Exploring the causality between gut microbiota and esophageal carcinoma: a two-sample Mendelian randomization analysis

Exploring the causality between gut microbiota and esophageal carcinoma: a two-sample Mendelian randomization analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the causality between gut microbiota and esophageal carcinoma: a two-sample Mendelian randomization analysis Bingxiao Lu, Xiangzeng Zuo, Jie Deng, Xiaoyu Gong, Na Wu, Yunxia Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4169602/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Esophageal cancer is a common tumor of upper gastrointestinal tract. However, the increasing evidence indicates that gut microbiota are closely related to the occurrence and development of esophageal cancer, the causal association between gut microbiota and esophageal cancer remains to be determined. As a consequence, in this paper, a two-sample Mendelian randomization analysis was used to evaluate and explore the causal relationship between gut microbiota and the risk of esophageal cancer, and identify specific pathogenic bacterial taxa. Methods Genetic instrumental variables for gut microbiota were identified from a genome-wide association study (GWAS) of 18,340 participants, esophageal cancer as the outcome variable was identified from a GWAS including 1091 cases and 410,350 controls. Using the inverse variance weighted (IVW) method as the primary analysis, and further (using) the weighted median method, MR-Egger regression, MR multi-directional residuals, and outlier tests were further performed to improve the robustness of the results. Results The IVW results showed that genus.DefluviitaleaceaeUCG011 (OR = 3.124, 95%CI 1.388–7.031, P = 0.006), genus.LachnospiraceaeUCG008 (OR = 3.964,95%CI 1.463–10.740, P = 0.007), family.Pasteurellaceae.id.3689 (OR = 2.022, 95%CI 1.046 − 3.909, P = 0.036)and order.Pasteurellales.id.3688༈OR = 2.022, 95%CI1.046 − 3.909, P = 0.036༉showing a positive causal relationship between gut microbiota and esophageal cancer.Conversely, genus.Peptococcus (OR = 0.525, 95% CI 0.286–0.961,P = 0.037) , genus.Ruminiclostridium5 (OR = 0.117,95%CI0.0310.442,P = 0.002), genus.RuminococcaceaeUCG009 ༈OR = 0.350, 95%CI 0.157 − 0.777, P = 0.01༉and genus.Ruminococcus1 ༈OR = 0.341, 95%CI 0.118 − 0.985, P = 0.047༉showed a negative causal relationship with esophageal cancer. No significant heterogeneity was found in the instrumental variable or horizontal pleiotropy. Conclusions Our MR analysis study revealed that the gut microbiota was causally associated with esophageal cancer and the risk of esophageal cancer, and also identified eight bacterial taxa with a causal relationship with the development and progression of esophageal cancer.That may be useful in providing new insights for further mechanistic and clinical studies of microbiota-mediated cancer. gut microbiota esophageal cancer Mendelian randomization single nucleotide polymorphisms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Esophageal cancer (EC) is a common upper gastrointestinal tumor with the 7th highest incidence among all tumors and is a serious threat to human health [ 1 ] . The occurrence and development of EC is a complex process. Despite differences in morbidity and mortality among countries, China contributes nearly half of the global EC cases due to its large population [ 2 ] . The most common histological subtypes of esophageal cancer are squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), of which esophageal squamous cell carcinoma accounts for more than 90% of esophageal cancer cases [ 3 ] with. Even though,the prognosis of EC has significantly improved with the continuous improvement of screening methods and the updating of treatment methods, most patients are still in the intermediate and advanced stages when diagnosed, which greatly reduces the survival rate of EC. According to relevant literature records that the 5-year survival rate of EC patients is less than 20% [ 4 ] . The human symbiotic microbial community consists of > 100 trillion microorganisms, mainly living on the surface of human epithelial cells, including the skin, digestive tract and respiratory tract [ 5 ] . Among them, the billions of microorganisms attached to the human intestine are collectively refered gut microbiota [ 6 ] . With the development of microbial analysis methods (e.g. 16SrRNA sequencing), high-throughput sequencing technologies (e.g. second-generation sequencing), and genomics methods [ 7 ] , the roles and mechanisms of gut microbiota in the host ecosystem have been more comprehensively characterized.The gut microbiota, known as the "forgotten organ", has gradually become a research hotspot in recent years. There is increasing studies have found that gut microbiota not only play an important role in maintaining physiological functions such as homeostasis, material metabolism, immune homeostasis, growth and development, and cognition and other physiological functions [ 8 ] , but also through the induction of DNA damage and the release of inflammatory mediators, interference with the immune system, regulating carcinogenic host pathway proteins, and generating carcinogenic metabolites mechanism to promote the occurrence and development of tumors [ 9 ] . As a common digestive tract tumor, the association and mechanism of intestinal microbiota and its occurrence and development have been gradually researched. For example, Zaidi et al [ 10 ] found in rat models and human clinical samples that Escherichia coli was significantly enriched in esophageal adenocarcinoma patients or rats compared with the healthy ones. Li et al [ 11 ] used a 16S rRNA method to analyze the differences in fecal flora of patients with esophageal squamous cell carcinoma, and found that Prevotella spp. significantly decreased in patients with squamous cell carcinoma, compared with healthy people in the control group. In addition, there is emerging evidence [ 12 – 18 ] from varying degrees which have shown that gut microbiota are gradually playing a central role in the development of esophageal cancer. While the growing evidence of an association between gut microbiota is closely related to the risk of esophageal cancer, but whether there is a causal effect remains unknown. Mendelian Randomization (MR) is a data analysis method using genetic variants strongly correlated with exposure factors as instrumental variables to evaluate the causal relationship between exposure factors and outcomes [ 19 ] .Most notably, two-sample MR analysis can utilize Single Nucleotide Polymorphism (SNP) exposure and outcome associations from independent GWAS and combine them into a single causal estimate to analyze the casual relationship between exposure and outcome [ 20 ] . Compared with conventional regression methods, this approach is less susceptible to measurement error, confounding and reverse causality. Using two-sample Mendelian randomization analysis to explore the causal relationship between gut microbiota and tumors has been increasingly studied, but more of them focus on colorectal cancer, breast cancer, gynecological cancer and other cancer. But whether there is a causal effect between gut microbiota and esophageal cancer remains unknown.Therefore, in our study, in order to explore the potential causal relationship between gut microbiota and esophageal cancer and to identify specific pathogenic bacterial taxa, we conducted a two-sample MR study based on genome-wide association study (GWAS) pooled data. Materials and Methods 1. Research design In this study, we use public GWAS summary statistics, at the same time the gut microbiota are used as the exposure, and esophageal cancer as the outcome to conduct MR analysis for evaluating the causal relationship between 199 gut microbiota and esophageal cancer. Finally, heterogeneity testing and gene-level multi-analysis are conducted. Validity testing and other quality controls verify the reliability and robustness of the causal relationship results. The data are publicly available, so that ethical approval was not required for this study. MR analysis was explored based on three hypotheses: (1) IVs are strongly related to exposure; (2) IVS are not related to relevant confounding factors; (3) IVs affect outcomes through exposure only. (Fig. 1 ) 1.1 Data source The summary statistics for human gut microbiota we used in this study were obtained from the most recent GWAS meta-analysis, a large and multi-ethnic study, which included 18,340 participants from 24 cohorts. Of the 24 cohorts, 22 were composed of adults or adolescents (N = 16,632), and two cohorts were composed of children (N = 1,708). This study combined data from the United States, Canada, Germany, 16S rRNA gene sequencing profiles and genotyping data from Denmark, the Netherlands, Belgium, Sweden, Finland, the United Kingdom and other countries, and related analyzes were conducted on age, gender, technical covariates and genetic principal components [ 21 ] . The GWAS data for esophageal cancer arise out of a large pan-cancer study that combined UK Biobank (408,786 individuals of European ancestry, age 40–69 years, mean age 56.5 years; 48,961 cancer cases) and Kaiser Permanente Genetic Epidemiological study of Adult Health and Aging (66,526 individuals of European ancestry, 18 to 100 + years, mean age 63 years; 16,001 cancer cases) two large, independent, prospective cohorts, including 1091 patients with esophageal cancer and 410350 healthy controls [ 22 ] . In order to reduce the population stratification differences, most of the outcome and exposure data we selected came from European populations. 