| Section ?Screening of Notchl related genes in AML patients based onTCGA analysisBackground:Acute myeloid leukemia(AML)is a highly heterogeneous molecular phenotype characterized by the malignant transformation of hematopoietic stem/progenitor cells.During the course of pre-leukemia and disease development,affected hematopoietic cells have gradually accumulated a series of molecular changes,including somatic mutations of cells,genomic genetic abnormalities,epigenetic changes and transcriptomics changes.Through next-generation sequencing,a variety of gene level changes,epigenetic changes and cytogenetic abnormalities have been identified in AML,and they have played an important guiding role in the clinical diagnosis and treatment of AML.In recent years,based on public database and bioinformatics analysis,the discovery of more AML-related molecular biological indicators has become a research hotspot in this field.It will help to improve the understanding of the pathogenesis of AML,and provide new prognostic biomarkers and targets for AML clinical diagnosis,treatment and prognosis.The cancer genome atlas(TCGA)project is a multi-center research supported by the National Institute of Health,aimed at mining major oncogenes by large-scale high-throughput sequencing and bioinformatics analysis in more than 30 human tumors.The database currently contains samples and clinicopathological information of about 20,000 cancer patients,and provides public cancer genome data to improve tumor diagnostic methods,treatment standards,and ultimately achieve the purpose of early diagnosis and treatment.The establishment of the TCGA database provides a rich treasure for the molecular biology research of tumors,which will help improve the understanding of tumor mechanisms.Notch signaling pathway is a highly conserved signaling pathway between species,which is of great significance for maintaining the normal function of cells.It consists of Notch receptors(Nocthl-4),Notch ligands(DSL),and CSL(a class of DNA binding proteins),etc.,and participates in many pathophysiological processes in the body through signal transmission between cells,such as cells maintenance of sternness,regulation of apoptosis and immune response.Among them,the Notchl receptor can be bound and activated by a ligand,which then causes the release of the intracellular domain(NICD)of Notchl cells.The NICD will transfer to the nucleus of the cell and bind to DNA and transcription factors,thereby causing changes in downstream gene expression.Studies have reported that Notchl receptor-ligand binding between bone marrow stromal cells(BMSCs)and hematopoietic stem cells(HSCs)affects the ability of HSCs to self-regenerate and impede cell maturation.More malignant osteosarcoma cells have higher Notchl level,and the down-regulation of Notchl can inhibit the invasion and metastasis of cervical cancer tissue.In a mouse model,activation of Notchl can induce T-ALL,demonstrating its important role in the pathogenesis of leukemia;At the same time,studies have shown that Notch1 can regulate the expression of chemokine receptor CCR7 and promote the central nervous system of T-ALL metastasis.Moreover,we previously reported that Notch 1/D114 pathway was activated in AML cells and could promote disease progression.Besides,we found that activation of Notchl pathway may indicate an unfavorable prognosis in AML.It can be seen that the Nocthl signaling pathway plays an important regulatory function in a variety of solid tumors and hematological malignancies.An in-depth discussion of the regulatory network of the Nocthl signaling pathway has important clinical implications for understanding the pathogenesis of AML and discovering new diagnostic and therapeutic targets significance.Objective:By downloading and analyzing the expression profile information and clinicopathological information of AML patients in the TCGA database,the genes related to Notch 1 expression were screened,and the genes related to risk status and prognosis of AML patients were screened to find potential molecular markers and targets of AML,and lay the foundation for the later related functional mechanism research.Materials and methods:1.Use the cBioportal tool(http://www.cbioportal.org)to download AML(non-APL)-related gene expression profile data and clinicopathological information in the TCGA database.2.Based on R X64 3.4.1 software for batch analysis of the data,and screen Spearman test for genes that are related to Notch 1 gene expression(correlation coefficient>0.5,P<0.05).3.Based on the risk status information of AML patients in TCGA database,use R X64 3.4.1 software to analyze the correlation between genes and patient risk status in batches,and screen genes related to risk stratification of AML patients.4.Based on the gene expression information,the median gene expression was selected,and the patients were divided into two groups of high and low expression,and the K-M survival curve of the two groups of patients was drawn based on the prognosis information of the patients.Univariate COX regression analysis was used to analyze the correlation between the expression of each gene and the prognosis of AML patients,and to screen genes that could prompt patients' prognosis.5.Cross-compare the genes selected by the three methods 2,3,and 4 above to select genes that are related to Notch 1 expression and have clinical significance.Results:1.By analyzing Notch 1-related genes in AML patients,a total of 172 genes satisfy the screening conditions with correlation coefficient? 