Lactobacillus spp. create a protective micro-ecological environment through regulating the core fucosylation of vaginal epithelial cells against cervical cancer

Abstract

Vaginal dysbiosis often occurs in patients with cervical cancer. The fucosylation of mucosal epithelial cells is closely related to microbial colonization, and play an important role in protecting the vaginal mucosal epithelial cells. However, no reports on the relationship between vaginal dysbiosis and abnormal mucosal epithelial cell fucosylation, and their roles in the occurrence and development of cervical cancer are unavailable. Here we report that core fucosylation levels were significantly lower in the serum, exfoliated cervical cells and tumor tissue of cervical cancer patients. Core fucosyltransferase gene (Fut8) knockout promoted the proliferation and migration of cervical cancer cells. In patients with cervical cancer, the vaginal dysbiosis, and the abundance of Lactobacillus, especially L. iners, was significantly reduced. Meanwhile, the abundance of L.iners was positively correlated with core fucosylation levels. The L. iners metabolite lactate can activate the Wnt pathway through the lactate-Gpr81 complex, which increases the level of core fucosylation in epidermal cells, inhibiting the proliferation and migration of cervical cancer cells, and have application prospects in regulating the vaginal microecology and preventing cervical cancer.

Fig. 1. Comparison of core fucosylation between healthy volunteers and cervical cancer patients.

A Immunofluorescence was used to analyze the core fucosylation level of cervical cancer and paracancerous tissues. B LCA blotting was used to compare the core fucosylation levels in exfoliated cervical cells. C LCA blotting was used to compare the core fucosylation levels in serum samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. The effect of de-core fucosylation on the occurrence and development of cervical cancer.

A LCA blotting was used to verify Fut8 gene knockout in SiHa cells. B An MTT assay was used to evaluate the effect of Fut8 gene knockout on the proliferation of SiHa cells. C A scratch assay was used to evaluate the effect of Fut8 gene knockout on the migration of SiHa cells. D Fut8^+/+ and Fut8^−/− SiHa cells were injected into nude mice to detect tumor formation in vivo. ***p < 0.001, ****p < 0.0001. E Histogram of differentially expressed genes in Fut8^−/− SiHa cells compared to Fut8^+/+ SiHa cells. F Bubble plots of GO enrichment analysis of differentially expressed genes in Fut8^−/− SiHa cells compared to Fut8^+/+ SiHa cells. G Bubble plots of KEGG enrichment analysis of differentially expressed genes in Fut8^−/− SiHa cells compared to Fut8^+/+ SiHa cells.

Fig. 3. Comparison of the vaginal microbiota structures of healthy volunteers and cervical cancer patients.

A The alpha diversity of the two groups (indicated by the Chao 1, Shannon, Simpson indices). B The beta diversity of the two groups (expressed based on PCA). C The composition of dominant microbiota in the two groups (expressed based on LEfSe analysis). D The relative abundance of the top ten microorganisms at the gene level in the two groups. E Comparison of the relative abundance of Lactobacillus spp. and L. iners in the two groups. F Beta diversity of the HPV- and HPV + groups (expressed based on PCA). ***p < 0.001, ****p < 0.0001.

Fig. 4. The effect of L. iners metabolites on the proliferation and migration of cervical cancer cells.

A MTT assay in SiHa cells stimulated with L. iners metabolites and bacterial lysates. B Scratch assay in SiHa cells stimulated with L. iners metabolites. ***p < 0.001, ****p < 0.0001. C KEGG pathways enriched in differentially expressed genes after the treatment of cervical cancer cells with L. iners metabolites.

Fig. 5. L. iners metabolites increased the core fucosylation of epidermal cells by activating the Wnt pathway.

A Correlation analysis between the relative abundance of L. iners and core protein fucosylation. B Histogram of differentially expressed genes in SiHa cells stimulated with L. iners metabolites. C L. iners metabolites stimulated the expression of glycosylation-related genes in SiHa cells. D The level of core fucosylation and expression of β-catenin after treatment with L. iners metabolites. E After SiHa cells were treated with L. iners metabolites, DKK-1 at different doses was added, and the core fucosylation of epidermal cells was measured. F ChIP-qPCR was used to verify the binding of TCF/β-catenin to the promoter of the Fut8 gene. G Dual-luciferase reporter gene assay to verify the regulatory effect of TCF to the promoter of the Fut8 gene. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig. 6. The L. iners metabolite lactate activates the Wnt pathway through the lactate-Gpr81 complex to increase the core fucosylation of epidermal cells.

A Comparison of vaginal pH in healthy volunteers and cervical cancer patients. B Comparison of vaginal lactate levels in the two groups. C Schematic diagram of the effect of lactate. D After SiHa cells were treated with L. iners metabolites, lactate and lactate and 3-OBA, the level of core fucosylation and expression of β-catenin, wnt3, and Gpr81 were determined. E Cellular immunofluorescence experiments were used to analyze the level of core fucosylation and expression of β-catenin after SiHa cells were treated with L. iners metabolites, lactate and lactate and 3-OBA. ***p < 0.001, ****p < 0.0001.

Fig. 7. The molecular mechanism by which Lactobacillus spp. regulate the core fucosylation of epithelial cellsthrough the Wnt pathway.

Lactobacillus iners can secrete lactate to increase the level of core fucosylation of epithelial cells by activating the Wnt pathway, which inhibits the proliferation and migration of cervical cancer cells.