| Objective:1.To investigate the expression level of BYSL gene in patients with acute myeloid leukemia and its correlation with clinicopathological characteristics such as age,gender,and to study the impact of BYSL expression level on the survival of AML patients.2.To study the effect and mechanism of knocking down BYSL on the proliferation,apoptosis,cell cycle,and clonal formation functions in AML cell lines,and to study the upstream regulatory mechanism of BYSL.3.To construct a Nomogram prognostic model that includes gene expression information,clinicopathological and survival information of AML patients,which is suitable for clinical applications.Part 1.Expression of BYSL in patients with acute myeloid leukemia and its correlation with clinicopathological characteristics and prognosisMaterials and Methods:1.The microarray data in the GEO database was downloaded,and the expression difference of BYSL between AML patients and normal controls was analyzed.2.The RNA-sequence data and clinicopathological information of 151 AML patients in the TCGA-LAML project were downloaded,we analyzed the differences in BYSL gene expression in different ages,genders,FAB subtypes,with or without specific fusion genes and gene mutations.We took the median value of BYSL gene expression as the Cut-off value,all patients were divided into high-BYSL group and low-BYSL group.The Kaplan-Meier curves were plotted according to the Log-rank method,and the impact of BYSL expression level on patients’ survival was analyzed.In the same way,we downloaded and analyzed the data of the AML project in the TARGET database,and analyzed the impact of BYSL expression level on the patients’ survival.3.We collected 93 cases of bone marrow or peripheral blood samples from patients with de novo non-M3 AML who were hospitalized in the Department of Hematology,West China Hospital,Sichuan University from January 2019 to October 2020,and collected peripheral blood samples of 10 healthy volunteers as control.Mononuclear cells were isolated,and the expression of BYSL gene in each patient was evaluated by RT-q PCR.Patients’ clinical information including age,gender,bone marrow examination information,remission status after chemotherapy and other information was collected.Similarly,all patients were grouped into high-BYSL and low-BYSL groups based on the median expression value of BYSL,and the relationship between BYSL expression level and various clinical factors(age,gender,karyotype,gene mutation,etc.)of the patients was explored.The patients were followed up by telephone,and the follow-up end point was February 1,2021.We analyzed the impact of characteristics such as age,gene mutations,and BYSL gene expression levels on patients’ survival.Results:1.By analyzing the data in the two datasets GSE114868 and GSE7186 of the GEO database,and comparing the expression of BYSL gene in AML patients and healthy controls,we found that the expression level of BYSL in AML was significantly upregulated.2.By analyzing the sequencing data in the LAML project in the TCGA database,we found that the BYSL gene expression of patients with CBFβ-MYH11 fusion gene was significantly lower than that of patients without CBFβ-MYH11 fusion gene.The BYSL expression was significantly upregulated in patients with TP53 gene mutation.When grouped according to whether there was FLT3-ITD mutation,FLT3-TKD mutation,CEBPA mutation,DNMT3 A mutation,RAS mutation,etc.,no significant differences in BYSL expression levels were found among the groups.In the TCGA-LAML dataset,according to the median value of BYSL,all patients were divided into high-BYSL and low-BYSL groups.We found that the overall survival(OS)and event-free survival(EFS)of patients in the high-BYSL group were shorter,with the P values were 0.030 and 0.020,respectively.In the TARGET-AML dataset,patients were also divided into high-BYSL and lowBYSL group.It was found that the patients in high-BYSL group had shorter OS,P=0.004,and there was no significant difference in EFS between the two groups.3.Subsequently,we analyzed the information of 93 patients with newly developed non-M3 AML collected from the Department of Hematology of West China Hospital.We found that the expression of BYSL gene in peripheral blood or bone marrow mononuclear cells of patients with de novo AML was significantly higher than that in mononuclear cells of healthy people,with P value of 0.0086.After excluding patients who did not meet the criteria,a total of 80 patients were analyzed,and the median value of BYSL expression was used as the Cut-off point.The results showed that patients in the high-BYSL group suffered more severe anemia,but there were no differences in other clinical characteristics such as age,gender,and cytogenetics.The median OS of the entire cohort of 80 patients was18.8 months,and the median EFS was 13.6 months.The OS and EFS of patients≥ 60 years of age were significantly shorter than those of younger patients,with P values of 0.0002 and 0.019,respectively.The OS of patients who achieved complete remission after the first course of chemotherapy was significantly better than that of patients who did not achieve complete remission,P=0.020.Hematopoietic stem cell transplantation could significantly improve patient survival,whether it was OS or EFS.Then,we analyzed the effect of BYSL gene expression level on the survival of patients,and found that there was no significant