Differential Analysis And Prognostic Significance Of Matrix Collagen Remodeling Characteristics In Deficiency And Excess Syndromes Of Colorectal Cancer

The incidence and mortality of colorectal cancer(CRC)is all ranked 3rd among all malignant tumors in the world.As a systemic disease with heterogeneous characteristics,personalized treatment based on individual characteristics of each patient is the focus to improve the prognosis of CRC patients.Based on the syndrome differentiation of the holistic concept,traditional Chinese medicine(TCM)evaluates the disease progression of patients from the overall level and gives individualized treatment plan,so as to achieve good clinical efficacy in the postoperative treatment of CRC and improve the prognosis quality of patients.But the mechanism behind the evaluation of tumor progression based on TCM syndrome classification is not clear.Medical research showed that tumor development depends on a complex interaction between tumor cells and their microenvironment.Macroscopically,this process can be expressed as various signs of TCM syndromes,and its microscopic and objective aspects can be expressed as the changes of tumor cells and their microenvironment.Therefore,it’s in line with the principle of the "holistic view" in the diagnosis and treatment of TCM,which focuses on tumor microenvironment as the starting points to analyze the essence of TCM syndromes of CRC.The previous study showed that TCM syndromes had variable influence on CRC development and the stiffness or length of colon tissues;and in different syndromes,the component of microenvironment was different.Matrix collagen is the main component of tumor matrix environment.The abnormal remodeling of alignment and distribution of collagen can constitute the unique tumor microenvironment,which will play a significant role in the disease progression and prognosis of CRC patients.Meanwhile,matrix collagen is also the important factor of regulating the physical properties of tissues like elasticity or stiffness.Alterations in collagen content can influence the stiffness and thickness of colon tissues.From these,we deduced that the characteristics of matrix collagen remodeling might be related to TCM syndrome classification,which could provide a direction for us to explore the potential relationship between TCM syndrome and CRC development.Deficiency syndrome and excess syndrome are the basic syndrome types of "Eight steel syndrome differentiation",and the important factor of "Syndrome differentiation and treatment" in CRC.Taking the deficiency syndrome and excess syndrome of CRC as an example in this research:①compare the histopathological features and the levels of related tumor markers in patients with deficiency and excess CRC,and to explore the relationship between syndrome classification and the degree of malignant progression of tumor;②the characteristics of matrix collagen remodeling(collagen expression,arrangement morphology,related collagen remodeling enzyme level and tumor-stroma ratio)of tissue specimens were detected by modern biological techniques,and the difference of matrix collagen remodeling characteristics between CRC deficiency and excess syndrome was analyzed;③ CRC was classified according to histopathological characteristics,and the relationship between matrix collagen remodeling characteristics and malignant degree of CRC was analyzed;combined with bioinformatics analysis,the significance of matrix collagen remodeling to the progression and prognosis of CRC was studied.In order to clarify the potential causes of different syndrome types and different malignant progression of tumors,and provide theoretical basis and experimental support for TCM syndrome differentiation and treatment of CRC,but also provide new ideas for the research of TCM in the treatment of other malignant tumors.ObjectiveBased on the difference of matrix collagen remodeling characteristics of CRC patients between deficiency and excess syndromes,and the significance of this characteristics for the progression and prognosis of CRC,to explore the potential factors of tumor deterioration among different syndromes,so as to provide a theoretical basis for TCM syndrome differentiation and treatment.MethodsThe cancer tissues and distal normal tissues and clinical data of 63 patients with CRC who were hospitalized for surgical treatment in the Anorectal Department of the First Affiliated Hospital of Guangzhou University of Chinese Medicine from July 2018 to February 2020 were collected.Analysis of clinical data:Analyze the relationship among the preoperative serum tumor markers CEA,CA19-9,AFP,clinicopathological characteristics and TCM syndromes.Histological analysis:The cancer tissue and distal normal intestine tissue of 63 patients with colorectal cancer were collected.Sirius red staining was used to analyze the arrangement,parameters(width,length,stiffness,bending angle and density)of matrix collagen,and the area of type