Discovery And Design Of Peptides As MMP9 Inhibitors For Targeted Mantle Cell Lymphoma Therapy

Objective: Mantle cell lymphoma is one of the invasive non-Hodgkin's lymphoma and usually has a poor prognosis.In recent years,the annual incidence of mantle cell lymphoma has increased to 1~2 per 100,000 people.Although the early stage of mantle cell lymphoma can also show better efficacy,the incidence of recurrence and drug resistance is higher with the progress of the disease.Meanwhile,high-dose chemotherapy increases the incidence of adverse reactions in patients and seriously affects the prognosis and quality of life of patients.Due to the influence of many factors,the mechanism of mantle cell lymphoma is still unclear.Currently,in-depth molecular biology research is needed to explore potential therapeutic targets for mantle cell lymphoma,a troublesome tumor,and to design new anti-tumor drugs against these targets.Protein-protein and peptide-protein complexes are abundant in the cell and are involved in all important biological processes.With the deepening of the study of molecular structure,the functions of these interactions are gradually revealed.The affinity of molecules to their target protein can be predicted by computer technology,and the identification process of molecules and their receptors can be simulated by geometric matching and energy matching.Molecular docking based on complex 3D structure is a method to predict the binding orientation of molecules and target proteins,which is not only very important for structural biology,but also shows important application value in drug design.In this study,mantle cell lymphoma was selected as the research object and MMP9 was selected as the target through bioinformatics analysis.The main research contents included three aspects:(1)Screening MMP9 as the key target of mantle cell lymphoma through bioinformatics,and investigating the correlation between MMP9 and the occurrence,development and prognosis of mantle cell lymphoma;(2)MOE software was used to screen,design and synthesize activated MMP9 targeted inhibitory peptides through molecular docking method.(3)Investigate the effect of MMP9 targeted inhibitory peptide on the biological activity of mantle cell lymphoma.The above study is intended to provide theoretical basis for the occurrence and development of mantle cell lymphoma and design new targeted drugs.Methods: 1.Differences in Gene Expression between mantle cell lymphoma tissues and normal lymphoid tissues were analyzed using GSE32018 and GSE9327 data sets in the Gene Expression Omnibus(GEO)database.GO analysis and Pathway analysis were used to predict the biological processes and pathways involved in differentially expressed genes(DEGs).The protein interaction network was constructed with STRING,and Cytohubba and MCODE plugins in Cytoscape 3.5.1 were used to analyze the protein interaction network and predict the target genes closely related to mantle cell lymphoma.Gene set enrichment analysis(GSEA)was conducted after grouping the samples in the data set of GSE32018 and GSE9327 according to the expression level of target genes to predict that the target protein involves pathways and functions.2.Immunohistochemistry the expression of MMP9 in mantle cell lymphoma and normal lymph node tissues was detected by immunohistochemistry.3.Cell model construction MMP9 silenced cell model was respectively constructed by transfection of MMP9 si RNA in the cell line of mantle cell lymphoma.4.q RT-PCR and Western Blot were used to detect MMP9 expression of mantle cell lymphoma cell lines.Western Blot analysis of protein expression changes of MMP9,E-cadherin,Vimentin,Cyclin D1,p-STAT3 and STAT3 after MMP9 were silenced.5.Peptide design.We searched the crystal structure of target protein in PDB database,imported it into MOE software,selected template peptide segment,set up peptide segment and target protein,replaced key amino acids according to amino acid physical and chemical properties,modified template peptide segment,and optimized peptide segment conformation;The module of MOE was used to dock the template peptide and modified peptide with the target protein,respectively.According to the principles of geometric complementarity and energy complementarity,the peptides with the best binding to the target protein were screened.6.Detection of cell proliferation,invasion and metastasis ability.CCK-8 was used to detect the changes of cell proliferation activity of mantle cell lymphoma cells after MMP9 was silenced and addition of targeted inhibitory peptides.Transwell analysis