Fusobacterium Nucleatum Promotes Colorectal Cancer Metastasis By Modulating KRT7-AS/KRT7

Background and Aim Colorectal cancer(CRC)is the third most common cancer worldwide.About 90%of patients with colorectal cancer die from tumor metastasis.In-depth discussion of the mechanism of colorectal cancer metastasis has important scientific value for reducing the mortality of colorectal cancer.Gut microbiota is closely related to the occurrence of colorectal cancer.Fusobacterium nucleatum is a gram-negative anaerobic bacterium prevalent in the oral cavity which has gained significant attention for its potential role in colorectal cancer.Clinical data indicated that the abundance of Fusobacterium nucleatum is associated with lymph node metastasis and distant metastasis in colorectal cancer.However,it is not clear whether the abnormal colonization of Fusobacterium nucleatum drives the metastasis of colorectal cancer directly.This study will deeply explore the role of Fusobacterium nucleatum in colorectal cancer metastasis and its mechanism,which may provide potential new targets for the treatment of colorectal cancer.Materials and Methods1.We collected stool samples from patients with colorectal cancer and healthy people,detecting the abundance of Fusobacterium nucleatum in the stool of patients and healthy people by q PT-PCR,and analyzing the association of Fusobacterium nucleatum abundance and lymph node metastasis.2.After co-culturing Fusobacterium nucleatum and CRC cells,we determined the role of Fusobacterium nucleatum in cell migration by in vitro experiments(transwell migration assay and wound healing assay)and in vivo experiments(intravenous injection of CRC cells into nude mice via tail vein).3.RNA sequencing was performed to analyze the effect of Fusobacterium nucleatum on the long non-coding RNA(lnc RNA)and m RNA expression profiles of CRC cells.The q RT-PCR,western blot and immunofluorescence experiments were performed to verify the RNA sequencing results.The antisense lnc RNA KRT7-AS and m RNA KRT7 were selected to be further studied.4.After knockdown or over-expression of KRT7-AS or KRT7 respectively,the interaction between KRT7-AS and KRT7 was analyzed by q RT-PCR,western blot,and the function of KRT7-AS and KRT7 on cell migration and metastasis were determined by in vitro(transwell migration assay and wound healing assays)and in vivo(intravenous injection of colon cancer cells into nude mice via tail vein)experiments.5.We analyzed the expression levels of KRT7-AS and KRT7 in CRC patients in Sir Run Run Shaw Hospital and TCGA database and their correlation with lymph node metastasis.6.Software packages(TESS and FIMO)combined with RNA sequencing results were used to predict the transcriptional factor binding to the promotor region of KRT7-AS.The q RT-PCR,western blot and dual luciferase reporter genes assays were further performed to verify the transcriptional regulation of KRT7-AS by this transcription factor.Results1.We performed q RT-PCR to quantify the abundance of Fusobacterium nucleatum in stool samples from CRC patients and healthy controls.The results indicated an enrichment of Fusobacterium nucleatum in the stool of CRC patients compared with healthy controls.Meanwhile,CRC patients with positive lymph nodes metastasis showed more abundance of Fusobacterium nucleatum in stool compared to CRC patients without lymph nodes metastasis.2.Transwell and wound healing assay results indicated that Fusobacterium nucleatum infection significantly enhanced CRC cells migration.However,heat-killed Fusobacterium nucleatum was unable to promote CRC cells migration.In accordance with the results in vitro,Fusobacterium nucleatum-treated cells induced more metastatic nodules in lung than PBS-treated cells in nude mice.3.The results of RNA sequencing showed that anti-sense lnc RNA KRT7-AS and m RNA KRT7 were significantly up-regulated after Fusobacterium nucleatum infection.The q RT-PCR,western blot,and immunofluorescence assays further confirmed that Fusobacterium nucleatum could up-regulate lnc RNA KRT7-AS and m RNA KRT7.4.lnc RNA KRT7-AS could up-regulate KRT7 expression;conversely,KRT7 cannot regulate KRT7-AS expression.The results of in vivo and in vitro functional experiments proved that KRT7-AS and KRT7 could promote the metastasis of CRC cells.Over-expression of KRT7 restored the cell migration function inhibited by the knockdown of KRT7-AS,suggesting that KRT7-AS promoted CRC cell metastasis by up-regulating KRT7.Fusobacterium nucleatum treatment significantly induced more lung metastatic nodules.However,depletion of KRT7-AS attenuated Fusobacterium nucleatum-induced CRC lung metastasis,suggesting that Fusobacterium nucleatum promotes CRC metastasis by regulation of KRT7-AS.5.The expressions of KRT7-AS and KRT7 were significantly increased in CRC tissues compared with adjacent normal tissues.Moreover,the KRT7-AS expression was positively associated with KRT7 expression.High expression level of KRT7-AS and KRT7 were associated with lymph node metastasis in CRC patients.6.RNA sequencing results showed that Fusobacterium nucleatum could activate the NF-?B signaling pathway in CRC cells,and the result was confirmed by western blot and immunofluorescence assays.Software prediction results showed that there was a binding site of NF-?B p65 in the promoter region of KRT7-AS.The application of NF-?B inhibitor and knockdown of NF-?B p65 by si RNA both inhibited the expression of KRT7-AS.Dual luciferase reporter gene assay confirmed that NF-?B could regulate the transcriptional activity of KRT7-AS promoter.After inhibiting the NF-?B signaling pathway,Fusobacterium nucleatum infection could not effectively up-regulate KRT7-AS.Conclusions1.Fusobacterium nucleatum promoted metastasis of CRC.2.Fusobacterium nucleatum promoted the expression of antisense lnc RNA KRT7-AS,leading to the up-regulation of m RNA KRT7,which enhanced the metastasis of CRC cells.3.Fusobacterium nucleatum activated NF-?B signaling pathway,and activated NF-?B could promote KRT7-AS expression through transcriptional regulation.