Study On The Mechanism Of TRPA1 Regulating The Heterogeneity Of Cancer-associated Fibroblasts To Mediate Colorectal Cancer Metastasis

Objectives:Cancer associated fibroblasts(CAF)play a promoting role in the metastasis of colorectal cancer.Based on literature,it has been found that there are two different subtypes of inflammation associated cancer fibroblasts and tumor associated myofibroblasts,with TRPA1(Transitional receptor potential Ankyrin1,TRPA1)being highly expressed in both CAF subtypes.Different subtypes of tumor related fibroblasts exhibit differences in function and phenotype,which promote the metastasis of colorectal cancer.This study aims to explore:(1)whether TRPA1 mediates CAF in promoting tumor metastasis in colorectal cancer;(2)Exploring the mechanism of action of TRPA1 in different subtypes of CAF cells;(3)Can the regulation of CAF by inhibiting TRPA1 inhibit colorectal cancer metastasis.Methods:The primary CAF was extracted from human colorectal cancer tissue and verified by Western blot(WB)and ghost pen cyclopeptide test.After TRPA1 knockdown,WB assay,immunofluorescence assay(IF)and RT-q PCR were used for verification.Transwell assay,Western Blotting(WB),Flou-4AM assay,RT-q PCR,ghost cyclic peptide,collagen contraction and gelatin enzyme assay were used to observe the functional changes of CAF cells after TRPA1 knockdown.A mouse model of liver metastasis was injected into the spleen in vivo to observe the regulation of chemotherapeutic drugs combined with TRPA1 inhibitors on the function of promoting metastasis of CAF.Results:(1)In Inflammatory-like cancer-associated fibroblast(i CAF),TRPA1 gene was knocked down in vitro and found to inhibit the function of i CAF.Inhibition was demonstrated by the MAPK/NADPH/NF-κB signaling pathway.(2)In Myofibroblast-like cancerassociated fibroblast(my CAF),After knocking down TRPA1 gene in vitro,it was found that the collagen function of my CAF was reduced to inhibit the invasion of tumor cells,and it was verified that the inhibition of Ca N/NFATc3 signaling pathway mainly through Ca N/NFATc3 signaling pathway played an important role.(3)In vitro,i CAF and colorectal cancer fibroblasts simulated tumor microenvironment,and liver metastasis was verified by splenic injection,and it was found that the TRPA1 specific inhibitor HC030031 combined with oxaliplatin could inhibit colorectal cancer liver metastasis.(4)my CAF and colorectal cancer fibroblasts were used to simulate the tumor microenvironment,and liver metastasis was verified by splenic injection.It was found that TRPA1 specific inhibitor HC030031 combined with oxaliplatin could inhibit colorectal cancer liver metastasis.Conclusion:TRPA1 plays an important role in colorectal cancer metastasis by mediating CAF cells.The relevant mechanisms were identified: inhibition of TRPA1 in my CAF affected CAF cell function through the Ca N/NFATc3 signaling pathway;Inhibition of TRPA1 in i CAF affects CAF cell function via MAPK/NADPH/NF-κB signaling pathway.Both TRPA1 inhibitor and oxaliplatin can significantly inhibit tumor metastasis through in vivo splenic injection model.