To the best of our knowledge, this is the first two-sample MR analysis to investigate the causal relationship between GM and OCC. Using the aggregated GM data from the largest GWAS meta-analysis conducted by the MiBioGen consortium and the OCC data from the IEU Open GWAS project for analysis, we found genetic liability for 18 types of GM causally associated with OCC. In particular, both the IVW and MR-Egger methods showed a positive correlation between Bacillales and the incidence of OCC, with significant results. At least one method showed an association between 10 types of GM and a high risk of OCC, while 5 types of GM were associated with a low risk of OCC. In addition, there were two microbiota, Erysipelatoclostridium and Victivillaceae, whose MR-Egger-estimated causal relationship was opposite to that found by IVW analysis.
In the past, Mendelian randomization studies have investigated the causal associations between GM and various diseases, including lung cancer and periodontitis. The precise association between GM and OCC has not been the focus of dedicated research. Our study is the first to utilize a MR study to investigate the causal link between these two factors. In our study, due to the lack of pleiotropy of SNP, the causal relationship obtained by IVW was more accurate than that obtained by MR Egger[33]. Therefore, we considered using the IVW method to obtain results that indicated a positive correlation between Burkholderile, Bacillales, Betaproteobacteria, Desulfovibrio, Pasturellales, Pasturellaceae, Eubacteriumficatena, Alcaliginaceae and the risk of OSCC, while Butyrivibrio, Eggerthella, Clostridiumstricto1 are inversely correlated with OCC’s risk. This discovery may offer novel insights for the prevention and treatment of OCC.
The order Bacillales belongs to the phylum Firmicutes, and is a type of Gram-positive bacteria that primarily exists in the intestinal microbiota of humans and mammals[34]. Clinical studies have shown that an increase in the abundance of order Bacillales can lead to Graves' disease[35]. Furthermore, other research has indicated a strong positive correlation between increased abundance of order Bacillales and diseases such as pancreatic cancer[36] and endometriosis[37]. Additionally, retrospective studies have revealed that Bacillales expression accounts for 2% in patients with proliferative verrucous leukoplakia (PVL), a high-risk condition for OCC[38], and is enriched in patients with T1/T2 stage OSCC and non-lymph node metastasis[39]. Our study demonstrates the significant biological relevance of order Bacillales in the occurrence and development of OCC. This finding can provide new insights into the pathogenesis and treatment of OCC, and may lead to further research into the pathogenic mechanisms of the order Bacillales.
In addition to Bacillus, we discovered that seven types of GM were linked to OCC. These included order.Burkholderiale, class.Betaproteobacteria, family.Alcaligenaceae genus.Desulfovibrio, order. Pasteurellales, family.Pasteurellaceae, and genus..Eubacteriumfissicatena. It is worth noting that genus. Eubacteriumfissicatena belongs to the phylum Bacillota, while the other six groups belong to the phylum Pseudomonadota. Eubacterium is a fundamental genus in the human intestinal microflora, and its metabolite butyrate is crucial for colonic motility, immunomodulation, and reducing intestinal inflammation[40]. Research has shown that patients with HNCs who experience more severe psychoneurological symptoms (PNS) have a greater presence of Eubacterium fissicatena in their GM. This bacterium may worsen the PNS in patients by affecting neurological, immune, and endocrine signaling pathways, ultimately impacting the cancer treatment negatively[41]. However, the genus Eubacteriumfissicatena has not been extensively researched in connection to oral diseases, and there is currently no definite link to OCC.
The Pseudomonadota phylum is a significant part of the GM[42, 43]. The study by Radaic et al.[44] reported a significant increase in the Pseudomonadota phylum in the transition from normal oral mucosa to dysplasia and cancer, as evidenced by a gradual increase in the abundance of Pasteuriaceae and Burkholderiaceae in proportion to the degree of histological heterogeneous proliferation near the tumors, which is in accordance with our findings. Similarly, a study by Zhang et al.[45] arrived at similar conclusions, indicating that Ascomycetes were significantly more prevalent in OSCC tissues compared to saliva and mouthwash samples from OSCC patients, making up 52% of the taxonomic units. This suggests a potential association between Ascomycetes and the development of OSCC.
The family.Alcaligenaceae belonging to the Burkholderiale order, are part of the Betaproteobacteria class within the Pseudomonadota phylum. Chen et al.[46] discovered that the bacterial antigen from Burkholderia multivorans showed immune reactivity in an experiment aimed at identifying proteins that trigger the host immune response in the blood of OSCC patients. This suggests that the bacterial antigen could potentially serve as a serum biomarker for OSCC, aiding in the creation of early diagnostic tools for the disease. Additionally, Burkholderia has been observed to have a broader distribution and more classifications in oral disease states, indicating its potential contributory role in the development of oral diseases[47]. Conversely, certain Betaproteobacteria, such as Neisseria sicca, have been found to inhibit the progression of OSCC. Notably, Neisseria desiccata, identified as a periodontitis-negative associated bacterium with elevated levels in healthy populations, has been shown to regulate genome stability and impede the development of OSCC[48].