1.2 Screening of instrumental variables For the purpose of ensuring the inclusion of appropriate IVs, this study conducted IVs screening based on the core assumptions of MR. The specific steps for selecting IVs are as follows: (1) Among the exposure data already obtained, this study selected a relatively reliable threshold based on previous literature. P<1×10 − 5 is used as the standard to select SNPs that are strongly related to the abundance of gut microbiota; (2) To ensure that the selected IVs are independent of each other, the parameter r 2 < 0.001 was set, and the distance between SNPs which is within 10000Kb to exclude the SNP in linkage disequilibrium ensuring that SNPs are independent of each other; (3) Remove weak instrumental variables: Use the F statistic to evaluate the statistical strength of the correlation between SNPs and exposure. The formula of the F statistic is (F= \(\frac{{R}^{2}}{1-{R}^{2}}\bullet \frac{N-K-1}{K}\) ), where N is the sample size of the intestinal flora database, K is the number of SNPs, R2 represents the proportion of variation in the intestinal flora explained by SNPs, the calculation formula is R2=2 \(\bullet\) (1-EAF) \(\bullet\) EAF \(\bullet\) β2. Where EAF is the effect allele frequency of each SNP, and β is the effect value for the allele [ 23 ] . The F test value > 10 was used as the screening criterion to find SNPs without strong correlation but which is related to exposure factors; (4) Integration of the two sets of data: the SNPs related to the relative abundance of gut microbiota were project to the corresponding statistical parameters of esophageal cancer, and use the "harmonise_data" function to unify the data based on the gut microbiota abundance and the statistical parameters of the same sites in the GWAS results of esophageal cancer. 1.3 Data analysis methods After the final included IVs were determined according to the above IVs screening process, MR analysis was started. This study used 5 methods to estimate the causal effect: inverse variance weighted method, MR-Egger regression, weighted median method, simple mode method and weighted mode method. Previous studies have shown that the IVW method has higher testing efficiency than the other four methods, so the IVW method is used as the preferred causal effect estimation method in this study. So as to test the stability and reliability of the results additionally, this study conducted quality control on the MR results that remained statistically significant after FDR correction. The Cochran Q test was used to calculate heterogeneity, P < 0.05 indicated the presence of heterogeneity, MR-PRESSO analysis was used to remove outliers, MR-Egger regression intercept analysis was used to detect horizontal pleiotropy, and leave-one-out analysis reflected the robustness of MR results. And whether the causal effect was dominated by a certain SNP. All analyzes were performed in R software (version 4.313) using the Two-Sample MR package and the MRPRESSO package. (Fig. 2 ) Results 1.1 Instrument variable selection According to the screening criteria of instrumental variables for this study, 89 SNPs (Fig. 3 ) were finally included. The F statistics ranged from 18.49 to 33.84, all above 10, indicating that they were not affected by weak instrumental variables. 1.2 The two-sample MR results This study estimated the causal effect between the abundance of 199 gut microbiota and esophageal cancer, using the IVW method to find that 8 types of intestinal microorganisms are associated with esophageal cancer(Fig. 3 ; Fig. 4 ). The MR results of the correlation between all 199 bacterial characteristics and esophageal cancer risk are shown in Appendix 1( Supplementary Table 1 ). The results of our study showed that, genus. DefluviitaleaceaeUCG011 (OR = 3.124, 95%CI 1.388–7.031, P = 0.006) is positively associated with the risk of developing esophageal cancer. No abnormal values were found in the MR-PRESSO test. The intercept of MR-Egger regression also did not show potential horizontal pleiotropy (intercept p value = 0.863), no heterogeneity was detected in Cochran's Q heterogeneity test (P > 0.05); genus.LachnospiraceaeUCG008 (OR = 3.964, 95% CI 1.463 − 10.740, P = 0.007) was positively correlated with the risk of esophageal cancer. The MR-PRESSO test found no outliers, and the intercept of the MR-Egger regression did not show the potential horizontal pleiotropy (intercept p value = 0.563) as well, no heterogeneity was detected in Cochran's Q heterogeneity test (P > 0.05); family.Pasteurellaceae.id.3689 (OR = 2.022, 95%CI 1.046 − 3.909, P = 0.036) was positively correlated with the risk of esophageal cancer, and no outliers were found in the MR-PRESSO test. The intercept of the MR-Egger regression also did not show the potential horizontal pleiotropy (intercept p value = 0.328). No outliers were found in the Cochran's Q heterogeneity test (P > 0.05); order.Pasteurellales.id.3688 (OR = 2.022, 95%CI1.046 − 3.909, P = 0.036) was positively correlated with the risk of esophageal cancer, no outliers were found by MR-PRESSO test, the intercept of the MR-Egger regression also did not show the potential horizontal pleiotropy (intercept p value = 0.328), and no heterogeneity was detected in the Cochran's Q heterogeneity test (P > 0.05). On the contrary, genus.Peptococcus (OR = 0.525, 95%CI0.286-0.961, P = 0.037) is negatively correlated with the risk of developing esophageal cancer. No outliers were found in the MR-PRESSO test, and the potential horizontal pleiotropy was not shown by the intercept of the MR-Egger regression either. (intercept p value = 0.542), no heterogeneity was detected in Cochran's Q heterogeneity test (P > 0.05); genus.Ruminiclostridi-um5 (OR = 0.117, 95%CI 0.031 − 0.442, P = 0.002) was positively correlated with the risk of esophageal cancer. The MR-PRESSO test found no outliers, and the intercept of the MR-Egger regression did not show the potential horizontal pleiotropy (intercept p value = 0.960 ), no heterogeneity was detected in Cochran's Q heterogeneity test (P > 0.05); genus.RuminococaceaeUCG009 (OR = 0.350, 95%CI 0.157 − 0.777, P = 0.01) was positively correlated with the risk of esophageal cancer, no outliers were found in the MR-PRESSO test, the intercept of the MR-Egger regression did not show the potential horizontal pleiotropy (intercept p value = 0.206), and no heterogeneity was detected in the Cochran's Q heterogeneity test sex (P > 0.05) either; genus.Ruminococcus1 (OR = 0.341, 95%CI 0.118–0.985, P = 0.047) was negatively associated with the risk of esophageal cancer. No outliers were found in the MR-PRESSO test. No potential horizontal pleiotropy (intercept p value = 0.385) was shown by the intercept of the MR-Egger regression as well, and no heterogeneity was detected in the Cochran's Q heterogeneity test (P > 0.05). 1.3 Quality control In the Cochran’s Q heterogeneity test, no heterogeneity was observed in the SNPs of the 8 bacterial traits ( Supplementary Fig. 1 ). MR-Egger analysis did not detect potential horizontal pleiotropic effects, indicating that the instrumental variables did not significantly increase the impact of pathways other than exposure on outcomes. The MR-PRESSO global test found no outliers, and the leave-one-out test analysis showed that no individual SNP had an impact on the overall causal relationship(Fig. 5 ). Discussion Although the two-sample Mendelian randomization analysis is increasingly implemented to study the causality between gut microbiota and cancer, but the researches on esophageal cancer are relatively scarce. Therefore, in this study, we used two-sample MR research, eight bacterial taxa were identified as having a causality with the risk of esophageal cancer, at the level of order, family and genus. It brings potential targets for early diagnosis, treatment, monitoring and prevention of esophageal cancer, and has certain implications for public health intervention aimed at reducing the risk of esophageal cancer. There are quantity of emerging studies have shown that the gut microbiota is bound up to the occurrence and development of esophageal cancer. There are differences between patients with esophageal cancer and healthy people in the gut microbiota with the level at order, family and genus. The results of this study show that the genus LachnospiraceaeUCG008 is an important risk factor in esophageal cancer, which belongs to the Clostridium group of Firmicutes and is a type of obligate anaerobic bacteria. It is generally recognized that Barrett's esophagus is a precancerous lesion of esophageal adenocarcinoma. Accordingly, Liu et al. [ 24 ] found that the Firmicutes accounted for 55% of Barrett's esophageal microbiota at the phylum level, and the relative abundance of Fusobacteria in the microbiota was higher than that of normal peopel. Mirco et al [ 25 ] found that the gut microbiota of patients with non-alcoholic fatty liver disease (NAFLD) is rich in Lachnospiraceae . Some studies have confirmed that metabolic syndrome is closely related to the occurrence of Barrett’s esophagus. NAFLD is a hepatic manifestation of metabolic syndrome, suggesting that NAFLD may be related to esophageal ulcer, Barrett's esophagus, and esophageal adenocarcinoma. South Korea reported a large cohort study based on the national population, which included 8120674 subjects, including 936159 NAFLD patients. During an average follow-up period of 7.2 years, a total of 3,792 subjects developed esophageal cancer. Among them, the risk of esophageal cancer in NAFLD patients is approximately twice than that of non-NAFLD patients (HR 2.10, 95%CI 1.88–2.35). Moreover, NAFLD significantly increases the risk of death from esophageal cancer (HR 1.46, 95%CI 1.28–1.67) [ 26 ] . By evaluating the relationship between gastrointestinal microbiota and body mass index (BMI), Ley et al. [ 27 ] found that Firmicutes