| Part Ⅰ Expression of PDLIM7 in Colon Cancer and its relationship with tumor function-related PathwayObjective: The objective of this study is to examine the expression of PDLIM7 in colon cancer and its association with clinicopathological characteristics.Additionally,this research aims to investigate the role of PDLIM7 in colon cancer through an analysis of PDLIM7 methylation,copy number variation,somatic mutation,single-cell sequencing,and the microbiome.Methods: In accordance with predetermined inclusion and exclusion criteria,this study obtained a total of 86 colon cancer tissue specimens and 86 corresponding normal colon tissue specimens from the Department of Gastrointestinal Surgery at Northern Jiangsu People’s Hospital,between June 2015 and June 2016.The investigation primarily focused on analyzing the expression of PDLIM7 in colon cancer,and examining its potential correlation with various clinicopathological characteristics.The study employed various analytical tools to analyze data from the TCGA-COAD cohort related to PDLIM7 expression,methylation,copy number variation,and somatic mutation in publicly available datasets.Online bioinformatics data analysis platforms,R software,and related software packages were utilized for this purpose.Additionally,single-cell sequencing and microbiology data were integrated to explore the functional relationship between PDLIM7 and colon cancer.Specifically,the Linked Omics online analysis platform was used to analyze the TCGA-COAD expression data,and the co-expression gene set of PDLIM7 was identified.These gene sets were further analyzed using GSEA to examinethe potential effect of PDLIM7 expression on functional pathways associated with colon cancer.The MEXPRESS online platform was used to examine the methylation profile of PDLIM7 and its potential relationship with PDLIM7 expression.In addition,the TIMMER2.0 platform was utilized to analyze copy number variation of PDLIM7 across various cancer types,while c Bio Portal was used to investigate copy number variation of PDLIM7 in colon cancer and its potential impact on the colon cancer genome.Furthermore,the "maftools" software package was employed to analyze and compare the somatic mutation rates of PDLIM7 across various cancer types,and to reveal the somatic mutation landscape of colon cancer.This study utilized TCMA and c Bio Portal,to analyze the somatic mutation of PDLIM7 and examine its potential impact on the enrichment of colon cancer microbiome.The functional enrichment analysis was carried out using the "cluster Profiler" software package.Additionally,TISCH2 was employed to analyze the single-cell data set GSE146771 of colon cancer,and to preliminarily investigate the expression of PDLIM7 and its potential effect on the functional status of cancer cells in Cancer SEA.Finally,the "survival" software package and Kaplan-Meier Plotter online analysis were utilized to visualize the impact of PDLIM7 on survival.Results: 1.In this study,we investigated the expression of PDLIM7 in surgically resected colon cancer specimens and its correlation with clinical and pathological features.Our results showed that PDLIM7 was highly expressed in colon cancer tissues and significantly correlated with tumor differentiation,AJCC stage,and the number of lymph node metastasis(p<0.05).Furthermore,PDLIM7 expression was significantly different between the tumor group and normal colon tissue(p<0.05).In the TCGA-COAD cohort,PDLIM7 expression was also significantly correlated with N stage,AJCC stage,nerve invasion,and lymphatic invasion(p<0.05).Moreover,we found that high expression of PDLIM7 was associated with poor prognosis of colon cancer(p<0.05).Finally,our Gene Set Enrichment Analysis(GSEA)revealed that the co-expression gene set of PDLIM7 was significantly enriched in the pathways of lymphatic development,extracellular matrix(ECM),ECM structural components,and extracellular matrix receptor interaction(FDR <0.05).2.The Cp G island of PDLIM7 is located in its promoter region and has been found to be de-methylated in colon cancer,which may contribute to the upregulation of PDLIM7 expression.Our correlation analysis revealed a statistically significant negative correlation between DNA methylation and the expression