The Clinical Significance And Biological Mechanism Of AKAP9 In Acute Myeloid Leukemia

Objective:Acute myeloid leukemia（AML）is a hematological malignancy with high molecular genetic heterogeneity.Relapse is the main obstacle to improve the therapeutic effect of AML,and a small amount of leukemia cells remaining in the body is the root of relapse.The monitoring of minimal residual disease（MRD）can not only be used for early recurrence warning and efficacy evaluation,but also can be used to guide the individualized treatment of patients.MRD has become one of the keys to reduce the recurrence of AML and improve the curative effect.Therefore,it is of great clinical significance to screen MRD-related prognostic key genes and elucidate their molecular mechanisms in AML recurrence.The purpose of this study is to screen the MRD-related prognostic genes of AML through bioinformatics methods,establish a prognostic risk model,and evaluate the value of the model in predicting the prognosis of AML patients.According to the results,AKAP9 was found to be one of the key genes in the prognostic risk model.Therefore,we further focus on AKAP9,clarify the expression and clinical significance of AKAP9in AML,and preliminarily explore the possible mechanism of AKAP9 in AML recurrence and drug resistance,so as to provide a new potential target for the treatment of AML.Methods:（1）Screening of MRD-related prognostic genes and construction of prognostic risk modelDownload gene expression profile data and clinical data of AML patients from the official websites of TCGA and TARGET database.First,AML patients in TARGET database were divided into two groups according to MRD detection results,and differential gene analysis was performed by edge R method;Then,the relationship between the above differential genes and the survival prognosis of patients was analyzed by univariate Cox analysis to screen MRD-related prognostic genes;Then,the obtained independent prognostic genes of MRD-related were included in Cox proportional risk regression model.An AML prognostic related risk model was constructed based on TARGET database.Kaplan Meier（K-M）survival curve and receiver operator characteristic curve（ROC）were used to evaluate the prediction performance of the model.The model performance was verified by AML in TCGA database;Finally,the relationship between prognosis risk model and clinical pathological factors was analyzed by univariate and multivariate Cox regression to explore whether the model was an independent risk factor for AML prognosis.（2）Expression and clinical significance of AKAP9 in AMLBone marrow samples were collected from a 3^rd-class hospital in Lanzhou from January 2017 to April 2019,including 110 patients with AML in the experimental group and 8 patients with non-malignant tumors in the blood system in the control group.Mononuclear cells in bone marrow samples were isolated by density gradient centrifugation.Total RNA was extracted by Trizol method and reverse transcribed into c DNA;Then,the m RNA expression of AKAP9 in AML and control group was detected by RT-PCR;Finally,the difference of AKAP9 expression between AML patients and controls and the relationship between AKAP9 expression and risk stratification were analyzed.At the same time,the expression level of AKAP9 in AML was further verified by GSE2191 dataset of GEO database,GEPIA database and R2 database.Taking 285newly diagnosed and de novo AML patients in TARGET database as the research object,the prognosis differences between high expression group and low expression group of AKAP9 were compared by K-M curve method.Univariate and multivariate Cox analysis was used to analyze whether the expression of AKAP9 was an independent risk factor for overall survival（OS）and event free survival（EFS）of AML patients.（3）Effect and mechanism of AKAP9 on biological function of AML cellsFirstly,stable AKAP9 silenced AML cell lines THP1 and HL60 were constructed by lentivirus vector mediated sh RNA technology.Then the effect of AKAP9 silencing on the proliferation of AML cells was detected by CCK8 method,the effect of AKAP9silencing on the apoptosis and cell cycle of AML cell line was detected by flow cytometry,the effect of AKAP9 silencing on the migration ability of AML cell line was detected by Transwell method,and the effects of AKAP9 silencing on adriamycin resistance of K562/ADM and HL60/ADM cell line were detected by CCK8 respectively.Secondly,RT-PCR and Western Blot were used to detect the effects of AKAP9 silencing on the expression ofβ-catenin and stem cell markers（ALDH1A1 and CD133）of AML cells.