abundance increased (p value = 0.002) and Bacteroidetes decreased accordingly (p < 0.001), which was associated with high BMI. Furthermore,Patients with a BMI of 30 or higher had about twice the risk of developing EC as those with a BMI of < 25, and there was a dose-response relationship between increased waist circumference and the risk of developing EC [ 28 ] ,which supports our findings. The results of this study show that there is a positive causal relationship between gut microbiota of the genus DefluviitaleaceaeUCG011 and the incidence of esophageal cancer. Oral bacteria can migrate to the esophagus and stomach through swallowing, and the ecology of the esophageal flora will also be different in people with different oral health conditions [ 29 ] .The genus Defluviitaleaceae is more commonly found in the human oral cavity, and there is relevant evidence that it plays a key role in the occurrence and development of periodontitis. For example, in a comparative study by Chen Bin et al. [ 30 ] on the bacterial flora of people with aggressive periodontitis, chronic periodontitis and healthy periodontitis, it was found that compared with those with chronic periodontitis and healthy periodontitis, DefluviitaleaceaeUCG011 was significantly increased in patients with aggressive periodontitis (P < 0.05). Similarly, in another study on aggressive periodontitis and chronic periodontitis [ 31 ] , it was also found that the abundance of DefluviitaleaceaeUCG011 was significantly different between the two types of study populations, and was found to be related to periodontal pockets. There is a negative correlation with detection depth. In addition to being associated with the risk of periodontitis, more and more studies have found that DefluviitaleaceaeUCG011 is also widely present in the intestine and is associated with cardiac anti-fibrotic protection [ 32 ] and rheumatoid arthritis [ 33 ] , lipid metabolism [ 34 ] , social frustration disorder, childhood urticaria [ 35 ] and other diseases are related. It is worth noting that in oral microorganisms, The increased abundance of DefluviitaleaceaeUCG011 is a risk factor for periodontal disease, but in gut microbiota, the decreased abundance of DefluviitaleaceaeUCG011 is the causative factor for the development of the above diseases. Some studies have used kelp and Gastrodia elata extracts to increase this the abundance of bacterial taxa, thereby achieving the purpose of treating diseases, also further proves the pathogenic role of this bacterial taxa in related diseases. The results of this study show that there is a negative causal relationship between gut microbiota of the genus Ruminococcus1 and family RuminococcaceaeUCG009 and the incidence of esophageal cancer. Research shows that Ruminococcus can produce short-chain fatty acids, which provide an energy source for intestinal cells, improve intestinal barrier function, and also have anti-inflammatory effects. Short-chain fatty acids are carboxylic acids with 1 to 6 carbon bond atoms. It is formed by the metabolism of complex carbohydrates into oligosaccharides by the gut microbiota and fermentation. It can be directly absorbed by the intestinal mucosa, and there are many types, mainly butyrate, propionate, and acetate. Smith et al. [ 36 ] have shown that the high abundance of Ruminococcus produces a large amount of short-chain fatty acids that can regulate the homeostasis of colonic Treg cells, improve the re-conversion of immune tolerance, and inhibit the inflammatory response in colitis mice, while butyric acid and The specific anti-inflammatory mechanism of acetic acid is to inhibit G protein-coupled receptor 43 and then inhibit histone deacetylase in Treg cells to play an anti-inflammatory effect [ 37 – 38 ] . Some studies have found that periodic intake of yogurt can effectively increase the probiotics in the intestines, significantly increase the levels of short-chain fatty acids, and thereby accelerate the apoptosis of colorectal cancer cells [ 39 ] . Mueller et al. [ 40 ] studied that after metformin treatment, the relative abundance of Ruminococcus torque group increased and improved overweight/obese adults, which means that Ruminococcus torques can alleviate obesity. Feng et al. [ 41 ] also proved that Ruminococcaceae can alleviate obesity. Studies have shown that Ruminococcaceae_UCG-013 is positively correlated with serum HDL-C levels and negatively correlated with serum TC, TG and LDL-C levels, and can be identified as the most important biomarker for reducing obesity. Ma et al [ 42 ] used MR analysis to show that Ruminococcaceae has a negative causal relationship with primary liver cancer, which has potential significance for the prevention and control of liver cancer. Ruminococcaceae_UCG-013 was shown to be more abundant in the gut microbiota of mice with a lean phenotype than in mice with an obese phenotype. The above studies reveal their beneficial effects in human diseases and support our findings. The genus Rumiclostridium is one of the first gastric bacteria discovered and plays a vital role in metabolism. Relevant studies have shown that increases and decreases in the abundance of Ruminiclostridium may lead to the occurrence and development of some diseases. The occurrence and development of inflammatory bowel diseases such as Crohn's disease, irritable bowel, and ulcerative colitis are closely related to this bacterial group.Wang et al.'s study [ 43 ] found that compared with healthy people, the abundance of Ruminiclostridium increased in patients with irritable bowel, and after multi-factor regression analysis, it was found that this bacterial group is a key indicator for the pathogenesis of irritable bowel. Similarly, a series of studies have also found that Rumiclostridium plays a role in the occurrence and development of children's eczema [ 44 ] , diabetes [ 45 ] , asthma [ 46 ] , spondyloarthritis [ 47 ] , and so forth. Among them, it is worth emphasizing that Yu et al. conducted a two-sample MR study on Rumiclostridium and spondyloarthritis, and the results showed a causal relationship between this bacterial group and spondyloarthritis, which provides new insights into the Potential treatments of spondyloarthritis. The genus Peptococcus is a normal flora found in the mouth, intestines, female reproductive tract, skin, etc. The role of Peptococcus in the occurrence and development of diseases is more concentrated in livestock production, and the use of antibiotics, fecal transplants and other means can promote the development of livestock production. For example, the study by Maltecca et al. [ 48 ] showed that the abundance of Peptococcus was related to the increase in growth rate and obesity of pigs, describing for the first time the value of microbial communities in pork production systems. There are also a few studies showing that Peptococcus also plays an important role in acute urticaria in children, cognitive impairment in diabetic patients, and colorectal cancer, but there are still few studies at present. In addition to the potential causal relationship between the above bacterial taxa and the occurrence of esophageal cancer at the genus level, the family Pasteurellaceae and the order Pasteurellales also have similar causal relationships. The association between Pasteurellaceae bacterial groups and diseases is mostly concentrated in the epidemic of poultry cholera, sepsis, inflammatory bowel disease, etc. In the article published by Upadhyay et al [ 49 ] , the results showed that the abundance of Pasteurellaceae is related to the pre-treatment A positive association with disease status in children with newly diagnosed Crohn's disease provides further support for this microbiota as a driver of inflammatory bowel disease progression. Overall, in our study, we found a potential causal relationship between these eight specific bacterial taxa and the occurrence of esophageal cancer. For a long time, gut microbiota have continued to appear as an important component of body health, disease status, and treatment response, bringing potential targets for the diagnosis, treatment, detection, and prevention of many diseases, especially tumors. Analysis and targeting of the microbiota in the gut and other niches will also become part of comprehensive cancer care as well as the management of other diseases, ultimately integrating into precision individual health management. Through regular detection of relevant carcinogenic gut microbiota, it can be changed early through antibiotics, prebiotics, fecal microbiota transplantation, probiotics, etc., so as to achieve the purpose of preventing the occurrence and development of tumors and even carrying out anti-tumor treatment.In addition, gut microbiota serve as research on biomarkers for tumor treatment has also been gradually developed, and has been widely studied in chemotherapy, radiotherapy, targeted therapy and immunotherapy. Gut microbiota are gradually playing an increasingly important role in the diagnosis and treatment chain of tumors. Most of previous studies have also shown that gut microbiota can affect the occurrence and development of esophageal cancer and the efficacy of treatment. However, previous studies were mostly small samples. Cross-sectional studies cannot fully demonstrate the causal relationship between gut microbiota and esophageal cancer. Although there are also randomized controlled trial studies that can help establish causal relationships, due to objective factors such as technical research and methods, there are still limitations in the screening of intestinal microbial groups involved in early diagnosis, treatment, and prognosis [ 50 ] . Therefore, in this study, we analyzed the causal relationship between intestinal microorganisms