detected the changes of invasion and metastasis ability of mantle cell lymphoma cells after MMP9 was silenced and addition of targeted inhibitory peptides.7.Cell cycle and apoptosis detection.Flow cytometry detected changes in cell cycle and apoptosis of mantle cell lymphoma cells after addition of targeted inhibitory peptides.8.Peptide affinity assay.Flow cytometry and immunofluorescent were used to detect the affinity between mantle cell lymphoma cells and targeted inhibitory peptides.9.Detection of peptide inhibition of MMP9 activity.ELISA was used to detect the content of MMP9 in cell culture medium of mantle cell lymphoma after adding MMP9 targeted inhibitory peptides.The activity of MMP9 in cell culture medium of mantle cell lymphoma was detected by gelatinase assay.10.Data analysis.SPSS20.0 software was used for statistical analysis(SPSS Inc.,Chicago,IL,USA).The data were expressed as mean ± standard difference(±SD)of three independent experiments.T test and chi-square test were used for analysis;Wilcoxon rank sum test was used to analyzed the immunohistochemical data;Pearson's chi-square test and Fisher's test were used to compare the correlation between expression level and clinicopathological parameters.The relationship between expression level and survival was analyzed by univariate or multivariate analysis.Kaplan-meier method was used for survival analysis.All the results were significantly different with p<0.05.xResults: 1.Bioinformatics analysis predicted that MMP9,as a key target gene,would play an important role in the occurrence and development of mantle cell lymphoma.(1)GSE32018 and GSE9327 were selected from GEO database,including 62 cases of mantle cell lymphoma specimens and 14 cases of normal lymph node specimens.The GEO2 R program in GEO database was used to compare the gene expression differences between tumors and normal samples,and 84 DEGs were selected.Compared with normal tissues,MMP9 was highly expressed in mantle cell lymphoma tissues(p<0.05).(2)In gene enrichment analysis,GO analysis suggested that 84 DEGs were mainly concentrated in "signal transduction"(ontology: BP),"cell exosomes"(ontology: CC) and "protein binding"(ontology: MF).Meanwhile,KEGG pathway analysis showed that DEGs was rich in 24 pathways,such as "tumor pathway","PI3k-AKT signaling pathway","cytokine receptor interaction pathway","Rap1 signaling pathway","NF-? B signaling pathway" and "leukocyte transendothelium migration pathway".(3)We established 84 DEGs protein-protein interaction(PPI)networks through STRING,including 52 nodes and 95 edges.The top ten genes of Cytohubba plugin were VIM,MMP9,CDH1,CCND1,ITGB1,SPP1,CXCL12,IGF2,TNFSF11 and FGF7.VIM,MMP9,CDH1 and CCND1 were the key genes(Degree ?10).The most significant clusters in MCODE cluster analysis included 7 nodules and 17 edges,and included four key genes VIM,MMP9,CDH1 and CCND1.Among all tumor-related genes,MMP9 has a high degree in PPI network,and the three-dimensional X-ray crystal structure of MMP9 can be obtained from PDB database(ID: 1L6J).Therefore,MMP9 is selected as the target for subsequent studies.(4)According to the expression of MMP9,the 62 mantle cell lymphoma specimens of GSE32018 and GSE9327 data sets were divided into high expression of MMP9 and low expression group.According to GSEA KEGG database enrichment analysis software,results showed that the high expression of MMP9 group mainly occurred in 9 kinds of signaling pathways,one of the most significance for cellular adhesive molecular pathways(NSE = 1.74,p = 0.008,FDR = 0.065).(5)Further Oncomine analysis revealed that MMP9 expression was up-regulated in a variety of lymphoma tissues,including Burkitt lymphoma,diffuse large B cell lymphoma,follicular cell lymphoma and Hodgkin's lymphoma.Immunohistochemical results from human protein atlas data suggested that MMP9 expression was increased in non-Hodgkin's lymphoma compared to normal lymphoid tissue.2.MMP9 is highly expressed in human mantle cell lymphoma,promotes tumor cell proliferation and invasion and is associated with poor prognosis.(1)The expression of MMP9 in mantle cell lymphoma was detected by immunohistochemistry.MMP9 was highly expressed in 59 of the 88 tumor samples(67%).In contrast,MMP9 was highly expressed in only 12(30%)samples from 40 normal lymphoid tissues.MMP9 expression was significantly correlated with clinical stage staging,bone marrow infiltration and LDH level(p=0.010,p=0.033,p<0.001). MMP9 expression was not correlated with other clinicopathological parameters of mantle cell lymphoma.