Order.Pasteurellales and family.Pasteurellaceae are classified within the same class of Gammaproteobacteria and the phylum Pseudomonadota. Hoppe et al.'s study[49] demonstrated that the bacterium Pasteurellaceae, Aggregatibacter actinomycetemcomitans, can induce apoptosis in oral cells and indirectly promote the proliferation of oral tumor cells by modulating the expression of carcinogenic-related α-defensin genes. Additionally, Pseudomonas aeruginosa in the Gammaproteobacteria phylum has been found to possess certain oncogenic properties. Pseudomonas aeruginosa, an opportunistic pathogen, is a major cause of infections such as ventilator-associated pneumonia, urinary tract, bloodstream, and chronic infections[50].
Genus.Desulfovibrio, a Gram-negative bacterium belonging to the Desulfovibrio family, has been linked to conditions such as obesity, metabolic syndrome, and inflammation in humans[51]. Several investigations have demonstrated an elevated presence of Desulfovibrio in hepatocellular carcinoma[50], colorectal carcinoma[52], and gastric carcinoma[53], suggesting a potential association with disease advancement. However, there is currently a lack of research or documentation regarding its involvement in OCC.
Genus Butyrivibrio, genus Eggerthella, and genus Clostridium sensustricto1 collectively exhibit a noteworthy trend in MR analysis, suggesting that an augmented abundance of these taxa correlates with a diminished incidence of oral cancer. The genus Butyrivibrio, classified within the order Clostridiales, serves as a salient example of a beneficial bacterium proficient in the production of butyric acid and other short-chain fatty acids[54]. This microbial cohort plays a pivotal role in intestinal health and metabolism, particularly serving as indispensable degraders of plant polysaccharides. Notably, genus Butyrivibrio displays a conspicuous reduction in abundance in Parkinson's disease (PD), demonstrating a negative correlation with pulmonary arterial hypertension, while concurrently manifesting a positive correlation in the context of gallstones according to MR studies[55–57]. Although the potential influence of the genus Butyrivibrio group in various diseases is currently under exploration, its specific role remains to be elucidated.
Genus.Eggerthella is a non-spore group of anaerobic gram-positive bacteria that are part of the normal human microbiome[58]. Clinical investigations reveal an enrichment of Eggerthella in individuals afflicted with inflammatory bowel disease, establishing positive correlations with colorectal cancer and multiple sclerosis[59–61].
Moving further, Genus.Clostridium sensu stricto1 is a group of Gram-positive anaerobic bacteria belonging to the family Clostridiales and Clostridiaceae, capable of producing spores. This particular genus exhibits a positive correlation with neurodegenerative conditions, including Alzheimer's disease, PD, and amyotrophic lateral sclerosis[62].
Notably, the current body of literature does not offer explicit reports linking these bacterial taxa to oral diseases. The groundbreaking observation posited by this study, elucidating the potential of genus Butyrivibrio, genus Eggerthella, and genus Clostridium sensu stricto1 in mitigating the occurrence of OCC, warrants further scrutiny. Future investigations should delve into the intricate mechanisms underlying the risk reduction associated with these microbial entities, thereby paving the way for targeted interventions aimed at preemptively controlling the onset of OCC.
But there are some limitations to our study. First of all, the databases included in the study all come from European ancestry, and further research is needed to expand the databases of other ethnic groups such as Asia and Africa to obtain more powerful evidence; Secondly, in order to obtain sufficient GM, the SNP used in the analysis did not reach the traditional GWAS significance threshold (P < 5×10− 8), we chose a more lenient threshold (P < 1×10− 5). Thirdly, we did not conduct the analysis of the GM and specific subtypes of OCC, which necessitates further research; Finally, the two sample MR method is a theoretical causal analysis method, and further proof of causal relationship needs to be carried out in animal tests to explore whether there is a causal relationship and its specific mechanism between the GM and OCC. Currently, we are conducting experiments simultaneously to prove this causal relationship.
Conclusion
This MR analysis method is less susceptible to confounding factors and evaluated the potential causal effect of GM on OCC and found a potential causal relationship between Bacillus orders and OCC, which provides new ideas for possible treatment as well as clues for the pathogenesis of OCC.