of PDLIM7(r=-0.39,p<0.001),suggesting that DNA methylation may play a role in the regulation of PDLIM7 expression.3.Our analysis of copy number variation(CNV)in PDLIM7 in colon cancer revealed that the predominant alterations were amplification and deletion,with no instances of deep deletion or high amplification.These findings suggest that large-scale changes in chromosome structure in the vicinity of PDLIM7 may not result in extensive alterations in gene expression.Furthermore,our results indicate that PDLIM7 expression in colon cancer is relatively unaffected by CNV.However,we found a significant correlation between PDLIM7 CNV and the activity of metallopeptidase in function-related pathways in colon cancer(p<0.05).Specifically,alterations in PDLIM7 CNV may affect the activity of matrix metalloproteinases and alter extracellular matrix remodeling in colon cancer.4.The somatic mutation rate of PDLIM7 in colon cancer was found to be 2.5%,with missense mutations being the only type of mutation observed.While this mutation rate is relatively low,PDLIM7 has a higher mutation rate in colon cancer compared to other cancers.In patients with colon cancer,missense mutations are the most common type of mutation observed,with SNP(single nucleotide polymorphism)being the predominant type of mutation detected,as opposed to INS(insertion)or DEL(deletion).Specifically,C>T is the most frequently observed type of single nucleotide mutation.Notably,PDLIM7 mutations have been associated with the enrichment of Streptococcus in colon cancer and with the activation of the PI3K-Akt signaling pathway,with statistically significant results(p<0.05).5.Analysis of PDLIM7 expression at the single-cell level revealed that it was primarily expressed in fibroblasts and was found to be significantly correlated with various functional aspects of tumor cells,including EMT(epithelial-mesenchymal transition),invasion and metastasis,and angiogenesis(p<0.05).Conclusion: 1.PDLIM7 is highly expressed in colon cancer and is associated with poor prognosis and malignancy.Its expression may be negatively regulated by DNA methylation.2.The somatic mutation of PDLIM7 was lower in colon cancer but higher than in other cancers.The copy number variation was mainly amplification and deletion,with no deep deletion and high amplification,and thus had little effect on its expression.3.PDLIM7 is closely related to the composition,structure and function of colon cancer ECM and is associated with EMT,invasion and metastasis,angiogenesis and other tumour cell functions.Part Ⅱ Preliminary study on PDLIM7 promoting EMT-induced colon cancer invasion and metastasis through ECM remodelingObjective: Previous studies have shown that PDLIM7 is closely associated with the extracellular matrix(ECM)and is involved in the regulation of various malignant biological functions of tumor cells,including epithelial-mesenchymal transition(EMT),invasion,and metastasis.The aim of this study is to investigate the molecular mechanisms by which PDLIM7 promotes ECM remodeling and EMT,ultimately contributing to invasion and metastasis in colon cancer.Methods: Firstly,the study investigated the correlation between PDLIM7 expression and the EMT signature score.The EMT signature score of each TCGA-COAD sample was calculated using the "GSVA" package in R software,and then correlated with PDLIM7 expression.Secondly,the study examined the correlation between PDLIM7 expression and common EMT biomarkers.Additionally,the study constructed a prognostic LassoCox risk model of EMT-related genes co-expressed by PDLIM7,and the risk model’s difference in prognosis and clinical parameters was further verified.The clinical variables were included in a Nomogram with clinical reference significance based on the results of univariate and multivariate analysis.Then we investigated the potential relationship between PDLIM7 and COL6A1 in colon cancer.Firstly,we explored the correlation between PDLIM7 and COL6A1 expression in the expression enrichment pathway hsa04512(extracellular-matrix receptor interaction)in the KEGG database.Subsequently,we performed immunohistochemical staining to examine the expression patterns of PDLIM7 and COL6A1 in different stages of colon cancer and metastatic tissues.Using