| Objective:Colorectal cancer(CRC)is a serious gastrointestinal cancer with high morbidity and mortality,and 40%of patients have KRAS(Kirsten rat sarcoma viral oncogene homolog)gene mutation.There is no effective treatment option for KRAS-mutant CRC.Since the KRAS gene is also a major biomarker for resistance to anti-EGFR therapy,it is necessary to explore the pathogenesis of KRAS-mutant CRC and find more reliable biomarkers and potential drug targets.Our study screened KRAS mutation related genes through bioinformatics methods by using transcriptome data,and explored the relationship between KRAS mutation,KRAS mutation related genes and clinical characteristics and prognostic value,which provided a basis for finding new biomarkers and potential targets of KRAS mutant CRC.Methods:(1)The KRAS mutation-related CRC expression profile dataset was downloaded from GEO(Gene Expression Omnibus).Two microarray datasets(serial numbers:GSE156915 and GSE75316)were finally selected.Differential expression analysis was performed between the KRAS mutant group and the wild group using the R limma package to obtain differentially expressed genes,and function enrichment analysis was carried out by online analysis tools to look for KRAS mutation-related pathways.The shared genes of the two gene sets were used as KRAS mutation-related genes for follow-up studies.The expression of selected KRAS mutation-related genes was verified in the c Bio Portal database,and the correlation of clinical characteristics and prognosis with KRAS mutation were also analyzed.(2)From 2019 to 2021,123 CRC inpatients of the Gastrointestinal Surgery Department of our hospital were collected,the clinical information and follow-up data onto these patients were also sorted out at the same time.Immunohistochemical staining was performed on the screened KRAS mutation-related genes to determine the protein expression.Using hypothesis testing,the association for protein expression levels of KRAS mutation-related genes with clinical characteristics and prognosis in CRC patients was investigated.Log rank test was used to compare the survival curves,and multivariate survival analysis was conducted for related genes to build a clinical prognosis prediction model.Results:(1)Shared genes of the differentially expressed genes were taken between the microarray dataset GSE156915 and GSE75316,and KRAS mutation-related genes KRT6A and CEMIP shared by the two microarrays were obtained.These two genes were significantly upregulated in KRAS-mutant CRC patients compared with KRAS-wild patients.The GSE156915dataset was enriched with 29 GO terms,including 1 GO term involving KRT6A and 3 GO terms involving CEMIP;the GSE75316 dataset was enriched with 34 GO terms and 1 KEGG pathway(hsa04310:Wnt signaling pathway),including 2 GO terms related to KRT6A and 3 GO terms related to CEMIP.The function of KRT6A was mainly enriched in the cytosol(GO:0005829),and the function of CEMIP was mainly enriched in the GO terminology of the extracellular region(GO:0005576),suggesting that the functions of two different genes play a regulatory role in the extracellular components of KRAS mutant CRC.(2)The expression levels of KRT6A and CEMIP in the c Bio Portal database in the KRAS-mutant group were higher than those in the KRAS-wild group,which further verified the results of the GEO data.KRAS mutation was associated with four molecular subtypes of CRC(P<0.05).The survival curves of progression-free survival(PFS)between the KRAS-mutation group and the KRAS-wild group has statistically difference(P<0.05),and KRAS mutation was a risk factor of the PFS;but for overall survival(OS),there was no significant difference in survival curve between KRAS mutation group and wild group.The PPI network formed by differential genes contained 410 nodes and 689 edges,the average connectivity was 3.36 with the PPI enrichment P value<1.0e-16.After using Cytoscape to visualize the functional interaction proteins related to KRT6A and CEMIP,it was found that there were 20 proteins that interacted with KRT6A and 2 proteins that interacted with CEMIP.(3)Through the analysis of 123 CRC patients,it was found that 60 patients have KRAS mutation,and the KRAS mutation rate was 48.78%,of which G12D was the most common mutation type,with a mutation rate of 41.67%.KRAS mutation was associated with gender,but not with age,TNM stage,or other clinic characteristics.The postoperative PFS survival curve of the KRAS-mutant group declined faster,and the prognosis was significantly worse than that of the KRAS-wild group.(4)The positive expression rate of KRT6A protein was significantly increased in CRC cancer tissues,and the expression difference between cancer and adjacent tissues was significant(X~2=4.75,P<0.05).KRT6A protein was related to N stage of TNM staging in CRC patients;The positive rate of KRT6A protein was higher in patients with lymph node metastasis(X~2=15,P<0.05).There was no statistical correlation between KRT6A protein expression and KRAS mutation,nor with other clinical features or prognosis.(5)The positive expression rate of CEMIP protein in CRC cancer tissue was higher than that in the adjacent cancer tissue,and the expression difference was significant(X~2=7.79,P<0.05).CEMIP was related to N stage and M stage in TNM staging of CRC patients,and the positive expression rate of CEMIP was higher in patients with lymph node metastasis and distant metastasis.Additionally,CEMIP protein expression was higher in KRAS mutant CRC patients than in KRAS wild-type CRC patients(X~2=5.4,P<0.05).The postoperative progression-free survival curves between CEMIP positive and negative expression groups were significantly different(X~2=25.28,Log rank P<0.05);the negative CEMIP expression group had a better prognosis,and the positive CEMIP expression was an independent risk factor for poor prognosis(postoperative PFS).Conclusion:The KRAS mutation-related genes KRT6A and CEMIP were screened out from the transcriptome data of colorectal cancer.Both of these two proteins were associated with TNM stage of CRC patients,and especially the positive protein expression of CEMIP indicates that patients with CRC may have a worse prognosis.These two genes may serve as target genes or potential therapeutic targets for KRAS mutant CRC.However,the mechanism of KRT6A and CEMIP in CRC,especially in KRAS mutant CRC,is still unclear and needs further study. |
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