Reproductive tract commensal bacterium Enterococcus faecium supernatant induces DNA damage associated stress responses and suppresses the growth of cervical cancer cells

Persistent infection with high-risk human papillomavirus (HPV) is closely associated with the progression of cervical cancer. Increasing evidence indicates that the vaginal microecology plays an important role in HPV infection outcomes and the development of cervical lesions; however, the direct effects of specific commensal bacteria in the reproductive tract and their underlying molecular mechanisms remain largely unclear. This study aimed to systematically investigate the effects of conditioned commensal bacteria Enterococcus faecium(E. faecium) supernatant on the malignant biological behaviors of cervical cancer cells and to elucidate the potential molecular mechanisms involved.

HeLa, SiHa, and C33A cervical cancer cell lines were used to evaluate the effects of E. faecium supernatant on cell proliferation, migration, cell-cycle distribution, and apoptosis. Immunofluorescence staining was performed to assess nuclear R-loop accumulation and the expression of the DNA damage marker γH2AX, thereby exploring E. faecium supernatant induced cellular damage responses. Furthermore, mRNA transcriptome sequencing was conducted in HeLa cells to identify key differentially expressed genes. Candidate genes were validated by RT-qPCR, Western blotting, and immunofluorescence, and siRNA-mediated knockdown experiments were performed to evaluate the functional roles of key genes in E. faecium supernatant mediated effects.

Treatment with E. faecium supernatant significantly inhibited the proliferation and migration of cervical cancer cells, induced cell-cycle arrest, and promoted apoptosis. In HPV-positive cervical cancer cells, E. faecium supernatant treatment led to abnormal accumulation of nuclear R-loops accompanied by enhanced γH2AX signaling. Transcriptomic analysis revealed substantial alterations in the global gene expression profile following E. faecium supernatant treatment, with differentially expressed genes predominantly enriched in stress response and DNA damage related pathways. Among these, the endoplasmic reticulum stress associated gene HSPA5 was markedly upregulated. Knockdown of HSPA5 further enhanced the inhibitory effects of E. faecium supernatant on cell proliferation and its pro-apoptotic effects in cervical cancer cells.

This study provides preliminary evidence that E. faecium supernatant inhibits the proliferation of cervical cancer cells and induces apoptosis, accompanied by nuclear R-loop accumulation and enhanced DNA damage signaling. HSPA5 may be involved in this process and may play a role in limiting the progression of cellular damage toward irreversible cell death in cervical cancer cells. These findings provide a new perspective for understanding the potential role of vaginal microecological factors in cervical cancer.