Screening And Identification Of Molecular Markers For Acute Myeloid Leukemia Based On Homoharringtonine Resistant Cell Lines

Background Acute myeloid leukemia(AML)is one of the most common and highly heterogeneous hematological malignancies in adults.It is characterized by impaired differentiation and maturation of blood cells,inhibition of normal hematopoiesis,and immature cells gathering in the bone marrow or peripheral blood,clinically leading to symptoms such as fever,infection,bleeding,and anemia.Even under the current advanced management and treatment,AML is still a disease with high recurrence and high death.The overall five-year survival rate is less than 50%,and it is less than 20% in elderly patients.With the popularization of sequencing technology,new mutations and molecular markers related to epigenetics and proteomics are constantly being discovered,which are the most important for the risk stratification and treatment of AML.Targeting drugs for FLT3-ITD mutations,IDH1/2 mutations,and BCL2 inhibitors have been used in clinical trials or clinical treatment of AML,and have achieved good results.Therefore,the discovery and identification of new AML molecular markers is very useful for the screening,diagnosis,prognosis,monitoring and treatment of AML,as well as predicting the possibility of an individual’s response to individualized treatment.Homoharringtonine(HHT)is an alkaloid extracted from plants,approved by the US FDA for the treatment of chronic myeloid leukemia(CML).HHT is also widely used in the treatment of AML in China.Our team innovatively proposed the "HAA" program.Through multi-center clinical research,it has become the first-line treatment program for low-and medium-risk AML patients in China.Accepting the HHT-based HAA program also faces the problem of drug resistance recurrence,and the problem of HHT resistance is still lacking in research.We constructed HHT-resistant cell lines,studied the transcriptome changes of HHT-resistant cell lines through high-throughput sequencing technology,and analyzed the differentially expressed genes between sensitive cell lines and drug-resistant cell lines to reveal the mechanism of HHT resistance,and explore new AML molecular markers for AML.Objective Through drug susceptibility experiments on 9 AML cell lines,select HHT-sensitive cell lines for constructing HHT resisitant cell lines,increasing HHT from low concentration to high concentration to establish AML cell lines with different HHT resistance indexes(RI).Compare HHT resistant cells and sensitive biological changes.Through the second-generation high-throughput sequencing RNA-sequence,the transcriptome changes of HHT-sensitive cell lines and different HHT-resistant cell lines were compared,and the possible mechanism of HHT resistance was revealed through bioinformatics analysis.Further use public big data combined with differentially expressed genes after HHT resistance to screen for new molecular markers with prognostic and/or therapeutic significance for AML,and further use our center’s AML sample library to further verify the newly discovered molecular markers for AML.We use molecular biology techniques to explore the regulatory effects and regulatory mechanism of newly discovered molecular markers on AML.In order to improve the prognostic stratification system of AML,provide new targets for the screening of targeted drugs for AML,and provide an experimental basis for the individualized management and treatment of AML in the future.Methods 1.Using MTS method to study the IC50 of HHT on 9 AML cell lines THP-1,KG-1,U937,MOLM13,MV4-11,HL-60,Kasumi-1a,OCI-AML3,NB4.Select the cell line most sensitive to HHT to induce HHT resistance.HHT is added to the cell culture medium from a low concentration,and the HHT concentration is gradually increased according to the arithmetic difference.When HHT can grow normally for 2 weeks in a specific HHT culture environment,adjust the HHT concentration.The resistance index(RI)was used to evaluate the resistance effect of HHT,and cells with different resistance indexes were frozen.When cells can grow normally and stably under the culture environment of HHT 50nm/L,stop the induction of drug resistance.Select the drug-resistant cell lines established under the culture conditions of 10nm/L,30nm/L,and 50nm/L to study the dynamics of HHT resistance.2.Using flow cytometry to detect apoptosis of HHT-sensitive cell lines and drugresistant cell lines,use MTS method to measure the growth curves of HHT-sensitive cell lines and HHT-resistant cell lines,and use flow cytometry to detect HHT-sensitive cell lines and HHT-resistant cells.The cell cycle of the strain and the expression of the multidrug resistance gene p-gp on the cell surface change.A nude mouse subcutaneous tumor-bearing model was used to observe the effect of HHT resistance in vivo.3.Performing high-throughput second-generation sequencing RNA-sequence(RNA-seq)on HHT-sensitive cell lines and drug-resistant cell lines,and compare the transcription of HHT-sensitive cell lines and cell lines with different drug RI through RNA-seq analysis.The RNA-seq results were verified by q RT-PCR.Furthermore,through GO enrichment analysis and Hub gene screening of differentially expressed genes,the relevant regulatory pathways and main