Study On The Mechanism And Clinical Application Of KCNAB2 As A Diagnostic Marker In Acute Myeloid Leukemia

BackgroundAcute Myeloid Leukemia(AML)is a malignant tumor of the hematopoietic system caused by malignant transformation of hematopoietic stem cells/progenitors.The clinical and molecular biology of AML is highly heterogeneous.The diagnosis of AML basically relies on the results of invasive bone marrow puncture.Therefore,screening new diagnostic markers for AML will help to make up for the shortcomings of the existing assessment criteria,achieve more accurate risk assessment while reducing the diagnostic cost,and facilitate the adoption of more appropriate treatment methods to promote the development of precision medicine for AML.It is great significance to study the pathogenesis of AML and search for biomarkers and new pharmacological targets for early diagnosis.Potassium ion channels are transmembrane proteins,which are the most abundant and widely distributed proteins in cell membranes.Studies have found that potassium ion channels are closely related to the occurrence and development of tumors,regulating not only the proliferation of tumor cells,but also cell cycle,apoptosis and metastasis.KCNAB2 gene is located in the 1p36 region,encoding voltage-gated potassium channel auxiliary subunit protein Kvβ2,which is a component of voltage-dependent potassium channel protein.Potassium channel blockers,which cause presynaptic membrane depolarization,open voltage-gated calcium channels and trigger the release of acetylcholine vesicles,enhance muscle signal transduction and improve muscle function.It has been studied that potassium channel blockers can inhibit the invasion and metastasis ability of oral carcinoma epithelial cells.In summary,potassium ion channels have significant advantages in cancer diagnosis and treatment,and also a hot spot in the diagnosis and treatment of malignant hematologic diseases.Since no highly specific diagnostic markers have been found for leukemia and the prognosis is poor,based on the current research progress in related fields and the preliminary experimental results of our group,we designed to study the effect of KCNAB2 on the biological function of AML,aiming to study its application value in the diagnosis of AML,screen new diagnostic and prognostic markers of AML with high specificity,explore the molecular mechanism of KCNAB2 mediating leukemia progression,and provide new methods and new targets for the clinical diagnosis and treatment of malignant hematologic diseases.Purposes1.To explore new molecular markers for early screening and diagnosis of AML,and reveal the biological functions and specific mechanisms of KCNAB2 in the occurrence and development of AML.2.The role and mechanism of potassium channel inhibitors in AML were systematically elaborated through cell level and animal experiments,providing new therapeutic targets and diagnosis and treatment ideas for acute myeloid leukemia.Methods1.Bioinformatics was used to screening and analysis differentially expressed genes in AML and verified by clinical samples.(1)Bioinformatics screening and correlation analysis of genes highly expressed in AML.(2)Peripheral blood samples of clinical AML patients were collected and gene expression was verified by qPCR.(3)To verify the feasibility of KCNAB2 as a biomarker for AML.2.Study on the biological function and mechanism of KCNAB2 in acute myeloid leukemia.(1)qPCR and Western blot were used to verify the expression of KCNAB2 in different cell lines.(2)Lentivirus transfection method was used to construct HL-60 cell line with stable knockdown of KCNAB2.(3)CCK8,flow cytometry and Transwell were used to detect the effect of KCNAB2 expression on the proliferation,apoptosis and invasion ability of acute myeloid leukemia cells.(4)The proteins interacting with KCNAB2 were screened by Co IP-MS assay and identified by mass spectrometry.3.Study on the effect and mechanism of potassium channel inhibitors on acute myeloid leukemia.(1)The effects of 4-AP on the growth,proliferation and apoptosis of HL-60 cells were detected by CCK8 and flow cytometry,and the IC50 value was determined.(2)Mass spectrometry was performed to analyze the enzymolysis peptide segments of proteins in the 4-AP treatment group and the control group by Label-free proteomic detection,and the differentially expressed proteins were identified.(3)The effect of potassium channel inhibitors on autophagy pathway proteins was verified by Western blot method.(4)AML mouse model was established,and the tumor volume was observed and recorded after intraperitoneal injection of 4-AP(1mg/kg/d).Results1.Bioinformatics was used to screening and analysis differentially expressed genes in AML and verified by clinical samples.(1)The differentially expressed genes in AML were analyzed by bioinformatics in TCGA database and GEO database,204 genes with high expression and 12 genes with low expression were found.