Mechanisms Of Fusobacterium Nucleatum Participating To The Tumorigenesis And Development Of Colorectal Cancer

BACKGROUND&AIMS: Fusobacterium nucleatum(F.nucleatum)has been shown to be closely related to the tumorigenesis and development of colorectal cancer(CRC),but the specific mechanisms have not been studied yet.Chemoresistance is a strait challenge in the treatment of patients with CRC.Improving our understanding about the mechanisms of chemoresistance could lead to strategies to increase patients’ response to therapy.In the present study,we investigated the roles of F.nucleatum in regulating autophagy by inhibiting microRNAs,and then inducing chemoresistance of CRC cells.We clarified the important role of F.nucleatum in colorectal cancer development,and these findings would provide a theoretical basis for the prevention and treatment of CRC and the study of microbial markers.METHODS: We detected autophagy in CRC cells HCT116 and HT29 after F.nucleatum infection by Western blot,confocal microscopy and transmission electron microscopy.Chemotherapy drugs Cisplatin and Irinotecan and autophagy inhibitor Chloroquine were added respectively to F.nucleatum-infected CRC cells,then we detected autophagy and apoptosis by cell proliferation assay,flow cytometry and Western blot analysis.We used mRNA chip of HT29 infected by F.nucleatum,CRC tissue chip about high / low abundance group of F.nucleatum and microRNAs target gene prediction software,and real-time PCR to screen microRNAs related to F.nucleatum and autophagy.We performed dual-luciferase reporter gene assay to detect whether microRNAs were involved in post-transcriptional regulation of target genes 3’UTR.After microRNA mimics or inhibitors transfected to F.nucleatum-infected CRC cells,we detected autophagy by real-time PCR,Western blot,confocal microscopy and transmission electron microscopy;then chemotherapy drugs Cisplatin and Irinotecan were added to the above pre-treated CRC cells respectively,and we detected autophagy and apoptosis by flow cytometry and Western blot analysis.We constructed xenograft model of colorectal cancer in nude mice,and we verified the autophagy and apoptosis of tumor tissue by Western blot,transmission electron microscopy and TUNEL staining.As for clinical specimens,we used real-time PCR to detect the correlation between F.nucleatum,candidate microRNAs and target autophagy genes mRNA in colorectal carcinoma tissue.RESULTS: Experiments in vivo and in vitro both showed that,F.nucleatum could promote CRC cell autophagy and induce chemoresistance,and the effects of chemoresistance were dependent on the increased levels of autophagy.After F.nucleatum infection,the expression levels of miR-18a* and miR-4802 in CRC cells were significantly reduced,while the expression of ULK1 and ATG7 mRNA were significantly increased.Overexpression of miR-18a* and miR-4802 could inhibit CRC cell autophagy induced by F.nucleatum,while the results were opposite when suppressing the microRNAs.In addition,overexpression of miR-18a* and miR-4802 could inhibit CRC cells resistance to chemotherapeutic drugs such as Cisplatin and Irinotecan by regulating autophagy target genes,which was induced by F.nucleatum infection.The abundance of F.nucleatum was negatively correlated with the expression of miR-18a* and miR-4802,and was positively correlated with the expression of ULK1 and ATG7 mRNA in human CRC tissues.Furthermore,the expression of microRNAs were negatively correlated with the expression of their autophagy target genes mRNA.CONCLUSION: F.nucleatum could promote autophagy by inhibiting the expression of miR-18a* and miR-4802 and increasing autophagy-related gene ULK1 and ATG7 mRNA in CRC cells,and then induce tumor resistance to chemotherapeutic drugs such as Cisplatin and Irinotecan.miR-18a* and miR-4802 may become valid candidate targets to improve the effects of chemotherapy in patients with CRC.