| Background:Colorectal cancer is one of the most common malignancies worldwide and one of the leading causes of cancer-related death.Surgical resection is the main choice for early-stage colorectal cancer,and chemotherapy is indicated for patients at risk of cancer recurrence as adjuvant therapy.Despite recent advances in targeted therapy in understanding the biology and development of malignant tumors,current therapeutic effects are still limited due to its high histological heterogeneity,and patient survival times remain suboptimal.Efficient and highly specific biomarkers and therapeutic targets are urgently needed.Therefore,it is necessary to further explore the relationship between new genes and its relationship with the occurrence and development of colorectal cancer and its malignant characteristics,so as to reveal the precise molecular mechanism of its occurrence and development,design reasonable therapeutic drugs and predict prognosis,and further improve the treatment level of colorectal cancer in our country.Objective:In this study,mass spectrometry proteomics technology combined with immunohistochemistry and public database mining was used to analyze the changes of protein expression profiles and related signaling pathways in colon cancer,and to deeply explore the key molecular markers of perineural invasion in colon cancer.At the same time,combined with immunohistochemistry and data from public databasets,we identified the expression changes of NENF in colon cancer and its clinical significance.Materials and methods:(1)10 pairs of colon cancer tissues and corresponding adjacent normal tissues that underwent surgical treatment without antitumor treatment before surgery were collected.Followed by tissue protein extraction,proteolytic digestion,and high-performance liquid chromatography peptide grading,the relative quantification of protein expression was performed by label-free quantitative LC/MS;(2)R-4.0.3 software was used to standardize and deal with missing values of proteome data,and paired student’s t-test was used to analyze and obtain differential proteins,and visualize.(3)Using a variety of bioinformatics methods:including GO,KEGG,GSEA to annotate the functions of differential proteins and explore the changes in related molecular signaling pathways.And STRING online website was use to analyze protein-protein interactions(4)For perineural invasion,the The included colon cancer tissues were subjected to subgroup differential analysis,protein function annotation and pathway analysis;(5)For key target molecules,download mRNA expression data from multiple datasets including TCGA and GEO,and verify its transcriptional level changes and effects.(6)138 colon cancer tissues with complete prognostic information were selected and tissue chips were made,including 75 colon cancer tissues and 63 adjacent normal tissues.Immunohistochemistry was used to identify the expression of NENF protein in colon cancer;(7)Statistical analysis and mapping in this study were using R-4.0.3 software or Prism 3.8.Result:1.The 10 pairs of colon cancer tissue samples and the corresponding adjacent normal tissue samples included in the study were sequenced by mass spectrometry,and a total of 9,695 proteins were identified.Paired t-test was used to analyze the differentially expressed proteins in the cancer and adjacent tissue samples.(|Log2FC|)>1 and pvalue<0.05 was used as the limiting condition for differential analysis.Finally,429 differentially expressed proteins were identified,of which 333 were significantly increased in colon cancer tissue samples.The expression of 96 proteins was significantly down-regulated in colon cancer tissue samples.KEGG analysis showed that the main enriched pathways of up-regulated proteins included:PI3K-AKT signaling pathway,phosphatidylinositol signaling system,phospholipase D signaling pathway,mTOR signaling pathway,MAPK signaling pathway.2.Analysis of perineural invasion(PNI)subgroups and calculation of log2FC and p value,a total of 517 proteins were differentially expressed,of which 447 proteins were highly expressed in the PNI-positive group,and 70 proteins were under-expressed in the PNI-positive group.GSEA enrichment analysis showed that compared with the PNI-negative group,the PNI-positive group mainly focused on the oxidative phosphorylation electron transport chain system in mitochondria,the GDNF/RET signaling pathway,the migration of neural crest cells during development,and the neural network in tumors.Enriched in pathways such as cristae cell migration.3.The expression changes of NENF transcription levels were verified in multiple colon cancer public datasets.The analysis showed that the expression of NENF mRNA was significantly increased in colon cancer tissues.Survival analysis showed that higher NENF mRNA expression was significantly correlated with the prognosis of patients.4.The expression of NENF was verified in colon cancer tissue by immunohistochemistry,and it was found that the protein expression of NENF was significantly up-regulated in colon cancer tissue,and the expression was higher in patients with stage Ⅲ and Ⅳ.The final survival analysis showed that the colon cancer with NENF expression Patients have shorter overall survival and worse prognosis.ConclusionIn this study,using label-free quantitative proteomics,immunohistochemistry,and bioinformatics analysis,we found that the protein expression profiles of colon cancer tissues and adjacent normal tissues were significantly different.Signal pathway enrichment analysis showed that PI3K-AKT signaling pathway,phosphatidylinos Classical signaling pathways such as alcohol signaling system,phospholipase D signaling pathway,mTOR signaling pathway,and MAPK signaling pathway are enriched in colon cancer and play an significant role in the occurrence and progression of tumors.Differential analysis of perineural invasion subgroups showed that there were certain differences in protein levels between the perineural invasion positive subgroup and the perineural invasion negative subgroup.The oxidative phosphorylation electron transport chain system in mitochondria,GDNF/RET signaling pathway,and the development Pathways such as neural crest cell migration and neural crest cell migration in cancer may play key roles in disease progression in the perineural invasion positive subgroup.At the same time,we found that the expression of NENF was significantly up-regulated in colon cancer tissue both in protein level and mRNA level,and was correlated with clinical stage,PNI and other pathological factors.It is expected to become a biomarker and therapeutic target for colon cancer. |
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