The Effect Of Epidermal Growth Factor Like Domain Protein-7 On The Biological Behavior Of Acute Myeloid Leukemia And Its Mechanism

Acute myeloid leukemia(AML)is a heterogeneous group of diseases characterized by maturation arrest accompanied by uncontrolled proliferation and accumulation of immature malignant cells.The end results of treatment of AML have improved before by reducing early mortality and refining disease risk stratification,with concurrent tailoring of treatment.However,the pathogenesis of AML is unclear,and with adverse effects of chemotherapy.With an ever-increasing understanding of the molecular pathogenesis of leukemia,molecular factors based on substantial clinical can be considered with prognostic associations.It may be used as a clinical treatment guide.Sequencing of AML genomes found that AML is genetically diverse and clonally heterogeneous,with multiple mutations acquired over time,and complex patterns of clonal evolution shaping disease progression and response to therapy.Among the gene mutations described,AML has both a common and a distinct repertoire of driver gene mutations,many of which fall into shared functional classes.Of its own panoply of specific oncogenic drivers,mutations that lead to aberrant regulation of DNA methylation and hydroxy methylation(DNMT3A,TET2,IDH1 and IDH2),altered messenger RNA splicing by the U2 complex(SF3B1,SRSF2,and U2AF1)modified chromatin architecture(ASXL1,EZH2,and KMT2A),and transcriptional deregulation(CEBPA,RUNX1,and WT1)are most frequent.Understanding the genes of AML represents an opportunity for us to rationalize diagnostic,prognostic,disease surveillance,and combination therapy protocols.Epidermal growth factor like domain protein-7(EGFL7),a member of the epidermal growth factor(EGF)like protein family,is a potent angiogenic factor expressed in many different cell types.Dysregulation of EGFL7 has been frequently found in several types of cancers,such as malignant glioma,colorectal carcinoma,oral and esophageal cancers,gastric cancer,hepatocellular carcinoma,pancreatic cancer,breast cancer,lung cancer,osteosarcoma,and acute myeloid leukemia.EGFL7 plays a vital role in controlling vascular angiogenesis during embryogenesis,organogenesis,and maintaining skeletal homeostasis.Accumulating evidence suggests that EGFL7 plays a crucial role in cancer biology by modulating tumor angiogenesis,metastasis,and invasion.Tumor necrosis factor receptor-associated protein 1(TRAP1)is a component of the heat shock protein 90(HSP 90)family responsible for protection from apoptosis and drug resistance,regulation of cell proliferation and protein homeostasis.TRAP1 contributes to tumor cell bioenergetics.TRAP1 is upregulated in several human tumors,for example,glioblastoma,colon,breast,prostate,and lung cancer.And high TRAP1 expression correlates with resistance.While TRAP1 is downregulated in the other tumors,such as ovarian,bladder,cervical and kidney cancer,which related to oxidative metabolism,TRAP1 plays different roles(oncogenic versus oncosuppressive)in human malignancies depending on tumor type.More studies need to explore its physiological mechanism.Recent studies suggested that TRAP1 involved in the maturation and stabilization of a variety of oncogenic proteins that are critical for tumorigenesis and malignant progression.TRAP1 protein involves in the activation of various oncogenic proteins and signaling pathways,and has a balanced function at tumor transformation.Therefore,the studies of TRAP 1 in tumorigenesis are bound to have a broader prospect.Our research is aim to explore the biological characteristic of EGFL7 in AML,and to study the underline mechanism of AML with EGFL7 and its binding protein TRAP1.It provides a new value for further in-depth study of the mechanism.PART I Expression and clinical significance of epidermal growth factor like domain protein-7 in acute myeloid leukemiaObjectiveTo detect the expression of epidermal growth factor like domain protein-7(EGFL7)in acute myeloid leukemia(AML).We retrospectively analyzed the correlation between EGFL7 and AML to find the new molecular prognostic indicators and supply new ideas for supervision of AML.Methods1.We tried to find the relationship between