regulatory genes of HHT resistance were analyzed,and preliminary verification was carried out.4.Using the online public databases UALCAN and GEPIA to analyze the prognosis of AML with significantly differentially expressed genes(DEGs)between HHT sensitive cell lines and HHT resistant cell lines.Select to meet the role of oncogene in AML,the prognostic P value of AML is less than 0.001 and has not been studied in AML.Making research on its expression,prognosis and regulatory mechanism in AML.5.Selecting a differentially high expression gene and a differentially low expression gene for research.Use our central AML sample library to analyze the expression level of differentially expressed genes in AML by q RT-PCR,and combine the patient’s age,gender,white blood cell,red blood cell,hemoglobin,platelet,bone marrow blast cell ratio,chromosome karyotype,gene mutation type,Treatment plan,treatment effect,survival time and other clinical data,analyze the expression characteristics of differentially expressed genes in AML,the treatment response of AML patients with different gene expression levels,overall survival time(OS),event-free survival time(EFS),and recurrence-free survival Time(RFS).Multivariate regression analysis was used to analyze the independent prognostic significance of DEG for AML patients.6.Using small hairpin RNA(sh RNA)targeted knockdown technology to knock down differentially expressed genes in AML cells,and verify the knockdown effect through flow detection of GFP expression,q RT-PCR and Western Blot.Observe the effects of DEGs on cell growth,apoptosis,cycle,surface antigen,differentiation and other biological functions after knockdown of AML cells.7.Using high-throughput second-generation sequencing to analyze the RNA-seq of AML cells that normally express differential genes and differential gene knockdown cells,and combine the TCGA database AML patient RNA-seq data to analyze the molecular mechanism of differentially expressed genes on the regulation of AML.Verification by western blot.8.Using AML animal models to study the regulatory effects of differentially expressed genes on AML in vivo.AML cells transfected and untransfected with sh RNA were injected into immunodeficient NCG mice through the tail vein.The success of the AML model was judged by the expression of CD45 in peripheral blood of mice or in vivo imaging.The survival time of mice was observed and recorded,and the tumor burden and survival time differences of mice in different treatment groups were compared.9.Using auto-dock computer model to analyze the targeting relationship between HHT and differentially expressed genes,and use this technology to find new targeted drugs for differentially expressed genes,and preliminarily judge the toxicity of targeted drugs to AML cells through proliferation and apoptosis experiments effect.Results 1.The concentration of HHT on AML cell lines was in the order of nmol/L,among which AML cell lines MV4-11 and MOLM13 were the most sensitive to HHT,which were 7.207 and 6.858 nmol/L respectively.By gradually increasing the concentration of HHT to induce culture,successfully established cell lines with different resistance indexes of HHT in MV4-11 and MOLM13 cell lines,named MV4-11R10,MV4-11R30,MV4-11R50,MOLM13R10,MOL13R30,MOLM13R50.The IC50 of MV4-11 R50 and MOLM13 R50 resistant cell lines cultured with FFT 50 nmol/L to HHT were 58.82 nmol/L and 109.9 nmol/L,respectively,and the drug resistance index(RI)were 15.81 and 28.11,respectively,which were high drug resistance level.The normal activity of drugresistant cell lines was proved by flow cytometry and the subcutaneous tumor in nude mice proved its drug resistance in vivo.2.Through growth,cellcycle and surface antigen analysis,it is found that the growth rate of HHT-resistant cell lines was slower than sensitive cell lines.There was a delay in the G0/G1 phase of the cell cycle and a decrease in the S phase.The surface of HHTresistant cell lines had increasing p-gp expression.Through the analysis of RNA-seq data,we found that immune regulation related pathways,especially G protein-coupled receptor-mediated immune signals,played an important role in HHT resistance.G protein-related proteins GNAI1 and CALCRL had prognostic significance for AML and may mediate HHT resistance.Further combining with significant DEGs and public online databases UALCAN and GEPIA,we found that differentially lowly expressed DPYSL2,C10orf128 and differentially highly expressed SPATS2 L,MAP4K1 had significant prognostic significance for AML,and their P values were both less than 0.001 and had not been studied in AML.3.It was confirmed by q RT-PCR and western that DPYSL2 was under-expressed in different HHT-resistant cells.Combining the data of AML patients in our sample bank and the data of the TCGA database,it was found that the expression level of DPYASL2 in AML patients was higher than that of normal cells.DPYSL2 expression decreased with the remission of treatment.Both the TCGA database and our center database showed that patients with DPYSL2 high expression of AML had shorter OS.Further analysis of our center data found that patients with high expression of DPYSL2 alsohad worse EFS and RFS.Multivariate analysis found that the high expression of DPYSL2 was an independent poor prognostic