| Objects:Multiple Myeloma(MM)is a group of hematological malignancies characterized by abnormal expansion of clonal plasma cells in bone marrow.It is a common hematological disease and occupies the second place in the incidence of hematological malignancies.MM has a number of complications.These complications seriously affect patients' quality of life.Until now,MM is still incurable.So,it is very important to find new treatment methods for MM and improve patients' quality of life.Currently,MM has also ushered in the era of immunotherapy,including targeting therapy.The ideal treatment targets are mainly expressed on the membrane of malignant cells,but not expressed in normal cells or tissues(or low expression).Finding ideal targets,designing targeting drugs and stimulating immune cells to taget fighting malignant cells are crucial for the treatment of MM.The main contents of this study include:(1)to screen new targets for the treatment of multiple myeloma(2)to explore whether new target have clinical prognostic value(3)to design high affinity target-binding peptides Methods:1.BioinformaticsThe data sets of MM with normal plasma cell as control in GEO(http://www.ncbi.nlm.nih.gov/GEO)were used to analysis.The differentially expressed genes between MM cells and normal plasmocytes were screened to find key gene in MM.2.To verify the expression of target's mRNA in clinicThe CD138+ cells were isolated from the bone marrow of patients with MM and normal controls.The expression of HLA-E mRNA was detected by qRT-PCR.3.To detect the expression of HLA-E protein in MM patients by Flow cytometry and analyze the clinical parametersCollecting new diagnosed MM patients and normal controls' bone marrow.The expression of HLA-E on CD138 positive MM cells were detected by flow cytometry.Analysis of the significance of different HLA-E expression in MM patients with clinical parameters.4.To detect the expression of HLA-E protein in MM patients by immunohistochemistry and analyze MM patients' prognosisCollecting the pathological sections of patients diagnosed as MM between Jan 2013 and Jan 2016 and non-malignant plasma cell disease.The expression of HLA-E was detected by immunohistochemical staining.Divided the MM patients into two groups according to the expression of HLA-E and then made a judgment about the clinical parameters and prognosis of the two groups.5.To design the high affinity target-binding peptides to HLA-E by MOEAnalyzing the tnteractions between targeting proteins and ligand,screening and designing model peptide.The peptide library was constructed based on the model peptide using the residue scan module in MOE software for randomly replacing of non-key amino acids.All predicting high affinity peptides with HLA-E were tested.6.To verify the affinity of target-binding peptides with HLA-EThe affinity peptide labeled with FITC,fluorescence intensity and positive percentage were measured to find the highest affinity peptide.And then,HLA-E high expressed 293 T cells was incubated with HLA-E blocking antibody before adding the highest concentration of FITC-labeled peptide to detect the fluorescence intensity and positive percentage.Fresh bone marrow samples from MM patients were incubated with CD138,HLA-E antibodies and FITC-labeled fluorescent peptide M and P3,respectively.The expression of HLA-E on the surface of CD138 positive myeloma cells was detected.And the fluorescence intensity and percentage of FITC labeled peptides were analyzed in HLA-E positive and negative cells respectively which could represent the ratio of affinity.7.Statistical AnalysisStatistical analysis was done by SPSS23.0 software and GraphPad PRISM7,p<0.05 was considered as statistically significant difference.Mann-Whitney U test was used to compare the difference of non-normal distribution data.And independent sample t test was used to compare the difference of normal distribution data.Pearson chi-square test was used to compare the correlation between HLA-E expression and clinicopathologic parameters.Kaplan-Meier survival curve and log-rank t test were used to analyze OS and PFS in MM patients.The cut-off value of HLA-E expression was determined by ROC curve and the median of immunohistochemical expression.HLA-E expression in MM patients was divided into high and low expression groups to perform prognostic analysis.Descriptive data are expressed as mean standard±deviation.Results:1.Identification HLA-E as a key target of Multiple Myeloma(1)Screening high expression genes in MM by GEO database.Two datasets containing both myeloma cells and normal plasma cells,including 182 myeloma patients and 11 normal patients,were included for further analysing.The cells of the two datasets were from the same source,and the combined analysis was reasonable.At last,a total of 134 high expressed genes in MM were screened from both of the datasets.