（4）The experiment in vivo verified the mechanism of AKAP9 in AML.The human AML cell xenograft models in nude mice was constructed by inoculating lentivirus infected HL60 cells under the armpit.The effect of AKAP9 silencing on the growth of transplanted tumor was observed.Immunohistochemistry and PCR were used to detect the effect of AKAP9 silencing on Wnt/β-catenin pathway and the expression of stem cell marker.Results:（1）Screening of MRD-related prognosis genes in AML and construction of prognosis risk modelThrough univariate Cox analysis,449 MRD-related prognosis genes in TARGET were screened,including 339 poor prognosis related genes and 110 good prognosis related genes.Based on the TARGET database,the AML prognostic risk model composed of five genes including AKAP9,MMP7,RAB3D,BICC1 and TCF15 was successfully constructed.According to the risk scoring model,AML patients were divided into high-risk group and low-risk group.The results of K-M survival analysis showed that the overall survival time of AML patients in high-risk group was significantly lower than that in low-risk group（P<0.001）.ROC curve analysis showed that prognostic risk model assessment had high sensitivity and specificity in predicting OS in patients with AML.Through the validation of TCGA database,it is also found that the model has good prediction performance for the prognosis of AML patients.Further analysis found that the prognostic risk model is an independent risk factor for poor prognosis of AML and can be used as an independent prognostic predictor of AML patients.（2）Expression and clinical significance of AKAP9 in AMLThe expression level of AKAP9 in AML patients with initial diagnosis,relapse or complete remission was significantly higher than that in the control group of hematological non-malignant tumors,and its expression was related to the prognostic risk stratification.The expression level of AKAP9 in AML patients was significantly higher than that in healthy controls.The positive rate of MRD in AML patients in AKAP9 high expression group was significantly higher than that in AKAP9 low expression group,while the complete remission（CR）rate in AKAP9 high expression group was significantly lower than that in AKAP9 low expression group.The time of OS and EFS in patients with AML in the high expression group of AKAP9 was shorter than that in the low expression group,and the difference was statistically significant（P<0.001）.（3）Effect and mechanism of AKAP9 on biological function of AML cellsAfter AKAP9 silencing,the proliferation of AML cell lines THP1 and HL60decreased significantly.AKAP9 silencing not only significantly prolonged the G0/G1phase of AML cell line,but also significantly reduced the G2/M phase,however,the S phase did not change significantly.After AKAP9 silencing,the apoptosis ratio of AML cell lines THP1 and HL60 increased.There was no significant difference in the number of cells in the upper and lower chambers of Transwell membrane between AKAP9silenced cell line and non-interference or negative control cell line.Compared with K562/ADM and HL60/ADM cell line or negative control group,the IC50 of K562/ADM and HL60/ADM to ADM decreased significantly after AKAP9 silencing respective（P<0.05）.AKAP9 silencing inhibited apoptosis in THP1 and HL60 cells.The expression levels ofβ-catenin,ALDH1A1 and CD133 also decreased significantly.（4）In vivo experiment results show that AKAP9 silencing can reduce the expression ofβ-catenin protein,down-regulate the expression of CD133 and ALDH1A1,the stem cell markers of AML,and then significantly inhibit the growth of AML transplanted tumors.Conclusion:（1）By screening the MRD-related prognosis genes of AML sequencing data in TARGET database,an AML risk prediction model composed of five genes is constructed.The model can be used to identify high-risk groups of AML and predict the prognosis and survival of AML patients.The model has reasonable design and high stability,so it has certain application value in predicting the prognosis of AML and guiding clinical practice.（2）The expression of AKAP9 was up-regulated in AML patients and was related to the prognostic risk stratification.The expression of AKAP9 was related to the positive rate of MRD in AML patients after treatment;High expression of AKAP9 is associated with poor prognosis in patients with AML;AKAP9 overexpression is an independent risk factor for poor prognosis in patients with AML.（3）AKAP9 silencing significantly inhibited the proliferation and cell cycle process of AML cells,promoted apoptosis,and reversed the resistance of K562/ADM and HL60/ADM cells to Adriamycin,but had no effect on cell migration;（4）The mechanism of AKAP9 in AML may be related to Wnt/β-Catenin signaling pathway and the stemness of AML;In conclusion,AKAP9 plays an important role in the recurrence and drug resistance of AML.AKAP9 may become an important index for predicting the prognosis of AML and a potential target for treatment.However,the research conclusions need to be verified by further experiments and primary cell studies.