I collagen(COLI)and type III collagen(COLIII).The expression of COLI,Ⅲ,Ⅳ and collagen regulatory enzymes(MMP-1,-2,-7,-9 and LOX,LOXL2)were analyzed by immunohistochemistry.The HE staining method was used to observe the tumor-stroma ratio in different parts.Through the grouping of deficiency and excess syndromes,the correlation between matrix collagen remodeling characteristics and TCM syndromes of CRC was explored.Through metastatic and non-metastatic grouping,analyze the significance of matrix collagen remodeling characteristics for the prognosis of CRC metastasis,and further explore the potential factors of the influence of TCM syndromes on the occurrence and development of CRC.Bioinformatics analysis:Using CRC data from Cancer Genome Atlas(TCGA)and Oncomine,the differential expression of genes encoding collagen remodeling between tumor and normal tissues was analyzed.The STRING protein database was used to obtain the protein interaction network between MMP-1,-2,-7,-9 and LOX and LOXL2.Through gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)function enrichment analysis,explore the biological functions of genes encoding collagen remodeling.Based on the GSE17536 and GSE39582 data sets,survival analysis software packages were used to detect the prognostic significance of collagen remodeling-related genes in CRC,construct a prognostic model,and verify the independent prediction of the model performance.To further verify the prognostic significance of matrix collagen remodeling characteristics for CRC.Results1.Clinical studies:the patients with deficiency syndrome CRC have a higher incidence of advanced tumors,and the serum CEA,CA19-9 level is significantly higher than excess syndrome patients with CRC(P<0.05).The elevation of serum tumor markers(CEA,CA19-9)was related to the late stage of tumor,the depth of tumor invasion and tumor metastasis(P<0.05).2.Histological examinations of deficiency and excess syndromes:the arrangement of collagen in CRC tissue had mild,moderate,and severe changes:except for higher density,the minor changes are similar to the arrangement of normal collagen;the density and straightness of collagen with moderate changes were higher than those with mild changes;the collagen with severe changes was irregular and densely arranged,and the cross-links between the collagens were in the form of plates.The proportion of moderate and severe remodeling in the deficiency-syndrome group was higher than that in the empirical group.In cancer tissues,the width,length and density of collagen in patients with deficiency syndrome,the expression and area of COLI,COLI/COLIII,COLI area/COLIII area,the expression of collagen regulatory enzymes(MMP-1,-2,-7,-9,LOX and LOXL2)and the TSR were higher than that of excess-syndrome patients(P<0.05).In normal tissues,COLI area/COLIII area of patients with deficiency syndrome is higher than that of excess-syndrome patients(P<0.05).3.The histological examination of the degree of tumor progression:in the metastatic CRC tissue,the proportion of collagen morphology was severely remodeled,and the width,length and density of collagen increased significantly(P<0.05).Compared with normal tissues,COLI and COLIII deposition,matrix ratio and collagen regulatory enzymes((MMP-1,-2,-7,-9,LOX and LOXL2)in cancer tissues were significantly increased(P<0.05),and were positively correlated with metastasis status(P<0.05).In tissues without distant metastasis,the expression level of COLIV was significantly higher than that of normal tissues(P<0.05),while in tissues with distant metastases,the expression of COLIV was significantly lower than that of normal tissues(P<0.05).4.Bioinformatics analysis showed that the up-regulation of genes related to collagen and collagen-regulating enzymes was associated with shorter overall survival time in CRC patients(P<0.05).The coding genes COL1A1,COL3A1,LOX,LOXL2,MMP2,COL4A3,COL4A5 and COL4A6 can be constructed a prognostic assessment model for CRC patients.In addition,the analysis results of GO and KEGG showed that the abnormal remodeling of matrix collagen may promote the development of CRC by activating platelets and other bursts.ConclusionThere were differences in malignant degree and matrix collagen remodeling characteristics between deficiency and excess syndrome in CRC.Compared with the excess syndrome,the malignant degree of deficiency syndrome CRC was higher,and it showed the more active remodeling characteristics with more obvious collagen deposition and cross-linking.The matrix collagen remodeling characteristics with excessive deposition and cross-linking indicated stronger invasive potential and poor prognosis of tumors.Therefore,the difference in the characteristics of matrix collagen remodeling is one of the substantive reasons for the different degrees of deterioration in deficiency and excess syndrome CRC,which also indicated that TCM syndromes have a certain prospective role in evaluating the development of tumors.