(2)Screening potential targets for MM therapy by bioinformatics.The protein-protein interaction network of 134 co-up-expressed genes was constructed and visualized by Cytoscape software.Three of the key clusters were obtained,which contained 31 key genes.The top 10 hub genes were screened by Cytohubba which pluged-in Cytoscape.Four genes,DDOST,HLA-E,NME1 and Canx,were obtained by crossing 31 genes in the key cluster and the top 10 hub genes in the protein-protein interaction network.(3)Identificaion HLA-E as treatment target of MM.The ideal therapeutic target of peptide drugs should be expressed mainly on the surface of MM cells,but little or no expressed in normal hematopoietic cells and tissues.Among these high expressed genes in MM,NME1 was an intracellular protein that did not qualified as a membrane protein.CANX,HLA-E and DDOST were all membrane proteins.To design affinity targetbinding peptides,the structure of the protein and it's interacted ligand was necessary.So far,HLA-E was the only one which had the structure and interaction ligand which seemed to be the only suitable target.(4)Validation of the differential expression of HLA-E mRNA between MM and nonmalignant patients.To confirm that HLA-E mRNA was over-expressed in MM patients,six bone marrow were collected from both newly diagnosed MM patients and control patients from ShengJing Hospital,respectivly.Myeloma and plasma cells were sorted by CD138 magnetic beads,and the mRNA expression was detected by PCR.The expression of HLA-E on six MM patients and six control patients was 0.1881±0.029 vs.0.048±0.016,respectively.There was a significant difference between the two groups(p <0.01).Verified the high expression of HLA-E mRNA in MM patients.2.High expression of HLA-E indicates poor prognosis of MM(1)Flow cytometry confirmed high expression of HLA-E protein in newly diagnosed MM patients.HLA-E expression was detected in 30 MM patients and 7 control patients.The expression of HLA-E antigen was 39.27±27.01(15.4-152.61)in MM and 11.28±0.79(8.82-14.33)in control patients,p<0.05.There was significant difference between the two groups.(2)HLA-E high expression indicated poor prognosis for MM.The 30 MM patients were divided into two groups according to the median expression of HLA-E.The relationship between age,gender,ISS stage and cytogenetics risk was investigated.The results showed that HLA-E expression was correlated with ISS stage of MM(p=0.025),that is,HLA-E expression was increased in ISS stage III MM patients.It was also associated with high-risk cytogenetics(p=0.000),that is,HLA-E expression was increased in high-risk MM patients.Thus,the increased expression of HLA-E in advanced and highrisk patients predicted that HLA-E was a poor prognosis marker.(3)The high expression of HLA-E was confirmed by immunohistochemical staining in patients with MM.The expression of HLA-E protein was detected and evaluated by immunohistochemical staining.The results showed that the expression of HLA-E was positive,158±58.63(60-270)in MM and nearly negetivge,43.5±20.82(20-90)in nonmalignant plasmacytosis.The expression of HLA-E in MM was significantly higher than that in control group(p<0.05).