and esophageal cancer using a two-sample Mendelian randomization study based on the independent data of GWAS. During the analysis, we used the F statistics of IVs. Meeting the threshold of > 10, which indicated that our analysis was unlikely to be affected by weak instrument bias, and finally suggested a causal relationship between gut microbiota and esophageal cancer, and a total of eight bacterial taxa were identified in terms of genus, family and order. However, this article also has limitations in its research. Firstly, we did not use Asian populations as research subjects. Environment and genes will affect disease symptoms and can make differences in gut microbiota. Therefore, the data we selected is not a excellent response to the status of intestinal microorganisms in the Chinese population and the relationship with esophageal cancer.Whether there is the same causal relationship as in the study conclusion is not yet certain. Secondly, stratified analysis of sub-strata was not performed. Different tissue types of esophageal cancer are different in terms of incidence groups, clinical manifestations, prognosis, etc. In this study, due to the limitations of the selected data, it was not possible to classify esophageal cancer patients and conduct further hierarchical analysis to more accurately study. Then in order to obtain enough Ivs, we selected the IVs of intestinal microorganisms with p < 1.0×10 − 5 , which are larger than the traditional genome-wide significance level ( p < 5 ×10 − 8 ), which may lead to some results were missed. Also, although the majority of patients in the GWAS summary data used in our study were European, there were a small number of gut microbiota data from other ethnic groups, which may bias the estimates and affect generalizability. Beyond this, the effects of the bacterial traits we reported were relatively weak, and there are no other independent GWAS of esophageal cancer patients with sufficient sample sizes to validate our conclusions. Conclusion Finally, in our two-sample MR study, a total of 8 gut microbiota were identified, including genus. DefluviitaleaceaeUCG011 , genus.LachnospiraceaeUCG008 , genus.Peptococcus , genus.Ruminiclostridium5 , genus.RuminococcaceaeUCG009 , genus.Ruminococcus1 , family.Pasteurellaceae , order.Pasteurellales. ,which have causalities with the risk of esophageal cancer. Declarations Interest declaration Conflict of Interest: Authors declare no conflicts of interest for this article. Acknowledgments We acknowledge GWAS database for providing their platforms and contributors for uploading their meaningful datasets. Funding Declaration This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Disclosure GWAS belongs to public databases. The patients involved in the database have obtained ethical approval. Users can download relevant data for free for research and publish relevant articles. Our study is based on open source data, so there are no ethical issues and other conflicts of interest. Ethical Statement The data are publicly available, so that ethical approval was not required for this study. References Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A.. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians . 2018, 68 (6), 394–424. Abnet, C. 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Association of gut microbiome and primary liver cancer: A two-sample Mendelian randomization and case-control study. Liver international : official journal of the International Association for the Study of the Liver. 2023, 43(1), 221–233. Wang, Y., Zhang, Y., Qian, Y., Xie, Y. H., Jiang, S. S., Kang, Z. R., Chen, Y. X., Chen, Z. F., & Fang, J. Y. Alterations in the oral and gut microbiome of colorectal cancer patients and association with host clinical factors. International journal of cancer. 2021, Orlova, E., Smirnova, L., Nesvizhsky, Y., Kosenkov, D., & Zykova, E. Acute urticaria in children: course of the disease, features of skin microbiome. Postepy dermatologii i alergologii. 2022, 39(1), 164–170. Zhang, Y., Lu, S., Yang, Y., Wang, Z., Wang, B., Zhang, B., Yu, J., Lu, W., Pan, M., Zhao, J., Guo, S., Cheng, J., Chen, X., Hong, K., Li, G., & Yu, Z. The diversity of gut microbiota in type 2 diabetes with or without cognitive impairment. Aging clinical and experimental research. 2021, 33(3), 589–601. Li, R., Guo, Q., Zhao, J., Kang, W., Lu, R., Long, Z., Huang, L., Chen, Y., Zhao, A., Wu, J., Yin, Y., & Li, S.. Assessing causal relationships between gut microbiota and asthma: evidence from two sample Mendelian randomization analysis. Frontiers in immunology. 2023, 14, 1148684. Yu, X. H., Yang, Y. Q., Cao, R. R., Bo, L., & Lei, S. F.The causal role of gut microbiota in development of osteoarthritis. Osteoarthritis and cartilage. 2021, 29(12), 1741–1750. Maltecca, C., Dunn, R., He, Y., McNulty, N. P., Schillebeeckx, C., Schwab, C., Shull, C., Fix, J., & Tiezzi, F. Microbial composition differs between production systems and is associated with growth performance and carcass quality in pigs. Animal microbiome. 2021, 3(1), 57. Upadhyay, K. G., Desai, D. C., Ashavaid, T. F., & Dherai, A. J. Microbiome and metabolome in inflammatory bowel disease. Journal of gastroenterology and hepatology. 2023, 38(1), 34–43. Long, Y., Tang, L., Zhou, Y., Zhao, S., & Zhu, H. Causal relationship between gut microbiota and cancers: a two-sample Mendelian randomisation study. BMC medicine. 2023, 21(1), 66. Supplementary Table 1 and Supplementary Figure 1 Supplementary Table 1 and Supplementary Figure 1 are not available with this version Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4169602","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288772704,"identity":"78055e39-3934-48b1-a48f-dabaf8b11893","order_by":0,"name":"Bingxiao Lu","email":"","orcid":"","institution":"the Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bingxiao","middleName":"","lastName":"Lu","suffix":""},{"id":288772705,"identity":"a0924487-f2b5-4890-9c3f-b70659ac4ad2","order_by":1,"name":"Xiangzeng 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11:54:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4169602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4169602/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54597065,"identity":"57f1dd5d-d5d7-4836-be03-142fdde7ae05","added_by":"auto","created_at":"2024-04-12 19:12:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":695293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign diagram of MR analysis method\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4169602/v1/3083c60be7b0e00700ed1178.png"},{"id":54597509,"identity":"60ff8713-240d-4df9-a6fc-cd0cf354bd70","added_by":"auto","created_at":"2024-04-12 19:20:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1392313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow chart of MR analysis\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4169602/v1/77d4b83b7cd4dd605bac963d.png"},{"id":54596382,"identity":"24fc2eb1-e177-4386-b45c-f014352d86fe","added_by":"auto","created_at":"2024-04-12 19:04:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":811743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMR results of the relationship between gut microbiota and esophageal cancer\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4169602/v1/597f025ce35ae3dd5f161c7f.png"},{"id":54596385,"identity":"3bc21e18-6b87-4872-995a-42e08e00404e","added_by":"auto","created_at":"2024-04-12 19:04:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1434008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScatter plot of the causal relationship between the gut microbiota and esophageal cancer\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4169602/v1/4cf30099136456aa73f2493c.png"},{"id":54596386,"identity":"f1ace770-44e7-4724-b102-05cd9d3f8f4d","added_by":"auto","created_at":"2024-04-12 19:04:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1053558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLeave-one-out diagram of the causal relationship between the gut microbiota and esophageal cancer\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4169602/v1/2611bfdcd5a38148e8d0d755.png"},{"id":64948738,"identity":"6b62f145-d48f-46bf-8c34-ae3aa20f817d","added_by":"auto","created_at":"2024-09-20 18:03:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6320061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4169602/v1/6737cc6c-ff1d-4633-8ce4-b56dcb984600.