EGFL7 expression in AML patients and the prognosis by cancer gene databases.2.Among these 182 de novo AML patients in our large cohort from January 2012 to April 2019.We measured the EGFL7 mRNA levels in all the AML patients by Real-time PCR.We analyzed the clinical features of the AML patients.3.The EGFL7 protein levels in AML patients were detected by Western blot.The expressions of soluble EGFL7 in the plasma of AML patients were detected by ELISA.4.In this study,a median was set as the cut-off value for EGFL7 mRNA expression.We analyzed the correlations between EGFL7 levels and the overall survival(OS),disease-free survival(DFS)of AML patients and their related risk factors by statistical methods.Results1.The databases showed the EGFL7 expression was related to the poor prognosis of AML patients.2.We enrolled 182 de novo AML patients in our center.The follow-up time was 94 months.According to the clinical characteristics,the platelets(P=0.008)and cytogenetics(P=0.019)have large impacts on the distribution of the patients with high EGFL7 level and low EGFL7 level.The distribution of two groups did not significantly depend on age(P=0.774),gender(P=0.298),bone marrow blast percentage(P=0.369),leukocyte level(P=0.949),hemoglobin level(P=0.089),European Leukemia Net(ELN)genetic groups(P=0.654),WHO subtype(P=0.807),induction chemotherapy regimen(P=0.143)and disease status(P=0.77).Furthermore,DNMT3A,KIT and NPM1 gene mutations were associated with EGFL7(P<0.05).3.It was showed that the EGFL7 mRNA levels in AML patients were higher than the control group by Real-time PCR(P<0.05),and the EGFL7 protein levels in AML patients were higher than that of control group by Western Blot.The expressions of soluble EGFL7 in the plasma of AML patients were also higher than control group tested by ELISA(P<0.05).4.Kaplan-Meier survival analysis showed high EGFL7 level AML patients have shorter OS and DFS than low EGFL7 level AML patients(22.6±9.1%vs 42.3±13.6%,P=0.008;DFS:19.1±7.7%vs 39.9±8.9%,P=0.012).According to the definition of intermediate-and adverse-risk AML groups in the ELN,high EGFL7 level AML patients have shorter OS and DFS than low EGFL7 level AML patients in the intermediate-and adverse-risk AML groups(26.3±10.3%vs 27.4±19.9%,P=0.032;DFS:23.3 ±8.7%vs 35.9±9.8%,P=0.031).The high EGFL7 level AML patients also have shorter OS and DFS than low EGFL7 level AML patients in cytogenetically normal acute myeloid leukemia(CN-AML)group(OS:16.4±8.6%vs 44.0±14.2%,P<0.001;DFS:15.0±7.8%vs 40.9±9.2%,P=0.001).Furthermore,high EGFL7 level CN-AML patients have shorter OS and DFS than low EGFL7 level CN-AML patients in the intermediate-and adverse-risk groups(OS:17.4±9.1%vs 28.4±20.7%,P<0.001;DFS:16.7±8.4%vs 36.7±10.1%,P=0.003).In the high EGFL7 level AML cohort,the patients who received allogeneic hematopoietic stem cell transplantation(allo-HSCT)have longer OS and DFS than those who did not receive allo-HSCT(OS:48.1±13.6%vs 9.4±8.1%,P=0.012;DFS:47.8±9.5%vs 5.5±5.2%,P=0.017).In the high EGFL7 level AML cohort of the intermediate-and adverse-risk groups,the patients who received allo-HSCT have longer OS and DFS than those who receive chemotherapy(OS:47.8±13.6%vs10.3±9.4%,P=0.012;DFS:47.7±9.5%vs 7.8±7.1%,P=0.014).In the high EGFL7 level CN-AML cohort,the patients who received allo-HSCT have longer OS and DFS than those who receive chemotherapy(OS:46.5±13.4%vs 0%,P=0.008;DFS:43.4±11.4%vs 0%,P=0.005).Moreover,among high EGFL7 level CN-AML cohort of the intermediate-and adverse-risk groups,the patients who received allo-HSCT also have longer OS and DFS than those who receive chemotherapy(OS:46.0±11.4%,P=0.005;DFS:43.0±13.4%vs 0%,P=0.003).5.The study constructed Cox proportion risk model including age,EGFL7,treatment and disease status.Multivariate Cox regression analysis revealed that high EGFL7 was an independent prognostic factor for OS and DFS in AML patients(OS:HR=1.832,95%CI:1.062-3.161;P=0.030;DFS:HR=1.699,95%CI:1.063-2.716;P=0.027).More important,the treatment of allo-HSCT has a positive impact on OS and DFS in high EGFL7 level AML patients(OS:HR=2.787,95%CI:1.590-4.883;P<0.001;DFS:HR=1.858,95%CI:1160-2.975;P=0.010).Conclusion1.EGFL7 level was high in bone marrow mononuclear cells and