0.5 and P<0.05.Among them,the expression of 148 genes was positively correlated with Notch 1?and the expression of 24 genes was negatively correlated with Notch 1.2.Based on the AML patients risk status information provided by the TCGA database,a total of 17(12.69%)were in the low-risk group,91(67.91%)were in the intermediate-risk group,and 26(19.40%)were in the high-risk group.The correlation between genes and risk status was analyzed,and a total of 5,931 genes related to risk status of AML patients were screened out.3.Further analyze the prognosis information of AML patients provided in TCGA,and screen for genes that can prompt patients' prognosis.A total of 1,584 genes related to prognosis were screened.4.The genes selected by the above three methods were cross-compared and intersected.A total of 19 genes related to Notchl expression,patient risk status and overall survival time of AML patients were screened.5.Further reading the literature revealed that a total of 6 genes(PACS2,PRKD2,BCL9L,SIPA1,PPM1F,RAPGEF3)may play a role in the occurrence and development of AML,as the object of our subsequent research.Conclusion:By downloading TCGA gene expression profile data and clinical pathological information data of AML patients,and using professional software for batch bioinformatics analysis,a total of 19 genes related to Notchl expression,patient risk status and prognosis of AML patients were screened.In the later period,through literature reading,six genes with potential functions in AML were further screened,which may play important regulatory functions in the occurrence and development of AML,and can be used as potential AML diagnostic indicators and therapeutic targets.Section ?Function and mechanism of PRKD2 in acute myeloid leukemiaBackground:Acute myeloid leukemia(AML)is a genetically and epigenetic highly heterogeneous hematological malignancy originating from hematopoietic stem/progenitor cells.It is characterized by the inability of bone marrow progenitor cells to proliferate.Controlled,resulting in abnormal accumulation of immature precursors and insufficient production of normal mature blood cells.AML can widely invade many important human organs such as liver,spleen and lymph nodes.Its inhibition of the body's normal hematopoietic function will cause symptoms such as anemia,infection,neutropenia and thrombocytopenia,which progress rapidly and are difficult to control.AML is the most common acute leukemia among adults,and its incidence has been increasing in recent years,with 38 cases per 100,000 adults,with a median age of onset of about 66 years.According to the National Cancer Institute,the number of new cases of AML in 2018 was 19,520,of which 10,670 died from the disease.In recent years,some progress has been made in the treatment of AML,and the patient's survival rate has improved.However,the currently widely used anti-leukemia drugs in AML treatment remains unsatisfactory.The 5-year survival rate for young patients(age<60 years)is only 30-40%,while the 5-year survival rate for patients>60 years is even lower,less than 10%.At present,the pathogenesis of AML is still not completely clear.The external environmental factors and internal genetic factors affecting its progress need to be further explored.Chemotherapy resistance and disease relapse are important scientific problems that need to be solved urgently in clinic.Therefore,elucidating the mechanism of AML occurrence,progression,chemotherapy resistance and recurrence after chemotherapy,discovering new treatments,and developing more effective anti-AML drugs are of great value for improving the prognosis of patients.The protein kinase D(PRKD)family belongs to the calcium/calmodulin-dependent protein kinase superfamily,including PRDK1,PRKD2,and PRKD3,which can be activated by reactive oxygen species,receptor tyrosine kinase,and hypoxia,thereby regulating the pathophysiological processes within the cell.Studies have shown that the expression of PRKD2 can serve as an important mediator in the gastrointestinal stromal tumor-endothelial cell communication process.Besides,it can play an important function in chronic myeloid leukemia(CML).The expression level and activity of PRKD2 are related to the differentiation level of lymphoma.It can perform important regulatory functions by activating NF-?B.In pancreatic cancer and glioma,high expression of PRKD2 can promote tumor cell growth,invasion and metastasis.PRKD2 is involved in the regulation of many tumors,but its role and mechanism in AML are not yet clear.This project aims to clarify the expression of PRKD2 in AML patients and its correlation with clinicopathological characteristics,and to verify its effect on AML cell proliferation and resistance in AML cell lines,as well as the specific molecular pathways that it regulates.This topic provides new ideas for the basic and clinical research of AML,and provides more reference biological targets for clinical diagnosis and treatment.Objective:The expression of PRKD2 gene in AML drug resistant and sensitive cell lines was detected,and the effect and molecular mechanism of PRKD2 on the proliferation and drug resistance of AML cells were