The Mechanism Of Long Non-coding RNA DUXAP8 Regulating The Proliferation Of Acute Myeloid Leukemia Cells Through The Wnt/β-catenin Pathway

Objectives:This study aimed to elucidate the expression level and function of long non-coding RNA DUXAP8 in acute myeloid leukemia(AML)patients,and to further explore its role in regulating the proliferation and apoptosis of acute myeloid leukemia cells.Methods1 The peripheral blood samples of 30 AML patients confirmed in theAffiliated Hospital of North Sichuan Medical Collegefrom January 2020 to December 2020 were collected as the study objects,and the peripheral blood samples of 30 healthy physical examination groups were collected as controls.The use of clinical specimens required for the study was approved by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College(2019ER(A)028).All patients with AML were tested according to the MCM classification and confirmed using the World Health Organization(WHO)2016 standard[1].Total peripheral blood RNA was extracted by TRIzol method,and the expression level of LncRNA-DUXAP8 in peripheral blood in AML patients and healthy people was detected by qPCR method.2 Taking the AML cell line THP1 cells as the research obj ect,theLncRN A-DUXAP 8 knockdown group(shDUXAP8)and overexpression group(pcDUXAP8)THP1 cell lines were constructed with commercial LncRNA-DUXAP8 knockdown and overexpression lentiviruses,respectively.In the control(shNC)and overexpression control(pcNC)THP 1 cell lines,the expression of LncRNA-DUXAP8 was detected by qPCR to verify the transfection effect.The proliferation and apoptosis of THP1 cells in shNC group、shDUXAP8 group、pcNC group and pcDUXAP8 group were detected by CCK8 method and flow cytometry.3The culture supernatant of THP1 cells in shNC group、shDUXAP8 group、pcNC group and pcDUXAP8 group was collected,and glucose consumption and lactate production levels were detected by glucose kit and lactate kit.Western Blot was used to detect the expression of Wnt/β-catenin pathway proteins Wnt5a、β-catenin、c-myc and Cyclin-D1 inTHP1 cells in shNC group、shDUXAP8 group、pcNC group and pcDUXAP8 group.Results1 The expression level of lncRNA-DUXAP8 in peripheral blood of AML patients was lower than that of normal people(P<0.0001).2In the functional study,we found that compared with the control group,THP1 cells in LncRNA-DUXAP8 knockdown group increased proliferation(96h:P<0.0001 144h:P<0.00011)and decreased apoptosis(P=0.0064),while LncRNA-DUXAP8 overexpression group THP1 Cell proliferation decreased(96h:P<0.0001 144h:P<0.0001)and apoptosis increased(P=0.0274).3 In the mechanism study,first,we found that compared with the control group,THP1 cells in the LncRNA-DUXAP8 knockdown group increased glucose consumption(1.29±0.24μmol/106cellsVS0.71±0.08μmol/106cells,P=0.0082),and increased lactate production(1.29±0.03μmol/106cellsvs0.67±0.1 μmol/106 cells,P<0.0001).In contrast,the overexpression group had decreased glucose consumption(0.37±0.06μmol/106cellsvs0.78±0.06μmol/106cells,P=0.0001)and decreased lactate production(0.55±0.04μmol/106cells vs 0.76±0.05p.mol/106cells,P=0.0004).In further pathway studies,we found that compared with the control group,the expressions of β-catenin、c-myc、Cyclin-D1andWnt5ain the LncRNA-DUXAP8 knockdown group were increased,while the expression levels of these proteins were decreased in the overexpression group.Conclusions1 Compared with the normal population,the expression level of LncRNA-DUXAP8 in AML patients was significantly reduced.2 Knockdown of LncRNA LncRNA-DUXAP8 can promote the proliferation of AML cells and inhibit apoptosis,while overexpression of LncRNA-DUXAP8 can inhibit the proliferation of AML cells and promote apoptosis.This suggests that the low-expressed LncRNA-DUXAP8 in AML patients is involved in the disease progression of AML.3Knocking down the expression of LncRNA-DUXAP8 can promote glucose uptake and lactate production by AML cells by upregulating the Wnt/β-catenin signaling pathway,while overexpressing LncRNA-DUXAP8 inhibits the Wnt/β-catenin signaling pathway and reduces glucose uptake and lactate production in AML cells.This suggests that LncRNA-DUXAP8’s regulation of the expression of the Wnt/β-catenin signaling pathway may be one of the mechanisms that affect glycolysis and participate in regulating AML cell proliferation and apoptosis.