single cell data set GSE146771 and TISCH2,we analyzed the expression of COL6A1 and cellular communication of various cell subsets under single cell resolution.We identified the most significant signal axis COL6A1-CD44 by linking the expressionenrichment pathway hsa04512.Finally,we searched the Cancer SEA data to elucidate the relationship between COL6A1 expression and functional status of tumor cells.Results: 1.The analysis revealed a significant positive correlation(p<0.05)between the expression of PDLIM7 and the majority of EMT characteristic gene sets(>60%,r>0.3).Specifically,PDLIM7 expression was negatively correlated with the epithelioid marker E-cadherin(r=-0.138,p<0.05),and strongly positively correlated with the expression of interstitial markers such as Fibronectin(r=0.652,p<0.05),Vimentin(r=0.715,p<0.05),and N-cadherin(r=0.701,p<0.05).These results suggest that PDLIM7 expression is associated with the EMT process in colon cancer,and may promote this process.2.We constructed a Lasso-Cox risk model based on the co-expression of PDLIM7 with EMT-related genes and drew a Nomogram based on the results of univariate and multivariate Cox analysis.The consistency index(C-index)of the Nomogram was found to be greater than 0.75,indicating its clinical predictive significance.Moreover,the AUC of the aforementioned models for 1-year,3-year and 5-year prognosis were all above 0.7,and after evaluation,they were determined to be robust.Furthermore,high risk score and low risk score were significantly correlated with TNM stage,AJCC stage,lymphatic invasion,vascular invasion and the number of positive lymph nodes(p<0.05),but not with age,sex,race,incisal margin,CEA level,nerve invasion,number of detected lymph nodes and history of colonic polyps(p>0.05).3.In the enriched extracellular matrix receptor interaction pathway(hsa04512)identified by gene set enrichment analysis(GSEA)of PDLIM7 coexpression genes,COL6A1 encodes type VI collagen,a critical molecule in regulating extracellular matrix(ECM)remodeling and biomechanical properties.The immunohistochemistry results demonstrated that PDLIM7 and COL6A1 were coexpressed in colon cancer,and their expressions were positively correlated(r=0.626,p<0.05).Strong m RNA expression correlation between PDLIM7 and COL6A1 was also found in the TCGA-COAD cohort(r=0.761,p<0.05).Furthermore,the expression of both PDLIM7 and COL6A1 varied in different clinical stages of colon cancer(p<0.05),with higher expression associated withlater tumor stages.PDLIM7 and COL6A1 were also detected in metastatic colon cancer tissues,indicating their role in the invasion and metastasis of colon cancer.Thus,the high expression of PDLIM7 may promote ECM remodeling by upregulating COL6A1 expression.4.At the level of single cells,the expression of PDLIM7 and COL6A1 was mainly observed in fibroblasts.The COL6A1-CD44 ligand receptor interaction,which is involved in fibroblast-tumor cell communication,was located in the hsa04512 pathway.The enrichment of this pathway in cell communication was investigated,revealing that the COL6A1-CD44 signal axis is the common signal axis of hsa04512 pathway and cell communication.This indicates that the COL6A1-CD44 signal axis is involved in fibroblast-tumor cell communication and modulates the hsa04512 functional pathway in colon cancer.Furthermore,PDLIM7 and COL6A1 were found to be co-expressed in this pathway,suggesting a potential PDLIM7-COL6A1-CD44 signal axis.Analysis of the Cancer SEA database revealed a moderate correlation(r=0.58,p<0.05)between the expression of COL6A1 and the EMT functional status of tumor cells.This finding suggests that the COL6A1-CD44 signal axis may promote the EMT process and contribute to tumor progression,invasion,and metastasis.Conclusion: 1.PDLIM7 is associated with EMT-related molecular markers and characteristic gene sets that promote the EMT process in colorectal cancer.2.Co-expression of EMT-related genes by PDLIM7 affects clinical prognosis and is associated with tumour progression,invasion and metastasis.3.PDLIM7 co-expresses COL6A1 remodelling ECM and may promote EMT process through COL6A1-CD44 signaling axis,leading to tumour progression,invasion and metastasis. |
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