(2)Patients with high MMP9 expression had poor prognosis.High MMP9 expression was significantly associated with shorter overall survival(OS)and progression-free survival(PFS)compared with low MMP9 expression in patients with mantle cell lymphoma(p=0.012,p=0.030).Multivariate Cox analysis suggested that MMP9 was an independent prognostic risk factor for OS in patients with mantle cell lymphoma(p=0.027).(3)After inhibiting the expression of MMP9 in Jeko-1 cells of mantle cell lymphoma,the phenotype of epithelial cells was reversed to mesenchymal cells,E-cadherin expression was up-regulated,and Vimentin expression was down-regulated.Cyclin D1 and proto-oncogene STAT3,both of which are molecular markers of mantle cell lymphoma,were down-regulated in Jeko-1 cells with MMP9 silenced.(4)CCK-8 experiment results showed that Jeko-1 cells showed decreased proliferation activity after inhibiting MMP9 expression(p<0.01).Transwell assay showed that the invasion and metastasis ability of Jeko-1 cells decreased after inhibition of MMP9(p<0.05).3.MOE software was used to screen MMP9 targeted inhibitory peptides.(1)MMP9 usually exists in the body in the form of pro MMP9.Its propeptide is partially inserted into the fissure of the active site,preventing zinc ions in the catalytic active center from binding to the substrate.When activated,the propeptide falls off,exposing the active site and activating the function of MMP9.According to this characteristic of propeptide,the amino acid sequence TPRCGVPDL containing the key amino acid 97~100 PRCG in the propeptide sequence was selected as the template peptide to inhibit the activation of MMP9.According to the physicochemical properties of amino acids,different sequences of amino acids were designed as candidate peptides.(2)The candidate peptides and MMP9 structures were introduced into MOE software,and MMP9 was used to dock with each candidate peptide respectively by molecular docking module,and targeted inhibitory peptides were screened according to S score and interaction analysis.Peptides M1,M2 and M3 could bind to zinc ions in active sites and inhibited enzyme catalytic activity.M3 could be connected with Pro193,His405 and Leu409 amino acid residues,and obtained the lowest S score.Therefore,M1,M2 and M3 sequences were selected for solid phase peptide synthesis for subsequent verification experiments.4.MMP9 targeted inhibitory peptide M3 has a good affinity and inhibition.(1)The results of flow cytometry showed that all the four peptides were compatible with Jeko-1 cells,and the affinity gradually increased with the increase of peptide concentration.(2)Gelatine enzyme assay results showed that MMP9's ability to decompose gelatins gradually decreased with the gradually increasing concentrations of template peptide(M0)and M3,and M3 had a stronger effect on MMP9 activity than M0.Meanwhile,ELISA experiment showed that MMP9 expression did not decrease with the increase of M3,indicating that M3 only inhibited MMP9 activity without affecting its expression.Cellular immunofluorescence was used to detect that M3 was indeed compatible with Jeko-1 cells.5.MMP9 targeted inhibitory peptide M3 can inhibit the proliferation,invasion and metastasis of mantle cell lymphoma cells.(1)In CCK-8 experiment,Jeko-1 cells proliferation decreased gradually with the increase of M3 concentration,presenting a dose-dependent relationship.Compared with the control group,the difference was most significant at 48 h(p<0.01).(2)Flow cytometry showed that the apoptosis rates of Jeko-1 cells after adding 50 and 100 ?M M3 were 12.42±1.06% and 20.31±0.69%.The apoptosis rate of MMP9 group was 25.87±1.53%.The results of cell cycle analysis indicated that the PI index of jeko-1 cells decreased from 0.50±0.015 to 0.49±0.012 and 0.45±0.012 with the increase of M3 concentration from 0 to 50 and 100 ?M after 48 hours of culture.(3)In the invasion experiment,50 and 100 ?M M3 could significantly inhibit the invasion ability of Jeko-1 cells(54.5% p<0.01,63.6% p<0.01).Jeko-1 cell migration was significantly inhibited in the migration experiment(27.3% p<0.05,36.4% p<0.01).Conclusion: 1.Bioinformatics indicated that MMP9 was a key gene of mantle cell lymphoma and affected the proliferation,invasion and metastasis of mantle cell lymphoma cells.MMP9 was selected as the target of drug design.2.MMP9 was highly expressed in mantle cell lymphoma and was closely related to poor prognosis.3.MMP9 targeted inhibition peptide M3 could inhibit the proliferation,invasion and metastasis of mantle cell lymphoma,which was expected to be a new drug for the treatment of mantle cell lymphoma.