Cervicovaginal microbiota disorder combined with the change of cytosine phosphate guanine motif- toll like receptor 9 axis was associated with cervical cancerization

Abstract

Background: Convincing studies demonstrated that cervicovaginal microbiota disorder and toll-like receptor 9 (TLR9) high expression were related to cervical carcinogenesis. However, the effects of cervicovaginal microbiota integration TLR9 in cervical cancerization are unclear. Based on the biological basis that unmethylated cytosine-phosphate-guanine (CpG) motifs of bacteria could activate TLR9, we explored the effects of cervicovaginal microbiota disorder and CpG motif-TLR9 axis change in cervical carcinogenesis.

Methods: A total of 341 participants, including 124 normal cervical (NC), 90 low-grade cervical intraepithelial neoplasia (CIN1), 78 high-grade cervical intraepithelial neoplasia (CIN2/3) and 49 squamous cervical cancer (SCC), diagnosed by pathology were enrolled in the study. Here, metagenomic shotgun sequencing was used to reveal cervicovaginal microbiota characteristics, and TLR9 protein was detected by western blotting.

Results: Our results showed that the diversity of cervicovaginal microbiota gradually increased along with the poor development of cervical lesions, showing the abundance of Lactobacillus crispatus and Lactobacillus iners decreased, while the abundance of pathogenic bacteria gradually increased. The level of TLR9 expression was gradually increased with cervicovaginal microbiota diversity increasing, the abundance of Lactobacillus decreasing, and we found a positive correlation dependency relationship (r = 0.384, P = 0.002) between TLR9 and GTCGTT motif content. Stratified analysis based on HPV16 infection, we found that the characteristics of cervicovaginal microbiota and increased TLR9 expression were also closely related to HPV16 infection.

Conclusions: Cervicovaginal microbiota dysbiosis might lead to the CpG motif increased, which was closely associated with TLR9 high expression, and ultimately might promote the progression of cervical lesions.

Keywords: Cervical lesion; Cervicovaginal microbiota; CpG motif; Metagenomic shotgun sequencing; TLR9.

Fig. 1

Abundance of the top 20 microbiota among different individuals is shown by the color code at the logarithmic scale: log10 (abundance) (a); the total number of species and the number of unique species in each group (b); alpha diversity in the progression of cervical lesions (c) and beta diversity (d)

Fig. 2

Cervicovaginal microbiota composition in the four groups (a) and different HPV16 infection status (b); characteristic bacteria in the four groups and different HPV16 infection status (c)

Fig. 3

TLR9 protein expression in the progression of cervical lesions (a) and the relationship between TLR9 protein expression and Shannon index (b); microbiota composition (c) and characteristic bacteria under different TLR9 expression conditions (d)

Fig. 4

Inter-relationships among microbiota community in TLR9 low expression group (a) and TLR9 high expression group (b); the top 20 genera with relative abundance, the correlation coefficient r > 0.4, and P < 0.05 were selected to draw a symbiotic network diagram. The nodes in the figure represent different strains, the size of the nodes represents the relative abundance of the strains, and the thickness of the lines between the nodes represents the correlation

Fig. 5

GTCGTT motif content of 10 main bacteria (a) and correlation analysis of GTCGTT motif content and TLR9 expression (b)

Fig. 6

Ranking of variable importance (a) and evaluation of the predictive effect of cervicovaginal microbiota on CIN2 + (b); ranking of variable importance (c) and evaluation of the predictive effect of cervicovaginal microbiota, TLR9 and HPV16 infection on CIN2 + (d)