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the causality between gut microbiota and esophageal carcinoma: a two-sample Mendelian randomization analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer (EC) is a common upper gastrointestinal tumor with the 7th highest incidence among all tumors and is a serious threat to human health \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The occurrence and development of EC is a complex process. Despite differences in morbidity and mortality among countries, China contributes nearly half of the global EC cases due to its large population\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The most common histological subtypes of esophageal cancer are squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), of which esophageal squamous cell carcinoma accounts for more than 90% of esophageal cancer cases\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003ewith. Even though,the prognosis of EC has significantly improved with the continuous improvement of screening methods and the updating of treatment methods, most patients are still in the intermediate and advanced stages when diagnosed, which greatly reduces the survival rate of EC. According to relevant literature records that the 5-year survival rate of EC patients is less than 20%\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe human symbiotic microbial community consists of \u0026gt;\u0026thinsp;100 trillion microorganisms, mainly living on the surface of human epithelial cells, including the skin, digestive tract and respiratory tract\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Among them, the billions of microorganisms attached to the human intestine are collectively refered gut microbiota\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. With the development of microbial analysis methods (e.g. 16SrRNA sequencing), high-throughput sequencing technologies (e.g. second-generation sequencing), and genomics methods\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, the roles and mechanisms of gut microbiota in the host ecosystem have been more comprehensively characterized.The gut microbiota, known as the \"forgotten organ\", has gradually become a research hotspot in recent years. There is increasing studies have found that gut microbiota not only play an important role in maintaining physiological functions such as homeostasis, material metabolism, immune homeostasis, growth and development, and cognition and other physiological functions\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, but also through the induction of DNA damage and the release of inflammatory mediators, interference with the immune system, regulating carcinogenic host pathway proteins, and generating carcinogenic metabolites mechanism to promote the occurrence and development of tumors\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs a common digestive tract tumor, the association and mechanism of intestinal microbiota and its occurrence and development have been gradually researched. For example, Zaidi et al\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e found in rat models and human clinical samples that \u003cem\u003eEscherichia coli\u003c/em\u003e was significantly enriched in esophageal adenocarcinoma patients or rats compared with the healthy ones. Li et al\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e used a 16S rRNA method to analyze the differences in fecal flora of patients with esophageal squamous cell carcinoma, and found that \u003cem\u003ePrevotella spp.\u003c/em\u003esignificantly decreased in patients with squamous cell carcinoma, compared with healthy people in the control group. In addition, there is emerging evidence \u003csup\u003e[\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e from varying degrees which have shown that gut microbiota are gradually playing a central role in the development of esophageal cancer. While the growing evidence of an association between gut microbiota is closely related to the risk of esophageal cancer, but whether there is a causal effect remains unknown.\u003c/p\u003e \u003cp\u003eMendelian Randomization (MR) is a data analysis method using genetic variants strongly correlated with exposure factors as instrumental variables to evaluate the causal relationship between exposure factors and outcomes\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.Most notably, two-sample MR analysis can utilize Single Nucleotide Polymorphism (SNP) exposure and outcome associations from independent GWAS and combine them into a single causal estimate to analyze the casual relationship between exposure and outcome\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Compared with conventional regression methods, this approach is less susceptible to measurement error, confounding and reverse causality.\u003c/p\u003e \u003cp\u003eUsing two-sample Mendelian randomization analysis to explore the causal relationship between gut microbiota and tumors has been increasingly studied, but more of them focus on colorectal cancer, breast cancer, gynecological cancer and other cancer. But whether there is a causal effect between gut microbiota and esophageal cancer remains unknown.Therefore, in our study, in order to explore the potential causal relationship between gut microbiota and esophageal cancer and to identify specific pathogenic bacterial taxa, we conducted a two-sample MR study based on genome-wide association study (GWAS) pooled data.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Research design\u003c/h2\u003e \u003cp\u003eIn this study, we use public GWAS summary statistics, at the same time the gut microbiota are used as the exposure, and esophageal cancer as the outcome to conduct MR analysis for evaluating the causal relationship between 199 gut microbiota and esophageal cancer. Finally, heterogeneity testing and gene-level multi-analysis are conducted. Validity testing and other quality controls verify the reliability and robustness of the causal relationship results. The data are publicly available, so that ethical approval was not required for this study.\u003c/p\u003e \u003cp\u003eMR analysis was explored based on three hypotheses: (1) IVs are strongly related to exposure; (2) IVS are not related to relevant confounding factors; (3) IVs affect outcomes through exposure only. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Data source\u003c/h2\u003e \u003cp\u003eThe summary statistics for human gut microbiota we used in this study were obtained from the most recent GWAS meta-analysis, a large and multi-ethnic study, which included 18,340 participants from 24 cohorts. Of the 24 cohorts, 22 were composed of adults or adolescents (N\u0026thinsp;=\u0026thinsp;16,632), and two cohorts were composed of children (N\u0026thinsp;=\u0026thinsp;1,708). This study combined data from the United States, Canada, Germany, 16S rRNA gene sequencing profiles and genotyping data from Denmark, the Netherlands, Belgium, Sweden, Finland, the United Kingdom and other countries, and related analyzes were conducted on age, gender, technical covariates and genetic principal components\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe GWAS data for esophageal cancer arise out of a large pan-cancer study that combined UK Biobank (408,786 individuals of European ancestry, age 40\u0026ndash;69 years, mean age 56.5 years; 48,961 cancer cases) and Kaiser Permanente Genetic Epidemiological study of Adult Health and Aging (66,526 individuals of European ancestry, 18 to 100\u0026thinsp;+\u0026thinsp;years, mean age 63 years; 16,001 cancer cases) two large, independent, prospective cohorts, including 1091 patients with esophageal cancer and 410350 healthy controls\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In order to reduce the population stratification differences, most of the outcome and exposure data we selected came from European populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Screening of instrumental variables\u003c/h2\u003e \u003cp\u003eFor the purpose of ensuring the inclusion of appropriate IVs, this study conducted IVs screening based on the core assumptions of MR. The specific steps for selecting IVs are as follows: (1) Among the exposure data already obtained, this study selected a relatively reliable threshold based on previous literature. P\u0026lt;1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e is used as the standard to select SNPs that are strongly related to the abundance of gut microbiota; (2) To ensure that the selected IVs are independent of each other, the parameter r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was set, and the distance between SNPs which is within 10000Kb to exclude the SNP in linkage disequilibrium ensuring that SNPs are independent of each other; (3) Remove weak instrumental variables: Use the F statistic to evaluate the statistical strength of the correlation between SNPs and exposure. The formula of the F statistic is (F=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{R}^{2}}{1-{R}^{2}}\\bullet \\frac{N-K-1}{K}\\)\u003c/span\u003e\u003c/span\u003e), where N is the sample size of the intestinal flora database, K is the number of SNPs, R2 represents the proportion of variation in the intestinal flora explained by SNPs, the calculation formula is R2=2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\bullet\\)\u003c/span\u003e\u003c/span\u003e(1-EAF)\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\bullet\\)\u003c/span\u003e\u003c/span\u003eEAF\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\bullet\\)\u003c/span\u003e\u003c/span\u003eβ2. Where EAF is the effect allele frequency of each SNP, and β is the effect value for the allele\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The F test value \u0026gt;\u0026thinsp;10 was used as the screening criterion to find SNPs without strong correlation but which is related to exposure factors; (4) Integration of the two sets of data: the SNPs related to the relative abundance of gut microbiota were project to the corresponding statistical parameters of esophageal cancer, and use the \"harmonise_data\" function to unify the data based on the gut microbiota abundance and the statistical parameters of the same sites in the GWAS results of esophageal cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Data analysis methods\u003c/h2\u003e \u003cp\u003eAfter the final included IVs were determined according to the above IVs screening process, MR analysis was started. This study used 5 methods to estimate the causal effect: inverse variance weighted method, MR-Egger regression, weighted median method, simple mode method and weighted mode method. Previous studies have shown that the IVW method has higher testing efficiency than the other four methods, so the IVW method is used as the preferred causal effect estimation method in this study.