The Mechanism Of Sanzi Formula Against Colorectal Cancer Based On CCDC68 And Wnt/β-catenin Signaling Pathway

Colorectal cancer(CRC)accounts for the third highest incidence of cancer in the world(10%)and the second highest mortality rate(9.4%),posing a serious threat to Chinese life and health in the world.Sangzi formula is an effective prescription for the treatment of early colorectal cancer and precancerous lesions(colorectal adenoma)in long-term clinical practice.In this paper,the curative effect of Sangzi Formula on CRC was firstly studied theoretically and literature.Secondly,based on TCGA database,bioinformatics analysis method was used to explore a new target(CCDC68)for CRC diagnosis and treatment.Thirdly,retrospective analysis of colorectal cancer cases in our hospital was performed to verify CCDC68.Finally,animal experiments were conducted to explore the possible mechanism of Sangzi formula against colon cancer in CT26 tumor-bearing mice based on CCDC68 and Wnt/β-catenin signaling pathway.Objective:In this paper,the prognostic effect of CCDC68 in colorectal cancer was discussed from three parts:bioinformatics analysis,clinical research and experimental research,as well as the inhibitory effect and related regulatory mechanism of Sanzi formula,on colorectal cancer,so as to provide objective basis for TCM treatment of colorectal cancer.Methods:1.Bioinformatics analysis:Based on TCGA database mining,the prognostic effect of CCDC68 in CRC was analyzed.(1)The difference of CCDC68 expression between colorectal cancer tissues and normal tissues.(2)Correlation between CCDC68 expression and clinicopathological factors and prognosis.(3)Gene set enrichment analysis(GSEA)to explore the functional pathways related to CCDC68 and CRC.(4)The correlation between CCDC68 expression and immune infiltration was analyzed by single sample gene concentration analysis(ssGSEA).2.Clinical research:Retrospective collection of paired cancer,adenoma and paracancourous tissue specimens from 73 patients undergoing colorectal cancer surgery in our hospital was performed to verify CCDC68.(1)The expression difference of CCDC68 in colorectal cancer,adenoma and paracancer tissues.(2)Correlation between CCDC68 expression and clinicopathological factors.(3)Correlation between CCDC68 expression and prognosis.3.Experimental research:Based on CCDC68 and Wnt/β-catenin signaling pathway,to explore the anti-colon cancer mechanism of Sangzi formula on CT26 tumor-bearing mice.(1)Model establishment and grouping:48 BALB/C mice were subcutaneously inoculated with CT26 cells(0.2 ml,1*107 cells/ml)in the right forelimb.After successful modeling,they were randomly divided into model group,positive drug group(oxaliplatin),combined group(oxaliplatin+Sanzi formula 1.7g/kg),Sanzi formula 1.7g/kg group,Sanzi formula 3.4g/kg group and Sanzi formula 6.8g/kg group.(2)The changes of transplanted tumor volume in mice after Sanzi formula administration were observed.(3)Histopathological changes of transplanted tumor in mice were observed by HE staining.(4)The expression of CCDC68 in transplanted tumor tissues of mice was determined by IHC.(5)The expression of Wnt/β-catenin signaling pathway related proteins(Wntl,Wnt3a,β-catenin,GSK-3β,MMP-7,Cyclin D1)in the transplanted tumor tissues were determined by IHC and Western Blot.Results:1.Bioinformatics analysis:(1)The expression of CCDC68 is decreased in colorectal cancer.(2)CCDC68 was associated with advanced clinicopathological features(lymph node metastasis,distant metastasis,pathological stage,tumor residue,tumor type,TP53 status),survival time and poor prognosis.(3)GSEA showed that chemokine signaling pathway,JAK-Stat signaling pathway,FcεRI mediated NF-κB activation,cell adhesion molecules,complement cascade,FcεRI mediated MAPK activation,intestinal secretory IgA immune network and Toll-like receptor signaling pathway were enriched in the high expression CCDC68 phenotype.The Wnt signaling pathway was significantly enriched in CCDC68 low expression phenotype.(4)ssGSEA showed that CCDC68 expression was positively correlated with Th2 cells and Th cells.2.Clinical research:(1)CCDC68 is differentially expressed in colorectal cancer tissues,adenoma tissues and paracancer tissues.(2)The expression of CCDC68 was correlated with T stage and tumor differentiation in colorectal cancer patients.(3)Univariate and multivariate Cox regression analysis showed that CEA was an independent prognostic factor for overall survival of colorectal cancer patients.3.Experimental research:(1)Sangzi formula inhibited the growth of tumor tissue in colon cancer bearing mice.(2)Immunohistochemistry showed that Sangzi formula could up-regulate CCDC68 expression.(3)Immunohistochemistry showed that Sangzi formula inhibited the expression of Wnt/β-catenin signaling pathway related proteins(Wntl,β-catenin,MMP-7)in tumor tissues of colon cancer bearing mice.(4)Western Blot showed that Sangzi formula inhibited the expression of Wnt/β-catenin signaling pathway related proteins(Wntl,Wnt3a,β-catenin,GSK-3β,MMP-7,Cyclin D1)in tumor tissues of colon cancer bearing mice.The expressions of Wntl,Wnt3a,GSK-3β,MMP-7 and Cyclin D1 were correlated with the dose of Sangzi formula.Conclusion:1.Low expression of CCDC68 in colorectal cancer.CCDC68 may be a potential prognostic molecular marker of poor survival in patients with colorectal cancer.2.Sangzi formula inhibited the tumor tissue growth of colon cancer bearing mice,up-regulated the expression of CCDC68,down-regulated the expression of Wnt/β-catenin signaling pathway related proteins(Wntl,Wnt3a,β-catenin,GSK-3β,MMP-7,Cyclin D1).Thus,the activation of Wnt/β-catenin signaling pathway was inhibited and tumor growth was inhibited,which may be one of the molecular mechanisms of Sangzi formula against colorectal cancer.