factor for AML patients with OS,EFS,and RFS.4.Knockdown of DPYSL2 expression in AML cells by sh RNA can inhibit the growth of AML cells and promote AML cell apoptosis,but had no effect on the differentiation of AML cells.Western blot found that knocking down DPYSL2 can increase the splicing bands of apoptosis regulatory proteins PARP and caspase3,and reduce the expression of phosphorylated BCL2,but had no effect on the total BCL2 expression.The mouse AML model also confirmed that DPYSL2 KD can reduce the tumor burden of AML and prolong the survival time of AML mice.5.By analyzing the RNA-seq data of DPYSL2 control and DPYSL2 KD,it was found that the PI3K-AKT signaling pathway was inhibited in DPYSL2 KD cells.Through the analysis of TCGA RNA-seq,it was found that the PI3K/AKT pathway and the upstream JAK/STAT3/5 signaling pathway were more active in the DPYSL2 high expression group.Further western blot confirmed that JAK2/STAT3/STAT5-PI3K/AKT was the main regulatory pathway of DPYSL2 to AML.Auto-dock computer docking simulation found that both HHT and dihydroartemisinin(DHA)can directly target DPYSL2 to exert a good anti-leukemia effect.6.It was confirmed by q RT-PCR and western blot that SPATS2 L was highly expressed in different HHT resistant cells.It was found by q RT-PCR that SPATS2 L was expressed in many hematologic malignant tumors,and the expression was highest in AML cell line THP-1 and multiple myeloma cell line MMIS.Confocal fluorescence reveals that SPATS2 L was localized in the nucleus of AML cells.Combining the data of AML patients in our sample database and the data of the TCGA database,it was found that the expression level of SPAT2 L in acute monocytic leukemia was higher than that of other types of AML,and the expression level of AML patients with intermediate and highrisk prognosis is higher than that of AML with good prognosis.The expression of SPATS2 L decrease with the remission of treatment.Both the TCGA database and our center database showed that patients with SPATS2 L overexpressing AML had shorter OS.Further analysis of our center data found that patients with high expression of SPAT2 L AML also had worse EFS and RFS.Multivariate analysis found that high expression of SPATS2 L was an independent poor prognostic factor of AML patients with OS,EFS,and RFS.SPAST2 L had better prognostic significance for RFS in AML patients.7.Targeted knockdown of SPATS2 L expression in AML cells by sh RNA can inhibit AML cell growth,promote AML cell apoptosis,and reduce surface p-gp expression but had no effect on the cellcycle of AML cells.Western blot found that knocking down SPATS2 L can increase the cleavage bands of the regulatory proteins PARP and caspase3,and reduce the expression of phosphorylated BCL2,but had no effect on the total BCL2 expression.The mouse AML model also confirmed that SPATS2 L KD can reduce the second-generation tumor burden of AML and prolong the survival time of AML mice.8.By analyzing the RNA-seq data of SPATS2 L control and SPATS2 L KD and the TCGA database RNA-seq data,it was found that the JAK/STAT3/5 signaling pathway was inhibited after SPATS2 L suppressed.Further western blot confirmed that JAK2/STAT3/STAT5 signaling was the main regulatory pathway of SPATS2 L to AML.Decreasing the expression of SPATSL2 in AML cells can promote the apoptosispromoting effect of AML chemotherapeutics.Inhibitors targeting STAT3/STAT5 can reduce the expression of SPATSL2 in AML cells andhad anti-leukemia effects.Conclusion 1.Successfully constructed AML cell lines with different resistance indexes of HHT.The delay of G0/G1 phase and the decrease of S phase of the cell cycle in HHT resistant cell lines.The increased expression of cell surface antigen p-gp may cause the cells to be resistant to HHT.Through RNA-seq,it was discovered that G protein-related molecule GNAI1,CALCRL-mediated immune activation may be the molecular mechanism of HHT resistance.2.DPYSL2 was a gene with low differential expression in drug-resistant cell lines.The expression of this gene in AML cells was significantly higher than that in normal cells.3.AML patients with high expression of DPYSL2 had poor prognosis.DPYSL2 was an independent molecular marker of poor prognosis of AML.4.DPYSL2 may play a regulatory role on AML through the JAK2/STAT3/STAT5-PI3K/AKT/GSK3 b axis.5.Through auto-dock computer molecular docking analysis technology,it found that both HHT and DHA may be the direct target drugs of DPYSL2.6.SPATS2 L was a differentially highly expressed gene located in the nucleus of AML cells.7.SPATS2 L was highly expressed in M5 and medium-to high-risk AML patients,and was associated with poor prognosis.It was a new independent molecular marker of poor prognosis for AML.8.SPATS2 L may play a regulatory role on AML through the JAK2/STAT3/STAT5 axis.In this study,we constructed HHT resistant cell lines with increasing RI and revealed the possible mechanism of HHT resistance.Based on HHT-resistant cell lines,we discovered and proved two new molecular markers for prognosis and treatment of AML,explored their targeted drugs and revealed the mechanism of the two markers in AML,which are of great significance for the improvement of the prognosis system of AML,the development of new drugs and the personalized treatment of AML.