\u003c/p\u003e \u003cp\u003eSo as to test the stability and reliability of the results additionally, this study conducted quality control on the MR results that remained statistically significant after FDR correction. The Cochran Q test was used to calculate heterogeneity, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated the presence of heterogeneity, MR-PRESSO analysis was used to remove outliers, MR-Egger regression intercept analysis was used to detect horizontal pleiotropy, and leave-one-out analysis reflected the robustness of MR results. And whether the causal effect was dominated by a certain SNP. All analyzes were performed in R software (version 4.313) using the Two-Sample MR package and the MRPRESSO package. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Instrument variable selection\u003c/h2\u003e \u003cp\u003eAccording to the screening criteria of instrumental variables for this study, 89 SNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were finally included. The F statistics ranged from 18.49 to 33.84, all above 10, indicating that they were not affected by weak instrumental variables.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e1.2 The two-sample MR results\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThis study estimated the causal effect between the abundance of 199 gut microbiota and esophageal cancer, using the IVW method to find that 8 types of intestinal microorganisms are associated with esophageal cancer(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The MR results of the correlation between all 199 bacterial characteristics and esophageal cancer risk are shown in Appendix 1(\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe results of our study showed that, \u003cem\u003egenus. DefluviitaleaceaeUCG011\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;3.124, 95%CI 1.388\u0026ndash;7.031, P\u0026thinsp;=\u0026thinsp;0.006) is positively associated with the risk of developing esophageal cancer. No abnormal values were found in the MR-PRESSO test. The intercept of MR-Egger regression also did not show potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.863), no heterogeneity was detected in Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); \u003cem\u003egenus.LachnospiraceaeUCG008\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;3.964, 95% CI 1.463\u0026thinsp;\u0026minus;\u0026thinsp;10.740, P\u0026thinsp;=\u0026thinsp;0.007) was positively correlated with the risk of esophageal cancer. The MR-PRESSO test found no outliers, and the intercept of the MR-Egger regression did not show the potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.563) as well, no heterogeneity was detected in Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); \u003cem\u003efamily.Pasteurellaceae.id.3689\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;2.022, 95%CI 1.046\u0026thinsp;\u0026minus;\u0026thinsp;3.909, P\u0026thinsp;=\u0026thinsp;0.036) was positively correlated with the risk of esophageal cancer, and no outliers were found in the MR-PRESSO test. The intercept of the MR-Egger regression also did not show the potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.328). No outliers were found in the Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05);\u003cem\u003eorder.Pasteurellales.id.3688\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;2.022, 95%CI1.046\u0026thinsp;\u0026minus;\u0026thinsp;3.909, P\u0026thinsp;=\u0026thinsp;0.036) was positively correlated with the risk of esophageal cancer, no outliers were found by MR-PRESSO test, the intercept of the MR-Egger regression also did not show the potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.328), and no heterogeneity was detected in the Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eOn the contrary, \u003cem\u003egenus.Peptococcus\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;0.525, 95%CI0.286-0.961, P\u0026thinsp;=\u0026thinsp;0.037) is negatively correlated with the risk of developing esophageal cancer. No outliers were found in the MR-PRESSO test, and the potential horizontal pleiotropy was not shown by the intercept of the MR-Egger regression either. (intercept p value\u0026thinsp;=\u0026thinsp;0.542), no heterogeneity was detected in Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); \u003cem\u003egenus.Ruminiclostridi-um5\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;0.117, 95%CI 0.031\u0026thinsp;\u0026minus;\u0026thinsp;0.442, P\u0026thinsp;=\u0026thinsp;0.002) was positively correlated with the risk of esophageal cancer. The MR-PRESSO test found no outliers, and the intercept of the MR-Egger regression did not show the potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.960 ), no heterogeneity was detected in Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); \u003cem\u003egenus.RuminococaceaeUCG009\u003c/em\u003e(OR\u0026thinsp;=\u0026thinsp;0.350, 95%CI 0.157\u0026thinsp;\u0026minus;\u0026thinsp;0.777, P\u0026thinsp;=\u0026thinsp;0.01) was positively correlated with the risk of esophageal cancer, no outliers were found in the MR-PRESSO test, the intercept of the MR-Egger regression did not show the potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.206), and no heterogeneity was detected in the Cochran's Q heterogeneity test sex (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) either; \u003cem\u003egenus.Ruminococcus1\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;0.341, 95%CI 0.118\u0026ndash;0.985, P\u0026thinsp;=\u0026thinsp;0.047) was negatively associated with the risk of esophageal cancer. No outliers were found in the MR-PRESSO test. No potential horizontal pleiotropy (intercept p value\u0026thinsp;=\u0026thinsp;0.385) was shown by the intercept of the MR-Egger regression as well, and no heterogeneity was detected in the Cochran's Q heterogeneity test (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Quality control\u003c/h2\u003e \u003cp\u003eIn the Cochran\u0026rsquo;s Q heterogeneity test, no heterogeneity was observed in the SNPs of the 8 bacterial traits (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). MR-Egger analysis did not detect potential horizontal pleiotropic effects, indicating that the instrumental variables did not significantly increase the impact of pathways other than exposure on outcomes. The MR-PRESSO global test found no outliers, and the leave-one-out test analysis showed that no individual SNP had an impact on the overall causal relationship(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough the two-sample Mendelian randomization analysis is increasingly implemented to study the causality between gut microbiota and cancer, but the researches on esophageal cancer are relatively scarce. Therefore, in this study, we used two-sample MR research, eight bacterial taxa were identified as having a causality with the risk of esophageal cancer, at the level of order, family and genus. It brings potential targets for early diagnosis, treatment, monitoring and prevention of esophageal cancer, and has certain implications for public health intervention aimed at reducing the risk of esophageal cancer.\u003c/p\u003e \u003cp\u003eThere are quantity of emerging studies have shown that the gut microbiota is bound up to the occurrence and development of esophageal cancer. There are differences between patients with esophageal cancer and healthy people in the gut microbiota with the level at order, family and genus. The results of this study show that the \u003cem\u003egenus LachnospiraceaeUCG008\u003c/em\u003e is an important risk factor in esophageal cancer, which belongs to the Clostridium group of Firmicutes and is a type of obligate anaerobic bacteria. It is generally recognized that Barrett's esophagus is a precancerous lesion of esophageal adenocarcinoma. Accordingly, Liu et al.\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003efound that the Firmicutes accounted for 55% of Barrett's esophageal microbiota at the phylum level, and the relative abundance of Fusobacteria in the microbiota was higher than that of normal peopel. Mirco et al\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e found that the gut microbiota of patients with non-alcoholic fatty liver disease (NAFLD) is rich in \u003cem\u003eLachnospiraceae\u003c/em\u003e. Some studies have confirmed that metabolic syndrome is closely related to the occurrence of Barrett\u0026rsquo;s esophagus. NAFLD is a hepatic manifestation of metabolic syndrome, suggesting that NAFLD may be related to esophageal ulcer, Barrett's esophagus, and esophageal adenocarcinoma. South Korea reported a large cohort study based on the national population, which included 8120674 subjects, including 936159 NAFLD patients. During an average follow-up period of 7.2 years, a total of 3,792 subjects developed esophageal cancer. Among them, the risk of esophageal cancer in NAFLD patients is approximately twice than that of non-NAFLD patients (HR 2.10, 95%CI 1.88\u0026ndash;2.35). Moreover, NAFLD significantly increases the risk of death from esophageal cancer (HR 1.46, 95%CI 1.28\u0026ndash;1.67) \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. By evaluating the relationship between gastrointestinal microbiota and body mass index (BMI), Ley et al.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e found that Firmicutes abundance increased (p value\u0026thinsp;=\u0026thinsp;0.002) and Bacteroidetes decreased accordingly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which was associated with high BMI. Furthermore,Patients with a BMI of 30 or higher had about twice the risk of developing EC as those with a BMI of \u0026lt;\u0026thinsp;25, and there was a dose-response relationship between increased waist circumference and the risk of developing EC\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e,which supports our findings.