(2)Differentially expressed genes were substituted into the GEPIA website to analyze the correlation between expression and survival.A total of six genes were found,which met high expression and were associated with decreased overall survival and the target gene KCNAB2 was screened.(3)The pan-cancer expression of KCNAB2 gene was analyzed.10 genes had the closest interaction with KCNAB2 protein were found through the STRING website,and then brought into the DAVID database for pathway analysis.It was found that KCNAB2 had clear co-expression relationship with MMP7 and ITGB2.(4)The volcano map of ion channel-related genes in AML samples was analyzed by TCGA,and a total of 47 up-regulated ion channel-related gene expression heat maps were screened out.According to the up-regulated 47 genes,the Lasso regression dimension reduction was performed to obtain 5 genes,and the score of the expression of 5 genes and survival prognosis of patients was analyzed.KCNAB2 high risk score was not conducive to prognosis,and the AUC area of ROC curve was greater than 0.75.(5)Genes co-expressed with KCNAB2 were negatively correlated with survival time by weighted gene co-expression network analysis(WGCNA).GESA analysis of KCNAB2 showed that it was related to the metabolism of phosphoinositol,arachidonic acid and carbohydrate degradation.(6)The expression of KCNAB2 was detected in 74 newly diagnosed AML patients and62 normal control patients.The results showed that the expression of KCNAB2 in the newly diagnosed AML group was significantly higher than that in the normal control group,with statistical significance.(7)The expression level of KCNAB2 in the initial diagnosis and relapse stage of AML was significantly higher than that in the complete remission group.The expression of KCNAB2 in 21 patients was tracked from the initial diagnosis to the complete remission stage,and it was found that the expression of KCNAB2 in the complete remission stage decreased than the initial diagnosis.(8)According to the NCCN guidelines,some patients were divided into low-risk,medium-risk and high-risk groups,and there was no statistically significant difference in the expression of KCNAB2 among all groups.(9)The sensitivity,specificity and accuracy of KCNAB2 as a diagnostic marker were75%,97% and 81.6%,and had good stability in the detection of jaundice,hemolysis,lipemia,different temperature,different treatment time and repeated freezing-thawing conditions.2.Study on the biological function and mechanism of KCNAB2 in acute myeloid leukemia.(1)The expression of KCNAB2 in various tumors was detected.The expression of KCNAB2 in HL-60 cells was significantly up-regulated.(2)Knockdown of KCNAB2 had no significant effect on proliferation,cell cycle and apoptosis in HL-60 cells.(3)Compared with the control group,the number of cell invasion after transfection with KCNAB2 RNAi decreased,and the difference was statistically significant.(4)The proteins interacting with KCNAB2 were screened by Co IP-MS assay,and three obvious bands of interacting proteins were detected and sent to mass spectrometry for detection.The protein enrichment analysis of detection results showed that the proteins interacting with KCNAB2 were mainly enriched in energy metabolism,glycolysis and other pathways.3.Study on the effect and mechanism of potassium channel inhibitors on acute myeloid leukemia.(1)After 24 h treatment with different concentrations of 4-AP,cell proliferation decreased with the increase of 4-AP concentration in a dose-dependent manner.These results indicate that potassium channel inhibitors affect the proliferation of HL-60 cells.After 24 h treatment of HL-60 cells with different concentrations of 4-AP,the proportion of apoptotic cells increased with the increase of 4-AP concentration.(2)Proteomic detection was performed on HL-60 cells before and after 4-AP treatment.A total of 311 up-regulated proteins and 182 down-regulated proteins were analyzed.Functional enrichment analysis was performed on differentially expressed proteins,and the results showed that the differentially expressed proteins were closely related to autophagy pathways.(3)Autophagy related proteins were in autophagy activated state after treatment of HL-60 cells with potassium channel inhibitors for 24 h.(4)Intraperitoneal injection of 4-AP(1mg/kg/d)was performed daily to measure tumor volume for 6 consecutive days.In the control group,tumor volume increased day by day,while in the 4-AP intraperitoneal injection group,tumor volume stopped increasing and decreased at about 3 days.Conclusion1.It has been preliminarily confirmed that KCNAB2 is specifically highly expressed in acute myeloid leukemia and is closely related to the course of the disease,which may become a novel molecular marker to guide the diagnosis of AML.2.KCNAB2 may be related to the invasion and metastasis ability of AML and interact with energy metabolism-related proteins.3.Potassium channel inhibitors can inhibit the proliferation of AML cells and promote apoptosis,and the mechanism may be related to the autophagy pathway,suggesting that potassium channel proteins may be a new target for AML treatment.