verified by in vitro experiments.Materials and methods:1.Use real-time quantitative PCR(qRT-PCR)technology to detect the expression changes of the six genes screened in the first part in AML resistant and sensitive cell lines,further screening the functional gene that play an important role in the progression and chemotherapy resistance of AML.2.AML cell lines were infected with lentiviruses carrying PRKD2 expression vector or negative control vector to establish PRKD2 overexpression cell line;siRNAs fragment against PRKD2 and negative control siRNAs were synthesized and transfected into AML cell lines.3.Selection of PRKD2 overexpressing cells:After transfection,puromycin was added to the cell culture medium of AML cells for not less than two weeks to establish a stable overexpressing PRKD2 cells.4.Use qRT-PCR and Western blot to detect mRNA and protein expression changes of PRKD2 after its overexpression and interference.5.Cell proliferation and drug sensitivity detection:Cell lines overexpressing and interferred with PRKD2 were seeded into cell culture plates,and CCK8 is added to detect cell proliferation rate;In addition,chemotherapeutic drugs were added and the CCK8 method was used to detect changes of cell drug sensitivity.6.Detection of apoptosis:chemotherapeutic drugs were used to treat AML cell lines overexpressing or interferred with PRKD2,and the proportion of apoptosis induced by chemotherapeutic drugs was detected.7.Use real-time quantitative PCR technology and Western blot technology to detect the expression of Notch 1 pathway-related genes after interference or overexpression of PRKD2 in AML cell lines.8.Based on the mRNA data of AML in TCGA,AML patients were divided into high expression group and low expression group according to the mean expression of PRKD2,and the differences in clinical pathological characteristics between the two groups were analyzed.9.Based on the TCGA database,analyze the correlation between PRKD2 expression combined with common AML mutations and overall survival of AML patients.Results:1.The relative expression levels of the six genes(PACS2,PRKD2,BCL9L,SIPA1,PPM1F,RAPGEF3)screened in the first part of the AML chemotherapy-resistant cell line K562/A02 and the parental cell line K562 were detected.The five genes PACS2,PRKD2,BCL9L,SIPA1,and RAPGEF3 had significant changes.Among them,PRKD2 was significantly up-regulated in K562/A02 cells and PRKD2 had the most remarkably change.2.The efficiency of interference and overexpression of PRKD2 was detected in AML cells,and the results showed significant changes at mRNA and protein levels.3.Functional changes after overexpression of PRKD2:After overexpression of PRKD2 in AML cell lines(KASUMI and U937),CCK8 experiments showed that the proliferation rate of AML cells was significantly increased.At the same time,overexpression of PRKD2 can increase the drug resistance of AML cell lines.Flow cytometry showed that the overexpression of PRKD2 could significantly reduce ADM-induced apoptosis.4.Functional changes after interference with PRKD2:After interference with the siRNAs of PRKD2 in AML cell lines(KASUMI,U937 and K562/A02),CCK8 assay and flow cytometry were used to detect the drug sensitivity of AML cells.The results showed that after interference with PRKD2 siRNAs,the drug sensitivity of AML cells was significantly increased,and the apoptotic proportion of AML cells was also significantly increased.5.Changes in Notch 1 pathway-related genes after overexpression and interference with PRKD2:Real-time quantitative PCR and Wetsern blot showed that the expression level of Notch 1-related genes was significantly changed and was positively correlated with the expression level of PRKD2.6.Correlation between PRKD2 expression and clinicopathological characteristics of AML patients:According to the mean PRKD2 expression,a total of 47 patients were included in the PRKD2 high expression group and 87 patients were included in the PRKD2 low expression group.Analysis showed that high expression of PRKD2 was closely related to age and risk status of AML patients.At the same time,the high expression of PRKD2 was closely related to the occurrence of common AML mutations(FLT3,NPMc).7.Correlation between PRKD2 expression combined with common AML mutations and overall survival of patients:Based on the TCGA database,the significance of PRKD2 high and low expression on the prognostic evaluation of patients with and without common AML mutations(FLT3,IDH1,RAS,NPMc)was analyzed.The results showed that in AML patients with and without mutations of FLT3 and NPMc,high expression of PRKD2 was closely related to poor survival of patients.For IDH1 and RAS mutations,PRKD2 expression showed prognostic significance only in patients who do not have the mutations.Conclusion:Our results indicated that PRKD2 can promote the proliferation and chemo-resistance of AML cells by regulating the Notch 1 signaling pathway,and the analysis found that PRKD2 expression is closely related to the patient's age,risk status,gene mutation and other clinical characteristics,and has a good prognostic value.This study will help us better understand the specific mechanisms of AML chemo-resistance and proliferation,and provide new biological targets for clinical diagnosis and treatment of AML patients. |
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