells.Part Two The regulatory mechanism of circPAN3 on miR-370-3p/TSPAN3Methods:1.Bioinformatics software predicts the targeting relationship between circPAN3 and miR-370-3p,miR-370-3p and TSPAN3,and the luciferase experiment and RIP experiment identify the targeting relationship.2.qRT-PCR and Western blot were used to detect the expression levels of miR-370-3p and TSPAN3 m RNA and TSPAN3 protein in AML patients,AML cells and normal bone marrow stromal cells.3.Transfected circPAN3 overexpression vector,circPAN3 si RNA,miR-370-3p mimic,miR-370-3p inhibitor in AML cells,and co-transfect circPAN3 si RNA and miR-370-3p inhibitor,circPAN3 overexpression vector and miR-370-3p mimic;qRT-PCR method was used to detect circPAN3,miR-370-3p and TSPAN3 m RNA expression levels;Western blot method was used to detect TSPAN3 protein expression levels.4.Using Pearson to analyze the correlation between the expression levels of circPAN3,miR-370-3p and TSPAN3.Results:1.circPAN3 and miR-370-3p are mutually targeted,and miR-370-3p and TSPAN3 are targeted.2.The expression of miR-370-3p in AML patients and AML cells was decreased,and the expression of TSPAN3 m RNA and protein was increased.3.In AML cells,transfected with circPAN3 overexpression vector or miR-370-3p inhibitor,the expression of miR-370-3p was decreased,and the expression of TSPAN3 m RNA and protein was increased;AML transfected with circPAN3 si RNA or miR-370-3p mimic,the expression of miR-370-3p in the cells was increased,and the expression of TSPAN3 m RNA and protein was decreased;the expression of TSPAN3 m RNA and protein of AML cells co-transfected with circPAN3 si RNA and miR-370-3p inhibitor was increased;TSPAN3 m RNA and protein expression of AML cells co-transfected with circPAN3 overexpression vector and miR-370-3p mimic was decreased.4.In AML patients,the expression levels of miR-370-3p and circPAN3 are negatively correlated;miR-370-3p and TSPAN3 m RNA expression levels are negatively correlated;circPAN3 and TSPAN3 m RNA expression levels are positively correlated.Part Three circPAN3 passes miR-370-The 3p/TSPAN3/Wnt/β-Catenin axis regulates the functional verification of AML Methods:1.Transfection of circPAN3 si RNA into AML cells,CCK-8 method to detect cell proliferation,PI single staining method to detect cell cycle distribution,Annexin V-FITC/PI double staining method to detect cell apoptosis changes,Western blot to detect cell cyclin D1,P21,Bax,Bcl-2,Cas-3 Cleavage protein expression changes.2.Co-transfected circPAN3 si RNA,miR-370-3p inhibitor,circPAN3 si RNA,miR-NC inhibitor in AML cells,CCK-8 method to detect cell proliferation,PI single staining method to detect cell cycle distribution,the changes of cell apoptosis were detected by Annexin V-FITC/PI double staining method,and the changes of cyclin D1,p21,Bax,Bcl-2,Cas-3 Cleavage protein expression in cells were detected by Western blot.3.Transfected miR-370-3p mimic in AML cells,co-transfected miR-370-3p mimic,pc DNA-TSPAN3,co-transfected miR-370-3p mimic,pc DNA-NC,CCK-8method to detect cell proliferation,PI single staining method to detect cell cycle distribution,Annexin V-FITC/PI double staining method to detect changes in cell apoptosis,Western blot to detect changes in cyclin D1,p21,Bax,Bcl-2,and Cas-3Cleavage protein expression in cells.4.Transfected circPAN3 si RNA,circPAN3 overexpression vector in AML cells,co-transfect circPAN3 si RNA,pc DNA-TSPAN3,co-transfected circPAN3 si RNA,pc DNA-NC,co-transfected circPAN3 overexpression vector,si RNA control,and co-transfected circPAN3 overexpression vector,TSPAN3 si RNA,Western blot detection of Wnt1,β-Catenin protein expression.5.Transfected circPAN3 overexpression vector into AML cells,treated with Wnt/β-Catenin signaling pathway inhibitor C59,CCK-8 method to detect cell proliferation,PI single staining method to detect cell cycle distribution,the changes of cell apoptosis were detected by Annexin V-FITC/PI double staining method,and the changes of cyclin D1,p21,Bax,Bcl-2,Cas-3 Cleavage protein expression in cells were detected by Western blot.Results:1.After transfection with circPAN3 si RNA or miR-370-3p mimic,the proliferation ability of AML cells was decreased,the ratio of cell G0/G1 phase was increased,cell apoptosis was increased,Bcl-2,cyclin D1 protein expression levels were decreased,Bax,Cas-3 Cleavage and p21 protein expression levels were increased.2.Compared with circPAN3 si RNA and miR-NC inhibitor,after circPAN3 si RNA and miR-370-3p inhibitor were co-transfected,the proliferation ability of AML cells was increased,the cell G0/G1 phase ratio was decreased,cell apoptosis was decreased,the expression levels of Bcl-2 and Cyclin D1 proteins were increased,and the expression levels of Bax,Cas-3 Cleavage and P21 proteins were decreased.Compared with miR-370-3p mimics and pc DNA,after miR-370-3p mimics and pc DNA-TSPANS were co-transfected,the proliferation ability of AML cells was increased,the cell G0/G1 phase ratio was decreased,cell apoptosis was decreased,the expression levels of Bcl-2 and Cyclin D1 proteins were increased,and the expression levels of Bax,Cas-3 Cleavage and P21 proteins were decreased.3.Wnt1 and β-Catenin protein expression levels in AML cells were decreased after transfection of circPAN3 si RNA;Wnt1 and β-Catenin protein expression levels were increased in AML cells after co-transfection with circPAN3 si RNA and pc DNATSPAN3;Wnt1 and β-Catenin protein expression levels were increased in AML cells co-transfected with circPAN3 overexpression vector and TSPAN3 si RNA.4.Wnt/β-Catenin signaling pathway inhibitor C59 can reverse the effects of circPAN3 overexpression vector on AML cell proliferation promotion,cycle promotion and apoptosis inhibition.Conclusion1.circPAN3 was highly expressed in AML.High circPAN3 expression was associated with lower survival rates,FAB typing,and cytogenetics in AML patients.2.Downregulation of circPAN3 could inhibit AML cell proliferation,block cell cycle,and induce cell apoptosis.3.Circ PAN3 can act as miR-370-3p sponges,thereby regulating the expression of its target gene TSPAN3.4.Circ PAN3 regulates the biological function of AML cells through Mir-370-3P /TSPAN3/Wnt/-catenin axis.