The Causal Role of Esophageal Cancer and Gut Microbiota: A Bidirectional Mendelian Randomization Study

The Causal Role of Esophageal Cancer and Gut Microbiota: A Bidirectional Mendelian Randomization Study | 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 The Causal Role of Esophageal Cancer and Gut Microbiota: A Bidirectional Mendelian Randomization Study Xiaoying Zhou, Wei Su, Han Chen, Bixing Ye, Xinmin Si, Weifeng Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3149703/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 Aims Esophageal cancer (EC) and gut microbiota are reported to be clinically relevant but their genetic association is unclear. We carried out a bidirectional MR analyses to assess the casual relationship between EC and gut microbiota from fecal samples. Methods The microbiome genome-wide association studies (GWAS) data of 18,340 individuals provided by MiBioGen consortium and the EC GWAS data (740 esophageal cancers cases and 372,016 controls) provided by UK Biobank were respectively utilized as exposure and/or outcome data. Reliable single nucleotide polymorphisms (SNPs) were obtained after rigorous screening. A bidirectional Mendelian randomization (MR) analysis was conducted using the inverse-variance weighted (IVW) method. The sensitivity analyses including the MR-Egger method, weighted median, MR-PRESSO and mode methods were performed to examine the stability, heterogeneity and pleiotropy of the results. Results Forward MR analysis revealed genetic liability to gut microbiota was associated with a higher risk of EC ( Coprobacter (OR 1.001, 95%CI 1.000-1.002); Ruminococcus1 (OR 1.001, 95%CI 1.000-1.002); Turicibacter (OR 0.999, 95%CI 0.998-1.000); Senegalimassilia (OR 1.002, 95%CI 1.000-1.003); Veillonella (OR 1.001, 95%CI 1.000-1.002)) or a significantly lower risk of EC ( Eubacterium oxidoreducens (OR 0.999, 95%CI 0.998-1.000); Lachnospira (OR 0.998, 95%CI 0.996-1.000); Romboutsia (OR 0.999, 95%CI 0.998-1.000)). Reverse MR analysis showed that genetic liability to EC was also causally linked to increased susceptibility of gut microbiota dysbiosis (genera Eggerthella (Beta 37.63, 95%CI 4.76–70.50); Coprococcus2 (Beta 23.90, 95%CI 1.65–46.15); ChristensenellaceaeR.7 (Beta 22.75, 95%CI 4.22–41.28); genera Intestinimonas (OR -33.24, 95%CI-54.90–11.58)). Conclusions Our findings supported a bidirectionally causal relationship between gut microbiota and EC, implying the potential of gut microbiota as novel biomarkers and therapeutic drugs for EC. Gut microbiota Esophageal cancer Mendelian Randomization Bidirectional Figures Figure 1 Figure 2 Figure 3 Introduction Esophageal cancer (EC) is one of the most common fatal cancers, which imposing significant worldwide health burdens. The global incidence and mortality of EC ranked tenth and sixth, respectively, in 2020 [ 1 ]. The majority of cases (85% [512,500 cases]) were Esophageal squamous cell carcinoma (ESCC) and Esophageal adenocarcinoma (EAC) accounted for 14% (85,700 cases). The distribution vary from regions and adenocarcinoma is the main type in in European and American countries. Moreover, the incidence of EAC has been rising at an alarming rate in Western world [ 2 ]. It is well recognized that host and environmental risk factors, such as obesity, gastroesophageal reflux diseases and Barrett's esophagus, contribute to EAC [ 3 – 5 ]. The symptoms of early esophageal cancer are often atypical and easy to be ignored, thus primary prevention and early screening play key roles in improving clinical outcome and survival of patients [ 6 ]. Despite researchers have done a lot of exploring, the exact pathogenic mechanism of esophageal cancer remains unknown to date, making it difficult to find potential noninvasive biomarkers and predictors. Gastrointestinal tract (GIT) is colonized by trillions of microorganisms that comprise the gut microbiota. Gut microbiota not only takes part in protecting against pathogens and maintaining gut ecosystem homeostasis but also involves in several cancers [ 7 ]. According to a recent study, about 13% of all cancer cases in 2018 can be attributed to infectious etiology [ 8 ]. During the past few years, much attention has been paid to the association between esophageal cancers and gut microbes. Gut microbiota has a crucial role in the onset, development, and treatment