The Virulence Protein FadA On Fusobacterium Nucleatum Promotes The Progression Of Colorectal Cancer Via Binding To CircPDCD11

Research background and aims:Due to the westernization of lifestyle,colorectal cancer has become one of the ten most common cancers in China,whose incidence is still rising.Fusobacterium nucleatum is a common opportunistic pathogen in the oral cavity.It has certain pathogenicity in a variety of diseases,especially colorectal cancer,which is closely related to its occurrence and progression,but the relevant carcinogenic mechanism has not been fully clarified.Studies have shown that FadA,a virulence protein on Fusobacterium nucleatum,plays a pivotal role in the process of colorectal cancer.Besides,circ RNA is the latest hot molecule in the field of cancer research,which participates in many of the same metabolic signaling pathways as Fusobacterium nucleatum in the process of colorectal cancer.So far,no relevant studies have explored the relationship and mechanism between Fusobacterium nucleate and colorectal cancer with circ RNA as the starting point.In this study,experiments in vivo and in vitro,were conducted to further reveal the biological functions of virulence protein FadA on Fusobacterium nucleatum during the progression of colorectal cancer.It is first confirmed that virulence protein FadA on Fusobacterium nucleatum can affect the progression of colorectal cancer via binding to circPDCD11(hsa＿circ＿0003366)pathway,in order to further elucidate the pathogenesis of colorectal cancer and find a new therapeutic target.Methods:1.FadA overexpression vector(LV003-3*FLAG-FadA-his)was constructed.After the transfection of LV003-3* FLAG-FadA-his in human colon cancer cells HCT-116,the transcriptional and protein levels of LV003-3*FLAG-FadA-his were detected by q PCR and Western Blot.2.LV003-3*FLAG-FadA-his was transfected into human colon cancer cell line HCT-116,and the biological effects of FadA were analyzed by MTS method and Transwell chamber assay to further reveal the biological function of FadA in the process of colorectal cancer.3.After the transfection of LV003-3*FLAG-FadA-his in human colon cancer cell HCT-116,RIP technology was used to precipitate Fada and its binding RNA simultaneously by using FadA-specific His tag antibody.After separation and purification,Western Blot was used to detect the expression of FadA,and q PCR was used to verify the expression of FadA-bound circPDCD11.4.Circ PDCD11 sequence interference plasmid(p Sico R-circPDCD11)was constructed.Two experimental groups were set,one group was transfected with LV003-3*FLAG-FadA-his in human colon cancer cells HCT-116,and the other group was transfected simultaneously with LV003-3*FLAG-FadA-his and p Sico RcircPDCD11 in human colon cancer cells HCT-116.The expression of FadA was detected by Western Blot,and the biological effects of FADA were analyzed by MTS and Transwell assay in vitro.5.The subcutaneous tumor bearing model of nude mice was established.Two experimental groups were set,with 6 nude mice in each group: One group was a stable strain of human colon cancer cell HCT-116 transfected with subcutaneous injection of LV003-3*FLAG-FadA-his,and the other group was a stable strain of human colon cancer cell HCT-116 co-transfected with subcutaneous injection of LV003-3*FLAG-FadA-his and p Sico R-circPDCD11,which was in order to compare their effects on tumorigenesis ability and tumor growth rate in vivo.Results:1.After LV003-3*FLAG-FadA-his was transfected into human colon cancer cell line HCT-116 for 48 hours,q PCR and Western Blot analysis showed that FadA overexpression vector could be successfully transfected into human colon cancer cell line HCT-116,and the virulence protein FadA was significantly and stably expressed.2.After the overexpression of FadA in human colon cancer cells HCT-116,the proliferation ability of the cells was detected by MTS method at 24 h,48h and 72 h,respectively.The results showed that after the overexpression of FadA the proliferation ability of human colon cancer cells HCT-116 was increased at 48 h and72h,compared with the negative control group.3.After LV003-3*FLAG-FadA-his was transfected in human colon cancer cells HCT-116 for 48 hours,Transwell chamber experiments showed that the ability of migration and invasion of human colon cancer cells HCT-116 overexpressing FadA was increased,compared with the negative control group.4.LV003-3*FLAG-FadA-his was transfected into human colon cancer cell line HCT-116 for 72 h.RIP,Western Blot and q PCR showed that the virulence protein FadA could bind to circPDCD11 in human colon cancer cell line HCT-116.Compared with the negative control group,circPDCD11 expression was significantly increased after human colon cancer cells HCT-116 overexpressed FadA.5.LV003-3*FLAG-FadA-his was transfected into human colon cancer cell HCT-116 for 72 h to overexpress virulence protein FadA.Besides,LV003-3*FLAG-FadA-his and p Sico R-circPDCD11 were co-transfected with human colon cancer cell line HCT-116 for 72 h.Western Blot showed that after circPDCD11 expression was interfered,the expression of virulence protein FadA would be affected and decreased.6.Human colon cancer cells HCT-116 transfected with LV003-3*FLAG-FadA-his significantly promoted proliferation at 48 h by MTS method.However,after co-transfection of LV003-3*FLAG-FadA-his and p Sico RcircPDCD11 into human colon cancer cell HCT-116,the proliferation effect of the cancer cells was inhibited to some extent.7.Transwell chamber experiment found that after LV003-3* FLAG-FadA-His and p Sico R-circPDCD11 were simultaneously transfected in human colon cancer cell line HCT-116 for 48 h,cancer cells are less able to migrate and invade.8.The subcutaneous tumor-bearing model of nude mice was established by different groups.The virulence protein FadA can significantly promote the malignant proliferation and tumorigenesis of colorectal cancer cells.However,interference with the expression of circPDCD11 reduced the tumorigenesis ability of cancer cells and significantly reduced the tumor volume.Conclusion:1.The virulence protein FadA on Fusobacterium nucleatum can promote the proliferation,migration and invasion of colorectal cancer cells.2.Via binding to circPDCD11,the virulence protein FadA promotes the proliferation,migration and invasion of colorectal cancer cells and enhances the ability of malignant proliferation and tumorigenesis.3.The silencing of circ CPDCD11 in colorectal cancer can inhibit the expression of virulence protein FadA.Circp DCD11 may be a therapeutic target for colorectal cancer patients with F.nucleatum enrichment and high FadA expression.