Targeting Cathepsin L In Peripheral T-Cell Lymphoma: Based On Bioinformatics And Transcriptomics Analysis

BackgroundPeripheral T-cell lymphoma（PTCL）is a group of highly heterogeneous non-Hodgkin’s lymphoma（NHL）originating from T cells after thymus or mature NK cells.The incidence rate of PTCL is significantly higher in Asian populations than it in Europe or America populations.Unfortunately,there is no specific or effective method for PTCL clinical treatment at present.Most patients are not sensitive to CHOP/CHOP-like regimen which is considered as the first-line chemotherapy regimen.The high recurrence rate and the poor prognosis bring the severe challenges to clinical treatment of PTCL.Although the development and application of some molecular targeted drugs have brought new dawn to the treatment of PTCL,the efficacy of various drugs is different and still lack of the support of large-scale population cohort research.Therefore,it is still important to explore more targets of new drug development for precise treatment and individualized treatment of PTCL in the future.Cathepsin L,a member of cysteine protease family,is widely involved in various physiological processes,such as protein hydrolysis,antigen presentation,apoptosis,T lymphocyte differentiation and so on.Human studies have found an increase expression of cathepsin L in lung cancer,colorectal cancer,breast cancer,melanoma,endometrial cancer and other malignant tumors,which may be linked to the tumor progression,tumor invasion,tumor metabolism,tumor recurrence and prognosis of these malignant tumors.Due to the finding that cathepsin L is only increased in tumor cells,while none expression or little expression in normal tissues besides tumor compared to cathepsin B,K,S,C and other family proteins,cathepsin L has considered as one of the most vital factors to promote the occurrence and development of malignant tumors,and thereby an ideal molecular target for the treatment of malignant tumors.However,no systematic study on the expression and the role of cathepsin L in PTCL has taken out as far as we know.ObjectiveTo this end,the present study was performed to identify the levels of cathepsin L in PTCL tumors and normal controls.We hope our results could preliminarily reveal the possible role of cathepsin L in PTCL.MethodsBioinformatic analysis and transcriptomics analysis was used in this study.Firstly,the public biological database was used to analyze the differential expression of cathepsin L in PTCL and control tissues.Then,the tumor samples of newly diagnosed PTCL patients were used to verify the expression of cathepsin L by immunohistochemistry.Secondly,two PTCL cell lines Jurkat and Hut78 with stable and low expression of cathepsin L were constructed by sh RNA interference in vitro,and the knockdown of cathepsin L was verified by western blot.Next,the different expression genes between the cathepsin L knockdown group and the control group were analyzed by transcriptome（RNAseq）and thereby verified by RT-PCR.To take insight to the change of phenotype,GO and KEGG enrichment analysis of annotated different expressed gene was performed by Phyper based on Hypergeometric test.Lastly,to assess the role of cathepsin L in apoptosis in PTCL tumor cells according to the RNAseq analysis,the apoptosis antibody array was used to test the differential expression proteins between cathepsin L knockdown Jurkat cell group and the control group,and thereby verified by ELISA test.ResultsAccording to the analysis of TIMER,oncomine and GEO database,we found that the expression of cathepsin L was significantly increased in pan-cancer,including PTCL.The immunohistochemistry test also found an increase of cathepsin L in PTCL tumor tissue.The analysis by HPA database and CIBERSORT tool showed that there was no expression or little expression of cathepsin L in normal T cell subsets.The changes of different immune cell infiltration constituted the microenvironment of PTCL tumor.The gene enrichment analysis found that the differential expression genes might be related to a various pathway including lysosome,apoptosis,autophagy,antigen processing and presentation in PTCL.In order to explore the role of cathepsin L in PTCL,RNAi technology was performed to edit the two PTCL cells Jurkat and Hut78.The data of fluorescence microscope and flow cytometry showed about 30%cell infection rate in Jurkat and Hut78 cells.The result of western blot analysis confirmed that the expression of cathepsin L was significantly decreased in Jurkat cell and Hut78 cell after RNAi knockdown.Therefore,our data supported that two PTCL cell（Jurkat cell and Hut78 cell）with a stable and low expression of cathepsin L were set up for the furter studies.Next,our RNAseq analysis found that 602 differentially expressed genes were found in CTSL^low Jurkat vs.vector Jurkat cell,and 32 differentially expressed genes were found in CTSL^low Hut78 vs.vector Hut78 cell.A total of 8 common genes,including PAK4、FUS、HSPD1、LEF1、RRM2、CHAC1、SLC7A5、SESN2,was found between the CTSL^low Jurkat vs.vector Jurkat cell data set and CTSL^lowHut78 vs.vector Hut78 cell data set.The data of RT-PCR confirmed the change of these 8 differentially expressed genes.The gene enrichment analysis indicated that these 8 differentially expressed genes were mainly involved in glutathione metabolism and p53 signaling pathway.The apoptosis antibody array detection also found 8 differentially expressed proteins,like IGFBP-6、Fas、sTNF-R1、Bax、IGFBP-1、IGF-1s R and therey confirmed by ELISA test.The GO and KEGG enrichment analysis confirmed that p53 sigalling was involved in cathepsin L knockdown Jurkat cell compared with vector Jurkat cell.ConclusionsTaken together,our data indicated an increase expression of cathepsin L in PTCL tumor.The increased cathepsin L might play a role in the pathogenesis of PTCL via p53 signaling pathway.In addition,we also successfully constructed two PTCL cell lines with a stable and low expression of cathepsin,which can provide a tool in vitro experimental for the follow-up research.