\u003c/p\u003e \u003cp\u003eThe results of this study show that there is a positive causal relationship between gut microbiota of the \u003cem\u003egenus DefluviitaleaceaeUCG011\u003c/em\u003e and the incidence of esophageal cancer. Oral bacteria can migrate to the esophagus and stomach through swallowing, and the ecology of the esophageal flora will also be different in people with different oral health conditions \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.The \u003cem\u003egenus Defluviitaleaceae\u003c/em\u003e is more commonly found in the human oral cavity, and there is relevant evidence that it plays a key role in the occurrence and development of periodontitis. For example, in a comparative study by Chen Bin et al. \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e on the bacterial flora of people with aggressive periodontitis, chronic periodontitis and healthy periodontitis, it was found that compared with those with chronic periodontitis and healthy periodontitis, \u003cem\u003eDefluviitaleaceaeUCG011\u003c/em\u003e was significantly increased in patients with aggressive periodontitis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, in another study on aggressive periodontitis and chronic periodontitis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, it was also found that the abundance of \u003cem\u003eDefluviitaleaceaeUCG011\u003c/em\u003e was significantly different between the two types of study populations, and was found to be related to periodontal pockets. There is a negative correlation with detection depth. In addition to being associated with the risk of periodontitis, more and more studies have found that \u003cem\u003eDefluviitaleaceaeUCG011\u003c/em\u003e is also widely present in the intestine and is associated with cardiac anti-fibrotic protection\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e and rheumatoid arthritis\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, lipid metabolism\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, social frustration disorder, childhood urticaria\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e and other diseases are related. It is worth noting that in oral microorganisms, The increased abundance of \u003cem\u003eDefluviitaleaceaeUCG011\u003c/em\u003e is a risk factor for periodontal disease, but in gut microbiota, the decreased abundance of \u003cem\u003eDefluviitaleaceaeUCG011\u003c/em\u003e is the causative factor for the development of the above diseases. Some studies have used kelp and Gastrodia elata extracts to increase this the abundance of bacterial taxa, thereby achieving the purpose of treating diseases, also further proves the pathogenic role of this bacterial taxa in related diseases.\u003c/p\u003e \u003cp\u003eThe results of this study show that there is a negative causal relationship between gut microbiota of the \u003cem\u003egenus Ruminococcus1\u003c/em\u003e and \u003cem\u003efamily RuminococcaceaeUCG009\u003c/em\u003e and the incidence of esophageal cancer. Research shows that \u003cem\u003eRuminococcus\u003c/em\u003e can produce short-chain fatty acids, which provide an energy source for intestinal cells, improve intestinal barrier function, and also have anti-inflammatory effects. Short-chain fatty acids are carboxylic acids with 1 to 6 carbon bond atoms. It is formed by the metabolism of complex carbohydrates into oligosaccharides by the gut microbiota and fermentation. It can be directly absorbed by the intestinal mucosa, and there are many types, mainly butyrate, propionate, and acetate. Smith et al. \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e have shown that the high abundance of \u003cem\u003eRuminococcus\u003c/em\u003e produces a large amount of short-chain fatty acids that can regulate the homeostasis of colonic Treg cells, improve the re-conversion of immune tolerance, and inhibit the inflammatory response in colitis mice, while butyric acid and The specific anti-inflammatory mechanism of acetic acid is to inhibit G protein-coupled receptor 43 and then inhibit histone deacetylase in Treg cells to play an anti-inflammatory effect \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Some studies have found that periodic intake of yogurt can effectively increase the probiotics in the intestines, significantly increase the levels of short-chain fatty acids, and thereby accelerate the apoptosis of colorectal cancer cells \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Mueller et al. \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e studied that after metformin treatment, the relative abundance of Ruminococcus torque group increased and improved overweight/obese adults, which means that \u003cem\u003eRuminococcus torques\u003c/em\u003e can alleviate obesity. Feng et al. \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e also proved that \u003cem\u003eRuminococcaceae\u003c/em\u003e can alleviate obesity. Studies have shown that \u003cem\u003eRuminococcaceae_UCG-013\u003c/em\u003e is positively correlated with serum HDL-C levels and negatively correlated with serum TC, TG and LDL-C levels, and can be identified as the most important biomarker for reducing obesity. Ma et al \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e used MR analysis to show that \u003cem\u003eRuminococcaceae\u003c/em\u003e has a negative causal relationship with primary liver cancer, which has potential significance for the prevention and control of liver cancer. \u003cem\u003eRuminococcaceae_UCG-013\u003c/em\u003e was shown to be more abundant in the gut microbiota of mice with a lean phenotype than in mice with an obese phenotype. The above studies reveal their beneficial effects in human diseases and support our findings.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003egenus Rumiclostridium\u003c/em\u003e is one of the first gastric bacteria discovered and plays a vital role in metabolism. Relevant studies have shown that increases and decreases in the abundance of Ruminiclostridium may lead to the occurrence and development of some diseases. The occurrence and development of inflammatory bowel diseases such as Crohn's disease, irritable bowel, and ulcerative colitis are closely related to this bacterial group.Wang et al.'s study\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e found that compared with healthy people, the abundance of \u003cem\u003eRuminiclostridium\u003c/em\u003e increased in patients with irritable bowel, and after multi-factor regression analysis, it was found that this bacterial group is a key indicator for the pathogenesis of irritable bowel. Similarly, a series of studies have also found that Rumiclostridium plays a role in the occurrence and development of children's eczema\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, diabetes\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, asthma\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e, spondyloarthritis\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, and so forth. Among them, it is worth emphasizing that Yu et al. conducted a two-sample MR study on Rumiclostridium and spondyloarthritis, and the results showed a causal relationship between this bacterial group and spondyloarthritis, which provides new insights into the Potential treatments of spondyloarthritis.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003egenus Peptococcus\u003c/em\u003e is a normal flora found in the mouth, intestines, female reproductive tract, skin, etc. The role of \u003cem\u003ePeptococcus\u003c/em\u003e in the occurrence and development of diseases is more concentrated in livestock production, and the use of antibiotics, fecal transplants and other means can promote the development of livestock production. For example, the study by Maltecca et al. \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003eshowed that the abundance of Peptococcus was related to the increase in growth rate and obesity of pigs, describing for the first time the value of microbial communities in pork production systems. There are also a few studies showing that Peptococcus also plays an important role in acute urticaria in children, cognitive impairment in diabetic patients, and colorectal cancer, but there are still few studies at present.\u003c/p\u003e \u003cp\u003eIn addition to the potential causal relationship between the above bacterial taxa and the occurrence of esophageal cancer at the genus level, the \u003cem\u003efamily Pasteurellaceae\u003c/em\u003e and the \u003cem\u003eorder Pasteurellales\u003c/em\u003e also have similar causal relationships. The association between \u003cem\u003ePasteurellaceae\u003c/em\u003e bacterial groups and diseases is mostly concentrated in the epidemic of poultry cholera, sepsis, inflammatory bowel disease, etc. In the article published by Upadhyay et al\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e, the results showed that the abundance of Pasteurellaceae is related to the pre-treatment A positive association with disease status in children with newly diagnosed Crohn's disease provides further support for this microbiota as a driver of inflammatory bowel disease progression.