difference in OS between the high-BYSL and low-BYSL groups,but the two survival curves had a tendency to separate.Therefore,we further conducted a subgroup analysis and found that among the 71 patients who only received chemotherapy,patients in the high-BYSL group had a shorter OS,P=0.037.Among the 36 patients with normal karyotype,patients in the high-BYSL group showed shorter OS,P=0.048.Conclusion:BYSL was abnormally highly expressed in AML patients,higher BYSL expression was associated with more severe anemia.In patients who underwent chemotherapy without hematopoietic stem cell transplantation and patients with normal karyotypes,the prognosis of those patients with higher BYSL expression was worse.Part 2.The effect and mechanism of BYSL gene knockdown on the biological characteristics of acute myeloid leukemia cells Materials and Methods:1.A total of 7 AML cell lines were used in the experiments,including HL60,NB4,Kasumi,Molm-13,U937,THP-1 and K562.Total RNA and protein was extracted,and the expression level of BYSL at the RNA and protein level was detected.The cell lines Molm-13 and U937 with high BYSL expression level were selected for subsequent experiments.2.In Molm-13 and U937 cell lines,si RNA technology was used to construct BYSLknockdown cell lines,and BYSL expression at RNA and protein levels was tested to verify the knockdown efficiency.CCK8 assay was used to evaluate the effect of BYSL knockdown on cell proliferation,flow cytometry was used to evaluate the effect of BYSL knockdown on cell apoptosis and cell cycle,Western Blot was applied to test the expression levels of cell cycle-related proteins Cyclin D1 and Cyclin A2.3.Lentivirus silencing BYSL was produced and subsequently infected Molm-13 and U937 cell lines,and BYSL expression at RNA and protein levels were tested to verify the knockdown efficiency.Soft agar clone formation assay was used to examine the effect of BYSL knockdown on cell clone formation.4.Western Blot was used to detect the regulation of PI3K/AKT pathway by knocking down BYSL gene in Molm-13 and U937 cell lines,and to examine the expression changes of PI3 K,p-PI3 K,AKT,and p-AKT.The PI3K/AKT pathway activator 740-YP was used for the pretreat for the cell line to detect the expression changes of the above-mentioned proteins.5.The binding site of c-MYC and BYSL gene promoter region was predicted by JASPAR website,we designed specific q PCR primers.In the Molm-13 cell line,CHIP-q PCR was applied to verify the binding of c-MYC to the BYSL gene promoter region.The c-MYC inhibitor 10058-F4 was used to treat Molm-13 and U937 cells,and Western Blot was used to detect the change of c-MYC and BYSL protein expression.Results:1.The expression of BYSL gene in 7 AML cell lines was tested,and it was found that the BYSL expression level of AML cell lines was higher than that of healthy human MNC.In different cell lines,BYSL expression level was higher in NB4,U937,and Molm-13,but lower in K562 and THP-1.Therefore,Molm-13 and U937 cell lines were chosen for the subsequent experimental research.2.Using si RNA technology to knock down BYSL gene in Molm-13 and U937 cell lines,RT-q PCR and Western Blot assays verified that the 2 si RNA could successfully knock down the BYSL level.3.After knocking down the BYSL in Molm-13 and U937 cell lines,cell proliferation was significantly inhibited,the proportion of apoptotic cells did not increase significantly,the cycle was blocked in G0/G1 phase,at the same time Western Blot detected decreased expression of cycle-related proteins Cyclin D1,Cyclin A2,the clone formation ability was significantly weakened.4.After knocking down the BYSL gene in Molm-13 and U937 cell lines,Western Blot assay showed that there was no significant change in the expression of PI3 K and AKT protein,while the expression of p-PI3 K and p-AKT protein was significantly reduced.When pretreated with PI3K/AKT pathway activator 740-YP,we found that 740-YP could up-regulate the expression of p-PI3 K and p-AKT protein,knockdown of BYSL could partially block the increase of p-PI3 K and pAKT expression caused by 740-YP,the same trend was detected in the expression levels of Cyclin D1 and Cyclin A2.5.We successfully predicted the binding of c-MYC and BYSL promoter region in JASPAR database.CHIP experiment was performed in Molm-13 cell line.The result of CHIP-q PCR showed that BYSL gene promoter DNA sequences were significantly enriched by c-MYC antibodies pulling down compared to that by negative Ig G antibodies.In Molm-13 and U937 cell lines,c-MYC inhibitor10058-F4 could significantly down-regulate the expression of c-MYC and at the same time down-regulate the expression of BYSL.Conclusion:Knockdown of BYSL gene could significantly inhibit the proliferation of AML cell lines Molm-13 and U937,block the cell cycle in G0/G1 phase,and weaken the ability of clone formation.BYSL gene may play a role through PI3K/AKT pathway.The upstream of BYSL is regulated by the transcription factor c-MYC.Part 3.Establishment of a clinically applicable Nomogram prognostic modelMaterials and Methods:1.We used the RNA-sequence data,clinicopathological and survival information of AML patients in the TCGA-LAML dataset as a development cohort for constructing the Nomogram model.All continuous variables such as age,white blood cell count,platelet count,hemoglobin concentration,bone marrow blast cell ratio and other factors were converted