Fusobacterium Nucleatum Promotes Colorectal Cancer Liver Metastasis By Regulating IL-8 Expression Through MiR-5692a

Objective: Colorectal cancer is intimately associated with the intestinal microbiota,and Fusobacterium nucleatum appears to be highly abundant in colorectal cancer tissues.The aim of this study is to explore the effect and mechanism of Fusobacterium nucleatum on colorectal cancer liver metastasis,and to provide new insights for the prevention and treatment of colorectal cancer liver metastasis.Methods: In this study,bioinformatics techniques were used to examine the variations in the quantity of Fusobacterium nucleatum in the feces of colorectal cancer patients and healthy individuals.Clone formation and EdU assay were used to detect the effect of Fusobacterium nucleatum on the proliferation of colorectal cancer cells.The effects of Fusobacterium nucleatum on the migration and invasion of colorectal cancer cells were examined by wound-healing assay and Transwell assay.A mouse model of colorectal cancer liver metastasis was constructed by spleen injection,and the degree of liver metastasis in colorectal cancer was assessed by in vivo bioluminescence imaging.Transcriptome sequencing was used to analyze gene expression changes in colorectal cancer cells after coculture with Fusobacterium nucleatum.Bioinformatics analysis was used to identify the mechanisms by which Fusobacterium nucleatum affects the expression of target genes.We screened the activated signaling pathways in colorectal cancer cells after co-culture with Fusobacterium nucleatum by pathway enrichment analysis.qRT-PCR and Western Blot assays were performed to detect the expression of related genes and proteins.Small molecule inhibitors were used to inhibit the activation of related proteins and validate their effects.Results: The abundance of Fusobacterium nucleatum in the feces of colorectal cancer patients was higher than that of the healthy population.The proliferation,migration,and invasion of colorectal cancer cells were significantly enhanced after co-culture with Fusobacterium nucleatum in vitro.In a mouse model,Fusobacterium nucleatum also promoted the development of liver metastasis in colorectal cancer.Transcriptome sequencing revealed that the expression of IL-8 in colorectal cancer cells was significantly increased after co-culture with Fusobacterium nucleatum,and the promotion of proliferation,migration,and invasion of colorectal cancer cells by Fusobacterium nucleatum was mediated by IL-8,as demonstrated by the use of IL-8 receptor inhibitors.Further experiments revealed that miR-5692 a is an upstream target gene that regulates IL-8expression,and Fusobacterium nucleatum was able to promote IL-8 expression by inhibiting miR-5692 a expression in colorectal cancer cells.After promoting IL-8 expression,Fusobacterium nucleatum activated the ERK signaling pathway,which in turn promoted the proliferation and metastasis of colorectal cancer.Conclusions: Fusobacterium nucleatum activates the ERK pathway in colorectal cancer cells and promotes proliferation and metastasis of colorectal cancer by suppressing miR-5692 a expression and causing an increase in IL-8 expressionPart Ⅰ: The effect and mechanism of Fusobacterium nucleatum promoting IL-8 expression on colon cancer proliferation and metastasisObjective: The purpose of this part is to investigate the role of Fusobacterium nucleatum in the proliferation and metastasis of colorectal cancer and to explore the molecular mechanisms by which Fusobacterium nucleatum affect colorectal cancer cells,providing new insights into the relationship between Fusobacterium nucleatum and colorectal cancer.Methods: The differences in the abundance of Fusobacterium nucleatum between colorectal cancer patients and the healthy population were investigated by bioinformatics using the GMrepo database.The effects of Fusobacterium nucleatum on the proliferation of colorectal cancer cells in vitro were examined by clone formation and EdU assay,and the effects of Fusobacterium nucleatum on the migration and invasion ability of colorectal cancer cells were examined by wound-healing assays and Transwell.qRT-PCR and Western Blot were used to examine the expression of molecular markers related to epithelial mesenchymal transition in colorectal cancer cells after co-culture with Fusobacterium nucleatum.The key differential genes between Fusobacterium nucleatum affecting colorectal cancer cells and control were screened using transcriptome sequencing.A small molecule inhibitor,reparixin,was used to interfere with the action of IL-8,and to verify whether Fusobacterium nucleatum affects the proliferation and metastasis of colorectal cancer cells through IL-8.A mouse model of colorectal cancer liver metastasis was constructed by spleen injection technique,and the effect of Fusobacterium nucleatum on liver metastasis in colorectal cancer in vivo was evaluated.Results: The results of GMrepo database analysis showed that the fecal levels of Fusobacterium nucleatum were significantly higher in colorectal cancer patients than in the normal group.In vitro experiments showed that the proliferation ability of colorectal cancer cells was enhanced after co-culture with Fusobacterium nucleatum,while the migration and invasion ability were increased.The expression of epithelial marker molecules decreased and the expression of mesenchymal marker molecules increased after co-culture with Fusobacterium nucleatum,indicating that epithelial mesenchymal transition occurred.Transcriptome sequencing results identified six key differential genes associated with Fusobacterium nucleatum,and combined with analysis of colorectal cancer data in TCGA showed that IL-8 gene was associated with colorectal cancer metastasis.The promotion of proliferation,migration,invasion and epithelial mesenchymal transition of colorectal cancer cells by Fusobacterium nucleatum was lost after blocking the effect of IL-8 with reparixin.The in vivo splenic injection metastasis model also showed that liver metastasis