\u003c/p\u003e \u003cp\u003eOverall, in our study, we found a potential causal relationship between these eight specific bacterial taxa and the occurrence of esophageal cancer. For a long time, gut microbiota have continued to appear as an important component of body health, disease status, and treatment response, bringing potential targets for the diagnosis, treatment, detection, and prevention of many diseases, especially tumors. Analysis and targeting of the microbiota in the gut and other niches will also become part of comprehensive cancer care as well as the management of other diseases, ultimately integrating into precision individual health management. Through regular detection of relevant carcinogenic gut microbiota, it can be changed early through antibiotics, prebiotics, fecal microbiota transplantation, probiotics, etc., so as to achieve the purpose of preventing the occurrence and development of tumors and even carrying out anti-tumor treatment.In addition, gut microbiota serve as research on biomarkers for tumor treatment has also been gradually developed, and has been widely studied in chemotherapy, radiotherapy, targeted therapy and immunotherapy. Gut microbiota are gradually playing an increasingly important role in the diagnosis and treatment chain of tumors.\u003c/p\u003e \u003cp\u003eMost of previous studies have also shown that gut microbiota can affect the occurrence and development of esophageal cancer and the efficacy of treatment. However, previous studies were mostly small samples. Cross-sectional studies cannot fully demonstrate the causal relationship between gut microbiota and esophageal cancer. Although there are also randomized controlled trial studies that can help establish causal relationships, due to objective factors such as technical research and methods, there are still limitations in the screening of intestinal microbial groups involved in early diagnosis, treatment, and prognosis\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, in this study, we analyzed the causal relationship between intestinal microorganisms and esophageal cancer using a two-sample Mendelian randomization study based on the independent data of GWAS. During the analysis, we used the F statistics of IVs. Meeting the threshold of \u0026gt;\u0026thinsp;10, which indicated that our analysis was unlikely to be affected by weak instrument bias, and finally suggested a causal relationship between gut microbiota and esophageal cancer, and a total of eight bacterial taxa were identified in terms of genus, family and order.\u003c/p\u003e \u003cp\u003eHowever, this article also has limitations in its research. Firstly, we did not use Asian populations as research subjects. Environment and genes will affect disease symptoms and can make differences in gut microbiota. Therefore, the data we selected is not a excellent response to the status of intestinal microorganisms in the Chinese population and the relationship with esophageal cancer.Whether there is the same causal relationship as in the study conclusion is not yet certain. Secondly, stratified analysis of sub-strata was not performed. Different tissue types of esophageal cancer are different in terms of incidence groups, clinical manifestations, prognosis, etc. In this study, due to the limitations of the selected data, it was not possible to classify esophageal cancer patients and conduct further hierarchical analysis to more accurately study. Then in order to obtain enough Ivs, we selected the IVs of intestinal microorganisms with p\u0026thinsp;\u0026lt;\u0026thinsp;1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, which are larger than the traditional genome-wide significance level ( p\u0026thinsp;\u0026lt;\u0026thinsp;5 \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), which may lead to some results were missed. Also, although the majority of patients in the GWAS summary data used in our study were European, there were a small number of gut microbiota data from other ethnic groups, which may bias the estimates and affect generalizability. Beyond this, the effects of the bacterial traits we reported were relatively weak, and there are no other independent GWAS of esophageal cancer patients with sufficient sample sizes to validate our conclusions.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eFinally, in our two-sample MR study, a total of 8 gut microbiota were identified, including \u003cem\u003egenus. DefluviitaleaceaeUCG011\u003c/em\u003e, \u003cem\u003egenus.LachnospiraceaeUCG008\u003c/em\u003e, \u003cem\u003egenus.Peptococcus\u003c/em\u003e, \u003cem\u003egenus.Ruminiclostridium5\u003c/em\u003e, \u003cem\u003egenus.RuminococcaceaeUCG009\u003c/em\u003e, \u003cem\u003egenus.Ruminococcus1\u003c/em\u003e, \u003cem\u003efamily.Pasteurellaceae\u003c/em\u003e, \u003cem\u003eorder.Pasteurellales.\u003c/em\u003e,which have causalities with the risk of esophageal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eInterest declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of Interest: Authors declare no conflicts of interest for this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge GWAS database for providing their platforms and contributors for uploading their meaningful datasets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGWAS belongs to public databases. The patients involved in the database have obtained ethical approval. Users can download relevant data for free for research and publish relevant articles. Our study is based on open source data, so there are no ethical issues and other conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are publicly available, so that ethical approval was not required for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., \u0026amp; Jemal, A.. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA: a cancer journal for clinicians\u003c/em\u003e. 2018, \u003cem\u003e68\u003c/em\u003e(6), 394\u0026ndash;424. \u003c/li\u003e\n\u003cli\u003eAbnet, C. C., Arnold, M., \u0026amp; Wei, W. Q. 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BMC medicine. 2023, 21(1), 66.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Table 1 and Supplementary Figure 1","content":"\u003cp\u003eSupplementary Table 1 and Supplementary Figure 1 are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"gut microbiota, esophageal cancer, Mendelian randomization, single nucleotide polymorphisms","lastPublishedDoi":"10.21203/rs.3.rs-4169602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4169602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEsophageal cancer is a common tumor of upper gastrointestinal tract. However, the increasing evidence indicates that gut microbiota are closely related to the occurrence and development of esophageal cancer, the causal association between gut microbiota and esophageal cancer remains to be determined. As a consequence, in this paper, a two-sample Mendelian randomization analysis was used to evaluate and explore the causal relationship between gut microbiota and the risk of esophageal cancer, and identify specific pathogenic bacterial taxa.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eGenetic instrumental variables for gut microbiota were identified from a genome-wide association study (GWAS) of 18,340 participants, esophageal cancer as the outcome variable was identified from a GWAS including 1091 cases and 410,350 controls. Using the inverse variance weighted (IVW) method as the primary analysis, and further (using) the weighted median method, MR-Egger regression, MR multi-directional residuals, and outlier tests were further performed to improve the robustness of the results.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe IVW results showed that \u003cem\u003egenus.DefluviitaleaceaeUCG011\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;3.124, 95%CI 1.388\u0026ndash;7.031, P\u0026thinsp;=\u0026thinsp;0.006), \u003cem\u003egenus.LachnospiraceaeUCG008\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;3.964,95%CI 1.463\u0026ndash;10.740, P\u0026thinsp;=\u0026thinsp;0.007), \u003cem\u003efamily.Pasteurellaceae.id.3689\u003c/em\u003e(OR\u0026thinsp;=\u0026thinsp;2.022, 95%CI 1.046\u0026thinsp;\u0026minus;\u0026thinsp;3.909, P\u0026thinsp;=\u0026thinsp;0.036)and order.Pasteurellales.id.3688༈OR\u0026thinsp;=\u0026thinsp;2.022, 95%CI1.046\u0026thinsp;\u0026minus;\u0026thinsp;3.909, P\u0026thinsp;=\u0026thinsp;0.036༉showing a positive causal relationship between gut microbiota and esophageal cancer.Conversely,\u003cem\u003egenus.Peptococcus\u003c/em\u003e(OR\u0026thinsp;=\u0026thinsp;0.525, 95% CI 0.286\u0026ndash;0.961,P\u0026thinsp;=\u0026thinsp;0.037) ,\u003cem\u003egenus.Ruminiclostridium5\u003c/em\u003e(OR\u0026thinsp;=\u0026thinsp;0.117,95%CI0.0310.442,P\u0026thinsp;=\u0026thinsp;0.002),\u003cem\u003egenus.RuminococcaceaeUCG009\u003c/em\u003e ༈OR\u0026thinsp;=\u0026thinsp;0.350, 95%CI 0.157\u0026thinsp;\u0026minus;\u0026thinsp;0.777, P\u0026thinsp;=\u0026thinsp;0.01༉and \u003cem\u003egenus.Ruminococcus1\u003c/em\u003e༈OR\u0026thinsp;=\u0026thinsp;0.341, 95%CI 0.118\u0026thinsp;\u0026minus;\u0026thinsp;0.985, P\u0026thinsp;=\u0026thinsp;0.047༉showed a negative causal relationship with esophageal cancer. No significant heterogeneity was found in the instrumental variable or horizontal pleiotropy.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur MR analysis study revealed that the gut microbiota was causally associated with esophageal cancer and the risk of esophageal cancer, and also identified eight bacterial taxa with a causal relationship with the development and progression of esophageal cancer.That may be useful in providing new insights for further mechanistic and clinical studies of microbiota-mediated cancer.\u003c/p\u003e","manuscriptTitle":"Exploring the causality between gut microbiota and esophageal carcinoma: a two-sample Mendelian randomization analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 19:04:03","doi":"10.21203/rs.3.rs-4169602/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fae20a5-c730-4c3b-88cf-29a50a34e2a7","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-20T17:55:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 19:04:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4169602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4169602","identity":"rs-4169602","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}