(4)HLA-E high expression indicated poor prognosis in MM.The Cutoff value of HLA-E determined by ROC curve was 145,and patients were divided into HLA-E highexpression group and low-expression group.Kaplan-Meier survival analysis showed that the OS of HLA-E high-expression group was significantly shortened(31 months vs.not reached),p<0.01.Similarly,PFS with high HLA-E expression was significantly shortened(17 months vs.not reached),p<0.01.Combined HLA-E and ISS staging system showed that low expression of HLA-E with ISS stage I and II had the best prognosis,both OS and PFS were the longest.Whereas,high expression of HLA-E with ISS stage III had the worst prognosis,both OS and PFS were the shortest.Thus,high expression of HLA-E was a poor prognostic marker for MM.3.Rational design HLA-E high affinity target-binding peptides by MOE(1)Identification of model peptide.The candidate of model peptide obtained from CD94 and NKG2 A by analyzing the interaction between HLA-E and CD94/NKG2 A.In order to optimize the stability of the peptide,the candidate amino acid sequence was performed by conformation searching.And then docking function in MOE software was used to exam the interaction between peptide to HLA-E.Peptide contained 12 amino acids obtained from CD94 was selected as the best candidate,and the amino acid sequence of the model peptide was NALDESCEDKNR.(2)Construction of a targeting HLA-E peptide library.The peptide library was constructed based on random mutating non-key amino in model peptides.All the peptides in the peptide library were docked to the key region of HLA-E.The high affinity targetbinding peptides were obtained by ranking according to the affinity and stability.We selected the best of the top three peptides and named them PEPTIDE1-3(P1-3).Among them,P3 was the most compatible and stable peptide,which interacts with HLA-E key region by forming the most bonds.And P3 has the lowest energy and stable conformation which could be considered as the best one.4.Verify the affinity of peptides to HLA-E(1)Verify the peptides' affinity with HLA-E by flow cytometry in HLA-E positive cell line.293 T cell line didn't express HLA-E protein on cell surface.And after HLA-E plasmid transfection,HLA-E protein could be detected by FCM.The fluorescence intensity and expression percentage of FITC confirmed that the template peptides M and P1-3 were not interacted with the 293 T cells before transfection for little FITC fluorescence could be detected.Whereas FITC fluorescence could be detected on 293 T cells after transfection with HLA-E,suggesting that both model peptide M and P1-3 could target HLA-E protein.At the meanwhile,the results showed that the percentage of positive expression cells increased with the increase of peptide concentration,suggesting the affinity increased.Among M and P1-3,P3 had the best affinity of 85.21±3.05%,which was consistent with the prediction by MOE.HLA-E blocking antibody was added to HLAE high expressed 293 T cells for 30 min and then incubated with 50?g/ml FITC labeled P3,the results showed that the percentage of FITC positive cells decreased to 2.27±0.63%,which confirmed that P3 had high affinity to HLA-E antigen.(2)Verify P3' affinity to HLA-E by confocal laser microscopy.U266 cell line was a common myeloma cell line.HLA-E protein could not be detected on the surface of U266 cell line by FCM.As previous reported,adding leader peptide and negative peptide pretreated U266 cells.HLA-E protein was detected on the surface of leader peptide pretreated U266 cells,but still could not be detected in negative peptide pretreated cells.Confocal microscopy was used to detect the affinity between P3 and HLAE high expressed U266 cells,suggesting that the affinity of target peptides increased with the P3' concentration.After incubated the HLA-E high expressed U266 cells with HLA-E blocking antibody,50?g/ml P3 was adding but nearly no fluorescence signal was detected.(3)Verify P3' affinity to HLA-E positive myeloma patients' bone marrow by flow cytometry.The bone marrow samples of MM patients were collected,and the CD138+ and HLAE+ MM cells were identified by Flow cytometry.Adding FITC labeled M and P3 respectively with CD138 and HLA-E antibodies into MM patient's bone marrow sample.HLA-E-positive myeloma cells were found to be able to detect FITC fluorescein,while in HLA-E negative myeloma cells,almost no FITC fluorescein was detected,suggesting that both template peptide M and P3 could target HLA-E positive MM cells,and the affinity of P3 was significantly increased.Conclusion:1.Bioinformatics analysis showed that HLA-E could be considered as a key marker of MM2.Clinical parameter analysis showed that high expression of HLA-E was one of the risk factors of MM3.High expression of HLA-E was a poor prognostic factor for MM4.HLA-E could be considered a therapeutic target for MM5.P3 was the high affinity target-binding peptide to HLA-E-positive myeloma cells... |
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