plasma of AML patients.2.The AML patients with higher EGFL7 expression have shorter survival times and worse prognosis.Multivariate analysis showed that EGFL7 can be used as independent prognostic factor in AML patients.3.EGFL7 may be an adverse prognostic factor in AML patients,while allo-HSCT could improve the survival of AML patients with high EGFL7 levels.PART Ⅱ The proliferation effect and mechanism of epidermal growth factor like domain protein-7 in acute myeloid leukemiaObjectiveTo explore the effects of phenotypes of AML with EGFL7 silenced and overexpressed in vivo and in vitro.Methods1.To detect the EGFL7 mRNA expression of 10 AML cell lines by Real-time PCR.2.To detect the EGFL7 protein expression of AML cell lines by Western blot.3.According to the encoding sequence of EGFL7 in GeneBank and principle of silencing and overexpression designing,specific RNAs were designed and synthesized.Lentiviral system was used in the establishment of plasmids.The stable EGFL7 down-regulated clones MV4-11/shEGFL7 and overexpressed clones SHI-1/EGFL7 were produced.The EGFL7 expressions were confirmed by Real-time PCR and Western blot.4.To investigate the phenotypes of MV4-11 cells after RNA silencing of EGFL7 and SHI-1 cells after RNA overexpression of EGFL7.1)Cell proliferation of AML cell lines was detected by CCK8 assay and cell colony forming assay.2)The apoptosis of AML cell lines was detected by flow cytometry detection technology.3)The cell cycle of AML cell lines was detected by Ki-67 and DAPI double staining combined with flow cytometry detection technology.5.Leukemia mouse model:AML cells of MV4-11/shEGFL7 or SHI-1/EGFL7 and their control groups of MV4-11/shCtrl and SHI-1/Ctrl were injected into NSG mice tail vein,respectively.The survival time of NSG mice was recorded.The CD45 expression was detected by flow cytometry,and the spleen,liver and bone marrow were extracted and weighted.Frozen tissues were stained by immunohistochemical staining to discover the expression of leukemia cells.6.The MV4-11/shEGFL7 cell lines and MV4-11/shCtrl cell lines were prepared for the high-throughout mRNA microarray analysis.The level of molecular involved in the pathway was detected by Real-time PCR and Western blot.Results1.The expressions of EGFL7 mRNA and EGFL7 protein in AML cell lines were detected by Real-time PCR and Western blot,respectively.2.Choose the target AML cell lines for gene silencing or overexpression after detecting EGFL7 mRNA in AML cell lines.EGFL7 was successfully silenced by lentiviral-mediated shRNA or overexpressed by lentiviral-mediated,respectively.The stable silencing MV4-11/shEGFL7 cells,MV4-11/shCtrl and stable overexpression SHI-1/EGFL7,SHI-1/Ctrl cells were successfully constructed.3.The results of proliferation,apoptosis,and cell cycle assays.1)It was showed that EGFL7 silencing was associated with reduced proliferation of MV4-11 cells compared with control group by CCK8 assay and cell colony forming assay,EGFL7 overexpression was associated with increased proliferation of SHI-1 cells compared with control group by CCK8 assay and cell colony forming assay.2)MV4-11/shEGFL7 cells exhibited higher apoptosis rate than MV4-11/shCtrl cells by flow cytometry detection technology(P<0.05),while SHI-1/EGFL7 cells exhibited lower apoptosis rates than SHI-1/Ctrl cells by flow cytometry detection technology.3)Ki-67 and DAPI double staining combined with flow cytometry showed that there was no difference in cell cycle between the two AML cells groups.4.The results of vivo studiesCompared with the control group,EGFL7 silencing prolonged the survival time of MV4-11/shEGFL7 NSG mice.EGFL7 silencing decreased the quality of liver,and spleen in MV4-11/shEGFL7 NSG mice.Compared with the control group,EGFL7 overexpression shortened the survival time of SHI-1/EGFL7 NSG mice.EGFL7 overexpression increased the quality of liver,and spleen in SHI-1/EGFL7 NSG mice.EGFL7 silencing inhibited CD45 expression in liver,spleen,and bone marrow of MV4-11/shEGFL7 NGS mice,while EGFL7 overexpression promoted the CD45 expression in liver,spleen,and bone marrow of SHI-1/EGFL7 NGS mice.5.EGFL7 regulated the expression of PI3K,AKT and mTOR in the PI3K/AKT and mTOR pathway.The S6 protein