occurred more in the Fusobacterium nucleatum co-culture group,and the use of reparixin inhibited the promotion of liver metastasis by Fusobacterium nucleatum in colorectal cancer.Conclusions: Fusobacterium nucleatum is highly expressed in the feces of colorectal cancer patients and promotes the proliferation,migration,invasion and metastasis of colorectal cancer cells.The effect of Fusobacterium nucleatum on colorectal cancer proliferation and metastasis is mainly through the regulation of IL-8 expression.Part Ⅱ: The effect and mechanism of IL-8 expression regulation by Fusobacterium nucleatum through miR-5692 a on colon cancer proliferation and metastasisObjective: In the first part of this study,Fusobacterium nucleatum was found to affect the proliferation and metastasis of colorectal cancer by promoting the expression of IL-8.However,the specific mechanism by which Fusobacterium nucleatum regulates IL-8 expression in colorectal cancer cells is still unclear.This part of the study aims to investigate the molecular mechanisms by which Fusobacterium nucleatum regulates IL-8 expression in colorectal cancer.Methods: The miRNAs that control IL-8 expression were recognized by databases.qRTPCR experiments were used to identify miRNAs associated with Fusobacterium nucleatum.Dual-luciferase assay was performed to detect whether miR-5692 a binds to the predicted binding site on IL-8 mRNA 3’UTR.The mimics and inhibitor of miR-5692 a were transfected into colorectal cancer cells to construct miR-5692 a overexpression and knockdown cell lines.qRT-PCR and Western Blot assays were used to detect the effect of miR-5692 a on IL-8 expression levels.Clone formation and EdU assays were used to detect the effects of transfection with miR-5692 a on the proliferation of colorectal cancer cells.Wound-healing assays and Transwell assays were used to detect the effects of transfection with miR-5692 a on the migration and invasion ability of colorectal cancer cells.Results: The database predicts that six miRNAs,miR-5692 a,miR-573,miR-4687-3p,miR-4312,miR-376a-2-5p,and miR-7114-3p,may regulate the expression of IL-8.After coculture with Fusobacterium nucleatum,only miR-5692 a expression was decreased in colorectal cancer cells.Dual-luciferase assay results indicated that miR-5692 a was able to bind to IL-8 mRNA 3’UTR.The expression of IL-8 was decreased in colorectal cancer cells after transfection of miR-5692 a mimics.And the expression of IL-8 in colorectal cancer cells increased after transfection with the inhibitor of miR-5692 a.Fusobacterium nucleatum’s enhancement of the metastasis of colorectal cancer was blocked by the transfection with mimics of miR-5692 a.Conclusions: The expression of miR-5692 a was reduced in colorectal cancer cells after coculture with Fusobacterium nucleatum.miR-5692 a was able to bind to IL-8 mRNA 3’UTR and inhibit the expression of IL-8.Fusobacterium nucleatum promoted IL-8 expression by inhibiting miR-5692 a in colorectal cancer cells,which in turn promoted the proliferation and metastasis of colorectal cancer cells.Part Ⅲ: The effect and mechanism of activation of ERK by Fusobacterium nucleatum induced IL-8 expression on colon cancer proliferation and metastasisObjective: In the first part of this study,we found that Fusobacterium nucleatum enhanced colorectal cancer progression via promoting the expression of IL-8.The mechanism by which Fusobacterium nucleatum-induced IL-8 expression causes colorectal cancer development is unclear.This part of the study aims to investigate the potential mechanism by which Fusobacterium nucleatum affect the proliferation and metastasis of colorectal cancer cells after promoting the expression of IL-8.Methods: KEGG pathway enrichment analysis was performed to screen the activated signaling pathways in colorectal cancer cells after co-culture with Fusobacterium nucleatum.Western Blot assay was used to verify the activation of the relevant pathways.AKT inhibitor MK2206,ERK inhibitor U0126,p38 inhibitor SB203580 and JNK inhibitor SP600125 were used to validate the role of related pathways in the promotion of colorectal cancer metastasis by Fusobacterium nucleatum.Clone formation and EdU assays were used to detect the effects of Fusobacterium nucleatum on the proliferation of colorectal cancer cells after inhibiting ERK pathway.Wound-healing assays and Transwell assays were used to detect the effects of Fusobacterium nucleatum on the migration and invasion of colorectal cancer cells after inhibiting ERK pathway.qRT-PCR and Western Blot assays were used to detect the effects of Fusobacterium nucleatum on the epithelial mesenchymal transition after inhibiting ERK pathway.To verify whether the promotion of epithelial mesenchymal transition in colorectal cancer cells by Fusobacterium nucleatum is mediated by ZEB1.Results: The results of KEGG pathway enrichment analysis showed that MAPK pathway and PI3K-AKT pathway were activated in colorectal cancer cells after co-culture with Fusobacterium nucleatum,which was further confirmed by Western Blot assay.Only ERK inhibitor U0126 was able to reverse the promotion of epithelial mesenchymal transition in colorectal cancer cells by Fusobacterium nucleatum.Fusobacterium nucleatum’s encouragement of the growth and metastasis of colorectal cancer was blocked by the application of ERK inhibitors.The expression of ZEB1 was increased in colorectal cancer cells after co-culture with Fusobacterium nucleatum,whereas Fusobacterium nucleatum failed to promote ZEB1 expression in colorectal cancer cells after the use of IL-8 inhibitors or ERK inhibitors.The promotion of proliferation,metastasis and epithelial mesenchymal transition in colorectal cancer cells by Fusobacterium nucleatum was lost after knocking down ZEB1 expression.Conclusions: Fusobacterium nucleatum activates the ERK pathway by inducing IL-8 expression in colorectal cancer cells and increases the expression of downstream ZEB1,thereby promoting proliferation,metastasis and epithelial mesenchymal transition of colorectal cancer cells.