| Background:Ovarian cancer is regarded as the most lethal gynecological malignancy,which is mainly aroused from fallopian tube cancer and peritoneal malignancy.Among all subtypes,Epithelial Ovarian Cancer(EOC)is the most prevalent one,accounting for over 150,000 deaths each year worldwide.In the United States alone,approximately 22,000 new cases are reported annually,posing a great burden for the entire health system.Currently,the standard treatments of EOC include cytoreductive surgery in combination with platinum-based chemotherapy,radiotherapy,and immunotherapy,from which the ovarian cancer patients have greatly benefited and treated,nevertheless,none of them leads to a complete cure,and the overall five-year survival rate still remains poor(around 30%-40%).Accumulating evidence has shown that microbiota is viewed as an increasing important factor in the initiation and progression of many diseases,including cancer.Nevertheless,whether the microbiota is present in ovarian tumors,and what is their role in the development of EOC is still an unclear question needs to be further explored.Objective:To explore the different composition of microorganisms in epithelial benign and malignant ovarian tumors and find potential strains related to the occurrence and development of EOC,and to elucidate the molecular mechanism of EOC progression,so as to provide innovative ideas for clinical treatment of EOC.Methods:1.High-throughput sequencing technology was used to study the composition and relative abundance of microbiota in epithelial ovarian benign tumors and ovarian malignant tumors.2.The collected human ovarian tumor specimens were cultured,isolated and identified by culture-omics technology.3.Ovarian cancer ID8 cells were cultured and injected into epithelial ovarian cancer mice.The experiment was divided into model group(M),antibiotic treatment group(M-A),BMBT+antibiotic treatment group(M-A-BMBT),and MMBT+antibiotic treatment group(M-A-MMBT).To evaluate the effect of mixed bacteria in epithelial ovarian tumor on mouse EOC model.4.Mixed strains of epithelial ovarian cancer and propionibacterium acnes(p.acnes)strains were injected into epithelial ovarian cancer mice,the mice were divided into model group(M),antibiotic treatment group(M-A),SBT+antibiotic treatment group(M-A-SBT),and MBT+antibiotic treatment group(M-A-MBT);QRT-PCR was used to detect the expression of inflammatory factors such as TNF-αand IL1-β in serum of mice.HE staining and immunohistochemistry were used to detect the expression changes of the indicators related to cell migration and invasion in each group,and the expression changes of Gli1 in the tumor were detected.Western Blot was used to detect the expression of Hh signaling pathway related genes in subcutaneous ovarian cancer xenografts.5.p.acnes strains of epithelial ovarian cancer were injected into epithelial ovarian cancer mice,the experiment was divided into model group(M),antibiotic treatment group(M-A),SBT+antibiotic treatment group(M-A-SBT),MBT+antibiotic treatment group(M-A-MBT),GANT61+antibiotic treatment group(M-Ag),and MBT+GANT61+antibiotic treatment group(M-A-MBT-G),SBT+GANT61+antibiotic treatment group(M-A-SBT-G);HE staining and immunohistochemistry were used to detect the expression changes of the indicators related to cell migration and invasion in each group,and the expression changes of Gli1 were detected.Western Blot was used to detect the expression of Hh signaling pathway related genes in subcutaneous ovarian cancer xenografts in mice,and to explore whether tumor microorganisms in different patients affect the expression of Hh signaling pathway related genes in ovarian cancer.Results:1.The microbial composition of epithelial benign ovarian tumour(EBOT)and EOC tissues is distinct.The Chao1 index and Shannon index,which measures species richness and evenness,was significantly higher in EOC tissues than in EBOT tissue.The weighted UniFrac principal coordinate analysis(PCoA)indicating that the EOC group’s microbial diversity was considerably different from that of the EBOT group.According to the Venn diagram,658 and 4144 OTUs were found in EOC and EBOT tissues,respectively,with 312 OTUs present in both categories.Then,we further analyzed the microbial composition at the phylum level and found that Firmicutes,Bacteroidetes and Acidobacteria were the dominant bacteria in EOC and EBOT tissues.2.The specific EOC related bacteria were cultured and identified,and 99 strains of bacteria were obtained,including 81 strains of p.acnes,10 strains of Bacillus,6 strains of Streptococcus,7 strains of Bacillus cereus,1 strain of Escherichia coli and 2 strains of Lactobacillus.The composition of microorganisms was analyzed at the genus level and Devosia,Chelativorans,Oceanicaulis and Thermus were found to be the dominant genera in the EOC group.3.Compared with group M,intratumoral injection of mixed bacteria EOC and EBOT significantly increased tumor volume,and compared with intratumoral injection of mixed bacteria EBOT,intratum oral injection of mixed bacteria EOC had a more obvious tumor-promoting effect.In addition,the weight of tumors induced by mixed intratumoral injection of EOC and EBOT was heavier than that of group M.Survival tests showed that all mice injected with EOC mixed bacteria died before day 70,while mice injected with EBOT mixed bacteria and treated with antibiotics had survival rates of 33.3%and 66.7%on day 87,respectively.4.Compared with group M,intratumoral injection of p.acnes strains with EOC increased tumor volume,but not as effective as intratumoral injection of EOC mixed bacteria.In addition,intratumoral injection of EOC into the p.acnes strain promoted heavier tumor weight than did the M group and the acne strain shortened the survival of mice compared with the M group.The levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)increased after intratumoral injection of p.acnes strains with EOC.In addition,the results of H&E staining showed that the p.acnes strain increased the number of tumor cells,and the tumor cells were more uniform,thicker,and darker than the M group.The results of immunohistochemistry showed that thep.acnes strain significantly increased the expression of Gli1,and Western blot analysis showed that the p.acnes significantly enhanced the expression of Shh,Smo,Ptch1,Gli1 and Gli2.5.The results of HE staining showed that GANT61 could increase the necrotic tumor cells and expand the necrotic area,which was greater than that of p.acnes injected with EOC.IHC results showed that GANT61 administration significantly decreased Gli1 expression.In addition,GANT61 decreased the expression of transcription factors Gli1 and Gli2 according to Western blot studies.At the same time,the expression level of Ptch,the downstream target of GLI,decreased.Conclusion:1.This study compares the similarities and differences of intratumour microbiota among patients with epithelial benign ovarian tumour and patients with EOC and demonstrated that the diversity and composition of the intratumor microbiota were significantly different between the EBOT and EOC tissues.2.The P.acnes is potential key strains in the process of EOC progression and figured out the underlying molecular mechanisms,which was associated with the increase inflammatory response to activate Hh signaling pathway through in vivo and in vitro experiments. |
PDF Full Text Request