into categorical variables according to commonly used Cut-off values.Other categorical variables including gender,cytogenetic risk stratification,whether hematopoietic stem cell transplantation had been performed,whether there was a specific gene mutation,and the BYSL expression level were carried out in the form of categorical variables for subsequent analysis.With OS as the outcome variable,all factors were included in a univariate Cox regression analysis,the survival package in R was used for computational analysis,the Log-rank test was used to compare survival rates,and the prognostic factors(P <0.1)were screened out and included in the multivariate analysis,prognostic factors with P <0.05 in multivariate analysis were included as prognostic-related factors into the constructing of Nomogram.2.The concordance index was used to measure the accuracy of the Nomogram.The receiver operating characteristic curve,the ROC curve was used to evaluate the predictive performance of the Nomogram.It was quantified by AUC,the area under the curve.Similarly,the closer the value is to 1,the stronger the prediction ability and the more accurate it is.The calibration plots were conducted to compare the consistency between the predicted survival results of Nomogram and the survival results calculated by the Kaplan-Meier method.And according to the new prognostic model,the patients were grouped according to the risk score,and the survival difference between the high-risk group and the low-risk group was compared,which reflects the discrimination of patients with different risk stratifications of the Nomogram.3.We collected the clinicopathological information of the non M3 AML patient in the previous part for external validation of the Nomogram.The patients in the validation cohort were evaluated by Nomogram,and the total points of each patient(the score after the independent variables were weighted according to the Nomogram)were included as an independent factor into the further analysis,and then the ROC curve,calibration chart were plotted to evaluate the accuracy of the model.The survival difference between the high-risk group and the low-risk group was compared,which reflects the accuracy and applicability of the Nomogram.Results:1.By analyzing the impact of various factors on OS,we found that the high BYSL expression,age ≥ 60 years,no haemopoietic stem cell transplantation had been applied,higher cytogenetic risk,with FLT3-ITD mutation,FLT3-TKD mutation,DNMT3 A mutation,TP53 mutation or RUNX1 mutation were the risk factors of OS,and these factors were subsequently included in the multivariate Cox analysis.The results showed that high BYSL expression,age ≥60 years,no haemopoietic stem cell transplantation had been applied,higher cytogenetic risk level,with FLT3-ITD mutation and without NPM1 mutation,with TP53 mutation were independent prognostic factors of OS(P value<0.05).2.We integrated the above 6 statistically significant prognostic factors through R and establish a Nomogram model.After calculation,the model C-index was 0.754,which indicated that the Nomogram has a high prediction accuracy.The 1-year and 2-year OS predicted by Nomogram were in good consistency with real OS,with AUC of 0.80 and 0.82,respectively.At the same time,the calibration curves showed that the predicted survival of AML patients had a high degree of fit with the actual survival,but the prediction accuracy of 5-year OS was poor with AUC<0.7,and the calibration curve was far away from the diagonal,indicating predictive performance is not very good.According to the risk score generated after calculating the weight of Nomogram score,TCGA-LAML patients were divided into low-risk group and high-risk group according to the median value of risk score.According to the new risk stratification,the Kaplan-Meier curve showed that the survival rate of patients in the high-risk group was significantly lower than that in the low-risk group,with the P value of <0.0001,indicating that the Risk score had a good risk discrimination for patients.3.Then the information of the de novo non M3 AML patient from the West China Hospital was used for external validation.The results showed that the ROC curve of the validation cohort showed that the AUC scores for predicting 1-year OS and2-year OS were 0.70 and 0.69,suggesting that the model also had pretty good accuracy in predicting 1-year OS in the validation cohort.The validation cohort was grouped according to Risk score.The Kaplan-Meier curve showed that the OS of patients in the high-risk group was significantly shorter(P = 0.0194),suggesting that the new Nomogram prognostic model can distinguish between high-risk and low-risk patients.The figure shows that the 1-year OS calibration plot was located slightly above the diagonal,indicating that the model prediction still had good accuracy in the verification,but the actual survival was slightly higher than the predicted survival.Conclusion:We used the patients’ information in the TCGA database and included 6independent prognostic factors related to OS to establish a Nomogram prognostic model that could accurately predict the 1-year and 2-year OS of de novo non-M3 AML patients,and collected AML patients’ information at West China Hospital,to verify the model,the results showed that the Nomogram had high accuracy and wide applicability. |
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