downstream of the mTOR pathway was also regulated by EGFL7 detected by Real-time PCR and Western blot.6.The phenotypic changes of SHI-1/EGFL7 cells were rescued after using the mTOR inhibitor rapamycin.Conclusion1.EGFL7 promoted the proliferation of AML cell lines,and inhibited the apoptosis of AML cell lines.There was no difference in cell cycle of AML cell lines.2.In vivo studies suggested that EGFL7 silencing prolonged the survival time of NSG mice.EGFL7 silencing decreased the quality of liver and spleen of NSG mice and inhibited CD45 expression in liver,spleen,and bone marrow of NGS mice.EGFL7 overexpression shortened the survival time of NSG mice.EGFL7 overexpression increased the quality of liver and spleen of NSG mice,and promoted CD45 expression in liver,spleen,and bone marrow of NGS mice.3.EGFL7 regulated the expression of PI3K,AKT,mTOR and S6 in the PI3K/AKT and mTOR pathway.PART Ⅲ The mechanism of epidermal growth factor like domain protein-7 and tumor necrosis factor receptor related protein 1 regulating the proliferation of acute myeloid leukemiaObjectiveWe tried to explore the mechanism of AML with EGFL7 and the function of the binding proteintumor necrosis factor receptor-associated protein 1(TRAP1)of EGFL7 during the AML cell lines growth process.Methods1.Construct the HEK 293T/EGFL7 cell lines with lentiviral infection system and explore the relationship between EGFL7 and TRAP 1 in AML cell lines by immunoprecipitation and Western blot.The co-localization of EGFL7 and TRAP1 in AML cell lines was observed by confocal microscope2.Cell proliferation of AML cell lines was detected by CCK8 assay using stable MV4-11/shTRAP1 cells with or without the addition of human recombinant EGFL7 protein.The apoptosis of AML cell lines was detected by flow cytometry detection using stable MV4-11/shTRAP1 cells with or without the addition of human recombinant EGFL7 protein.The expressions of total protein and phosphorylated protein of PI3K,AKT,mTOR and S6 were detected by Western blot using stable MV4-11/shTRAP1 cells with or without the addition of human recombinant EGFL7 protein.Results1.The binding proteins of EGFL7 were screened out by immunoprecipitation mass spectrometry.2.TRAP1 was found to bind to EGFL7,and TRAP1 co-localized on the cytoplasm and cell membrane with EGFL7.3.By reviewed the multiple cancer gene databases to explore the relationship of TRAP 1 expressions in AML patients,it was found that AML patients with high TRAP1 level had a poor prognosis.4.TRAP1 was successfully silenced by lentiviral-mediated shRNA.The stable knockdown MV4-11/shTRAP1 was successfully constructed.5.The results of proliferation,apoptosis,and cell cycle assays.1)It was showed that TRAP1 silencing inhibited the proliferation of MV4-11 cells compared with MV4-11/shCtrl cells by CCK8 assay and cell colony forming assay.2)Flow cytometry detection showed the apoptosis rate was higher in MV4-11/shTRAP1 cells compared with the MV4-11/shCtrl cells.3)By adding human recombinant EGFL7 protein,the proliferation rate of MV4-11/shTRAP1 cells was faster than the proliferation rate of MV4-11/shTRAP1 cells without adding human recombinant EGFL7 protein.By adding human recombinant EGFL7 protein,the apoptosis rate of MV4-11/shTRAP1 cells was lower than the proliferation rate of MV4-11/shTRAP1 cells without adding human recombinant EGFL7 protein.6.The expressions of the total protein and phosphorylated protein of PI3K,AKT,mTOR and S6 were down-regulated in MV4-11/shTRAP1 cells compared with MV4-11/shCtrl cells.The expressions of total protein and phosphorylated protein of PI3K,AKT,mTOR and S6 were up-regulated by adding human recombinant EGFL7 protein compared with MV4-11/shTRAP1 cells without adding human recombinant EGFL7 protein.Conclusion1.EGFL7 and TRAP1 co-localized on the cytoplasm and cell membrane.2.It was showed that TRAP1 silencing inhibited the proliferation and cloning ability of AML cell lines,TRAP1 silencing promoted the apoptosis of AML cell lines.The phenotypic changes of MV4-11/shTRAP1 cells were rescued by adding human recombinant EGFL7 protein.3.EGFL7 may affect the proliferation of AML cell lines through the PI3K/AKT and mTOR pathway and S6 downstream of mTOR by binding to TRAP1.