Fusobacterium nucleatum Promotes Colorectal Cancer by Up-Regulating Cdk1 through Intestinal Metabolite Sodium Butyrate

This study aimed to investigate the effects of intestinal microorganism Fusobacterium nucleatum on the occurrence and development of CRC (colorectal cancer) by regulating the metabolite NaB (sodium butyrate) and its molecular mechanism. RNA and protein were extracted from clinical tissue. RT-qPCR and Western blot were used to detect the expression of mRNA and protein of Cdk1 in tumor tissue and normal/paracancerous tissues. At the same time, the relative level of mRNA of Fusobacterium nucleatum was detected by RT-qPCR, and the correlation between mRNA and Cdk1 was analyzed. After the colorectal cancer cells were treated with Fusobacterium nucleatum for 24 hours, the expression levels of cycle related proteins Cdk1 and P21 were detected. The differential metabolites in the culture medium were detected by NMR (nuclear magnetic resonance). Different concentrations of metabolite NaB were used to treat colorectal cancer cells DLD-1, SW480 and HCT116. MTT and colony formation test were used to detect the proliferation ability of DLD-1, SW480 and HCT116. The cycle arrest sites of SW480 induced by NaB were detected by flow cytometry, and the cycle-related proteins Cdk1, P21 and C-myc were detected by Western blot. Flow cytometry was used to detect the apoptosis rate of SW480 after NaB treatment, and the apoptosis-related proteins Cleaved-Casepase3, Cleaved-PARP and Bcl-2 were detected by Western blot. MTT assay was used to detect the effect of Fusobacterium nucleatum with different MOI on the proliferation of colorectal cancer cells after 4, 8, 24 h. Colorectal cancer cells were co-cultured Fusobacterium nucleatum for 24 h and the related protein Cdk1, c-myc, Cleaved-Caspase3 was detected by Western blot. The results showed that the levels of Cdk1 protein and mRNA in tumor tissues were significantly higher than those in normal/paracancerous tissues, and there was a certain correlation between Cdk1 and mRNA expression of Fusobacterium nucleatum (P<0.05). The results of Western blot showed that the levels of Cdk1 and P21 protein increased after treatment of colorectal cancer cells DLD-1 and HCT116 with Fusobacterium nucleatum. The results of NMR showed that there was a significant difference in metabolic pattern between the Fusobacterium nucleatum treated group and the untreated group, and the relative content of metabolite NaB was significantly lower than that in the untreated group (P<0.01). The cell viability of colorectal cancer cell lines DLD-1, SW480 and HCT116 treated with 1 mmol/L NaB for 24 h were (89.18±1.92)%, (85.07±0.61)% and (83.59±2.18)%, respectively. The inhibition rate of NaB on cell activity increased gradually with the increase of NaB concentration. At the same time, after 1 mmol/L NaB treatment, the clone formation rate of DLD-1, HCT116 and SW480 cells decreased by (20.07±4.85)%, (36.47±5.31)% and (31.13±5.22)%, respectively. Flow cytometry showed that NaB caused S-phase arrest of colorectal cancer cells. After colorectal cancer cells were treated with NaB for 24 h, the expression of cycle-related protein P21 increased, while the expression of Cdk1 and C-myc decreased. Flow cytometry showed that NaB increased apoptosis of colorectal cancer cell SW480. After colorectal cancer cells were treated with NaB for 24 h, the expression of apoptosis-related proteins Cleaved-Casepase3 and Cleaved-PARP increased, while the expression of anti- apoptosis protein Bcl-2 decreased (P<0.05). After SW480, HCT116 cells were treated with Fusobacterium nucleatum (MOI=50) for 4 h, the proliferation rate of colorectal cancer cells increased by (4.45±0.25)% and (2.61±0.75)%, respectively. With the increase of the MOI and the prolongation of infection time, the proliferation rate of colorectal cancer cells increased gradually (P<0.05). SW480 and HCT116 were cultured with or without Fusobacterium nucleatum for 24 h. Western blot results showed that Fusobacterium nucleatum infection promoted the expression of cycle-related protein Cdk1, C-myc, which was greatly weakened by its co-treatment with NaB; NaB induced Caspase3 cleavage, resulting in an increase in Cleaved-Caspase3 expression, while Fusobacterium nucleatum infection weakened this effect. In conclusion, intestinal microorganism Fusobacterium nucleatum up-regulates Cdk1 by regulating intestinal metabolite NaB to promote the proliferation of colorectal cancer cells and inhibit the apoptosis of colorectal cancer cells, thus affecting the occurrence and development of colorectal cancer.

CSTR: 32200.14.cjcb.2021.05.0009