Expression And Biological Function Of KCNAB2 In Acute Myeloid Leukemia

Background:Acute myeloid leukemia is a malignant clonal disease originating from myeloid hematopoietic stem or progenitor cells.It is one of the most common types of leukemia.It is characterized by the progressive abnormal proliferation of one or more undifferentiated cells and can extensively infiltrate liver,spleen and other tissues.At present,the clinical treatment is mainly chemotherapy and supportive therapy,assisted by hematopoietic stem cell transplantation and other methods.At present,the pathogenesis of acute myelogenous leukemia is not fully clear.Many studies have found that many ion channels,including calcium and potassium channel may be involved in the proliferation,apoptosis,migration and invasion of tumor cells,which are considered as new targets involved in cancer diagnosis and treatment strategies.At the same time,ion channel blockers can affect the function of cells by regulating ion shannels.TTX,TEA,4-AP,etc.,have been widely discovered and applied in the study of leukemia and other tumors.4-AP is one of the potassium channel blockers.The KCNAB2 gene encodes the beta subunit Kvβ2 of voltage-gated potassium ion channels,and we found that KCNAB2is significantly overexpressed in acute myeloid leukemia.In this study,we detected the expression level of KCNAB2 in healthy control group and patients with different tumor cells and different types of primary leukemia,and constructed the acute myeloid leukemia cell line HL60 that stably knocked down KCNAB2,to preliminarily explore the expression and biological function of KCNAB2 in acute myeloid leukemia.To investigate the effect of K~+ channel blocker 4-AP on acute myeloid leukemia cells.and to explore the role of voltage-gated potassium channels in acute myeloid leukemia.Objective:.To determine the expression level of KCNAB2 in acute myeloid leukemia,construct and identify KCNAB2 knockdown lentiviral vector,and study the effect of knockdown of KCNAB2 on the biological function of acute myeloid leukemia cells.And the effect of K~+ channel blocker 4-AP on acute myeloid leukemia was explored,so as a new idea for the diagnosis and treatment of acute myeloid leukemia is provided.Methods:1.The gene KCNAB2 which was highly expressed in acute myeloid leukemia was screened by bioinformatics analysis,and the genes MMP7 and ITGB2(CD18)which had clear co-expression relationship with KCNAB2 were predicted.2.Mononuclear cells in human peripheral blood were extracted using lymphocyte isolates.KCNAB2 expression levels in healthy human peripheral blood mononuclear cells and acute myeloid leukemia cells,Burkitt’s lymphoma cells,thyroid cancer,breast cancer,colorectal cancer cells,non-small cell lung cancer cells were measured by qRT-PCR and Western Blot methods.3.The peripheral blood mononuclear cells(PBMCs)from primary patients with four types of acute myeloid leukemia and chronic myeloid leukemia were collected,and the expression level of KCNAB2 in PBMCs was detected by Western Blot compared with that of healthy people.4.The expression levels of MMP7 and CD18 in different tumor cells and primary leukemia cells were detected by Western Blot.5.The HL60 cell line with stable KCNAB2 knockdown was constructed by lentivirus, fluorescence inverted microscope and flow cytometry were used to detect the infection efficiency of lentivirus.The expression levels of KCNAB2 and CD18 in stably infected HL60 cell lines were detected by Western Blot.6.Flow cytometry was used to detect the effects of KCNAB2 expression on HL60 cell cycle and apoptosis.CCK8 assay was used to detect the effect on cell proliferation.7.Using CCK8 method to detect the effect of different concentrations of 4-AP on HL60 cell proliferation,and using flow cytometry to detect the effect of different concentrations of 4-AP on cycle and apoptosis.Results:1.qRT-PCR and Western Blot results showed that KCNAB2 expression was significantly upregulated in acute myeloid leukemia cells HL60 compared with healthy control group.Western Blot results also showed that the expression of KCNAB2 was up-regulated in patients with primary acute myeloid leukemia type M2, the difference was statistically significant.2.Lentivirus was used to construct HL60 cell lines with stable knockdown of KCNAB2,and the infection efficiency was verified by inverted fluorescence microscopy and flow cytometry.The GFP expression efficiency of NC-RNAi and KCNAB2-RNAi groups was close to 100%.Western Blot was used to detect the knockdown efficiency of KCNAB2 in lentivirus-stably infected HL60 cell lines,and the results showed a significantly decrease in KCNAB2 expression,and the difference was statistically significant.3.Bioinformatics analysis predicted a clear co-expression relationship between ITGB2(CD18)and KCNAB2.Western Blot results showed that the expression of CD18 in KCNAB2-RNAi was down-regulated compared with the Control group and the NC-RNAi group,consistent with the trend of KCNAB2.4.CCK8 assay showed no significant change in cell proliferation after KCNAB2 knockdown.5.The results of flow cytometry showed that there was no significant change in the proportion of KCNAB2 knockdown cells compared with the other two groups in each phase.Flow cytometry showed that KCNAB2 knockdown could not increase apoptosis,and there was no significant difference in the proportion of cells among the three groups.6.Flow cytometry showed that 4-AP could inhibit HL60 cell proliferation and promote apoptosis.Conclusions:1.KCNAB2 was highly expressed in acute myeloid leukemia.CD18 was highly expressed in primary acute myeloid leukemia of type M2,M4 and M5.It is proved that there is a correlation between KCNAB2 and CD18.2.KCNAB2 knockdown had no significant effect on cell proliferation,cell cycle and cell apoptosis.3.K~+ channel blocker 4-AP can inhibit proliferation of AML cell line HL60 and promote cell apoptosis in a dose-dependent manner.There was no significant effect on cell cycle.