| Backgrouds:Melanoma is a highly malignant skin tumor with high mortality and great harm,and its incidence is rising rapidly,posing a serious threat to human health.Therefore,it is particularly important to explore its potential driving oncogenes,pathogenesis and therapeutic strategies.This research project first discovered the potential oncogenetic role of CRKL gene in melanoma by whole exome sequencing.CRKL is only reported in acral melanoma currently.However,the relationship of CRKL in the malignant process of malignant melanoma has not been explored yet,and further experimental verification is needed.At present,CRKL has been reported as an oncogene in some types of carcinomas,and there are other studies reporting that overexpression of the CRKL gene has a tumor suppressor effect in some cancers.Whereas,a systematic perspective of CRKL provided by integrated analysis across cancer is also demanded.Objectives:The objective of our research project is to further verify the association between CRKL and the malignant process as well as clinical prognosis of melanoma from molecular,cellular,and clinical levels,and to explore its carcinogenic mechanism,its possibility as a melanoma molecular marker and new therapeutic target,its potential as a tumor prognostic marker as well as its functional role across cancers.Methods:First,we performed whole-exome sequencing on a large number of melanoma tissues.After screening the target gene CRKL,we used clinicopathological data,The Cancer Genome Atlas(TCGA),and samples from the Genotype Tissue Expression Project(GTEx)The information was statistically analyzed using R software v4.0.3 for the expression of CRKL in the samples and its association with genetic heterogeneity,tumor stemness,tumor mutational burden(TMB),etc.UCSC Xena was used to calculate the overall survival and disease-specific survival of cancer samples,and to evaluate the expression of CRKL in melanoma and its correlation with prognosis.Using the c Bio Portal database,the R software "maftools" tool was used to further dissect the CRKL mutation in melanoma.Secondly,the Gene MANIA(http://genemania.org/)database was used to identify genes with the same and similar functions as CRKL and construct a protein interaction network,and further we used the cluster Profiler package in R software to determine the genes related to CRKL through KEGG and GO analysis and identified potentially associated pathways.Through clinical tissues and melanoma cell lines,RT-PCR and immunohistochemistry were used to verify the expression of CRKL,and si RNA silencing technology was used to knockdown the CRKL of melanoma cell lines A375,WM451 and WM793.Through CCK8 experiments,clone formation experiments,Scratch test,transwell invasion test,flow cytometry and apoptosis test,we verified the biological behavior including proliferation,migration and invasion of melanoma in vitro.We further used lentivirus infection to construct the A375 cell line that stably silences CRKL,and conducted subcutaneous tumorigenesis experiments in nude mice for in vivo verification.Subsequently,Hoechst 33342/PI double-stained fluorescence detection was used for the A375 cell line with stable silencing of CRKL and the control group,and q RT-PCR and western blot were used to detect the expression of molecules related to apoptosis and pyroptosis pathways.Also,the cell supernatant was collected.LDH release assay and ELISA were used to explore the release of inflammatory factors in melanoma cells after CRKL silencing.We further used TIMER 2.0,x CELL,CIBSORT,ESTIMATE and other algorithms to explore the correlation of the target gene CRKL with immune cell infiltration and immune microenvironment in melanoma.Through the MSig DB database,R language was used to screen the differentially expressed genes(DEGs)of the target gene CRKL in SKCM,and GSEA enrichment analysis was performed to evaluate the potential signaling pathways of the target gene CRKL.In addition,we assessed the association between CRKL expression and melanoma immune response using the Tumor Immune Dysfunction and Rejection Index(TIDE),from which transcriptome profiles were obtained from melanoma patients receiving anti-PD-1 therapy by R.Results:The results of database analysis,tissue microarray IHC staining and PCR showed that the expression of CRKL was increased in melanoma tissues.The higher the expression of CRKL in melanoma,the worse the prognosis of the patients.Gene mutation analysis showed that missense mutations were the main type of CRKL mutations in SKCM.The genetic heterogeneity analysis demonstrated that among the RNA modifications associated with CRKL,melanoma was associated with 40 RNA modifications;in melanoma samples,CRKL expression was negatively correlated with DNMT1 and DNMT3 b.The results of tumor stemness analysis indicated that the expression level of CRKL in melanoma was positively associated with epigenetically regulated RNA expression-based genes stemness scores(EREG.EXPss).In addition,CRKL is also abnormally expressed in a variety of other tumors,which is also associated with genomic heterogeneity,epigenetic modification,cancer stemness,and affects the prognosis of patients to varying degrees.The protein interaction and enrichment analysis showed that CRKL was closely related to Ras signaling pathway,Erb B signaling pathway,and cytoskeleton in malignant tumors.In the in vitro test,CCK8,colony formation,scratch assay,transwell invasion experiments,and tumor formation experiments in nude mice suggested that after silencing the expression of CRKL,the proliferation,migration,invasion,and tumorigenic ability of melanoma cells were significantly inhibited.However,knockdown of CRKL had no clear effect on the cell cycle of melanoma cells.The flow cytometry assay suggested that CRKL knockdown induced cell death of human melanoma cells.Hoechst 33342/PI staining results showed that a large number of PI positive cells appeared after CRKL knockdown.After knockdown of CRKL,the secretion of IL-1β,IL-18 and LDH in the supernatant of melanoma cells increased(P < 0.05).The expression levels of various proteins including NF-κB p65,NLRP3,Caspase 1,GSDMD,ASC,cleaved-Caspase 1,GSDMD-N increased after CRKL silencing(P<0.05),the expression level of Cleaved-caspase 3 was slightly increased(P<0.05),and BAX and Caspase 3 proteins expression did not change significantly.The results of the immune infiltration study on CRKL and melanoma suggested that the expression of CRKL was negatively correlated with the infiltration level of CD8+ T cells and TH1 helper T cells.Meanwhile,ESTIMATE analysis indicated that CRKL gene expression level was negatively associated with Immune and ESTIMATE scores.The results of functional enrichment analysis of CRKL-related differential genes showed that CRKL was significantly related to the immune response and inflammatory response pathways of melanoma,as well as biological structures or functions such as intermediate filaments and cytoskeleton.In various types of cancers including melanoma,CRKL is also associated with a large number of immune checkpoints and immune regulatory molecules.TIDE analysis also demonstrated that PD-1-treated SKCM patients with low CRKL expression levels displayed more favourable survival rate and better prognosis.Conclusion:CRKL is significantly up-regulated in melanoma and is closely related to the prognosis of melanoma patients.Knockdown of CRKL inhibits the proliferation,migration and invasion of melanoma,and induces the activation of the Caspase 1-GSDMD-dependent pyroptosis pathway.The CRKL gene is closely related to epigenetic modification,tumor stemness,inflammatory response,tumor immune microenvironment,various immune regulatory factors and immunotherapy response in melanoma.50 figures,10 tables,142 references... |
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