efficacy of gastrointestinal malignancies, which may be caused by inflammation and immune disorders [ 9 ]. Several researches have revealed the different esophageal bacterial strains, from normal esophagus to ESCC and EAC [ 10 – 12 ]. Ajayi et al [ 12 ] showed that normal esophagus is mainly colonized by Streptococcus while esophagitis and BE are associated with gram-negative bacteria. Furthermore, certain G-bacteria, including Escherichia coli and Fusobacterium nucleatum, are linked to EA. Yamamura et al [ 13 ] revealed that Fusobacterium nucleatum aggravate the survival of ESCC patients. The limitation is, most of the sample were obtained from esophagus by invasive examination. It has been proven that intestinal microbiome involves in inflammatory bowel diseases [ 14 ],diabetes [ 15 ], colorectal cancer [ 16 ] and so on. Recently, Deng et al [ 17 ] demonstrated increased richness of gut microbes and significant change of the distribution proportion of some bacteria in EC compared with health individuals through 16S rDNA gene sequencing of fresh stool. While it is still unclear of the causal relationship between gut microbiota and esophageal cancer. Mendelian randomization (MR) is an original approach to exploring the causality between exposure or risk factor and outcome, which has been widely applied to research in various fields. It screens for genetic variants that can represent exposure as an instrument to examine the relationship between instrument and outcome. Compared to traditional observational studies, which are prone to biases such as reverse causality and residual confounding, MR rules out these biases due to the law of independent assortment [ 18 ]. In this text, we utilize the genome-wide association study (GWAS) summary statistics from UK Biobank and MiBioGen consortium. A two-sample MR is carried out to evaluate the causal relationship between gut microbes and esophagus. Methods Figure 1 shows the flowchart of the MR analysis. A bidirectional two-sample MR study was designed to explore the causal effect of gut microbiota and esophageal cancer. Data Sources We obtained the genetic variants datasets from public-available GWAS. The summary statistics of esophageal cancer was provided by UK Biobank. The project is a population-based cohort study involving in 500,000 middle-aged participants. The study consisted of 740 esophageal cancers cases and 372,016 controls, and almost of them are European [ 19 ]. The summary statistics of gut microbes was acquired from the large-scale meta-analysis of 24 cohorts including 18,340 participants conducted by MiBioGen consortium. In this study, fecal microbial composition was analyzed targeting the V4, V3-V4, and V1-V2 variable regions of the 16S rRNA gene and classified by direct taxonomic classification. To identify host genetic variants that was mapped to the genetic loci impacting the relative abundance or presence of microbial taxa, microbiome quantitative trait loci (mbQTL) mapping analysis was carried out. A total of 211 taxa (131 genera, 35 families, 20 orders, 16 classes, and 9 phyla) were involved [ 20 ]. Selection of the instrumental variables We set the threshold of genome-wide significance (p < 5*10 − 6 ) to filter the SNPs strongly associated with the exposure in the MR analysis. Then, on the basis of the European ancestral individuals from the 1000 Genomes Project, we assessed the linkage disequilibrium to ensure the independence ,and appropriate SNPs within 10,000kb clumping window with an r2 < 0.001 were kept [ 18 ]. In addition, Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) Test was implemented to test the presence of horizontal pleiotropy, the p value in each MR analysis should be greater than 0.05 [ 21 ]. Before harmonizing the exposure and outcome datasets, we excluded the palindromic sequence due to the uncertainty of the direction for exposure and outcome. Moreover, F-statistics was calculated to evaluate the strength of IVs by the formula: F = R2(n − 1−k)/(1 − R2) k, (R2 represents the proportion of variation explained by each IV, k represents the number of eligible SNPs, and n represents the sample size) [ 22 ]. When F statistics > 10, the IVs was considered as strongly-correlated instruments. Statistical analysis In this study, we performed the analysis in R 4.1.0 using TwoSampleMR [ 23 ] and MRPRESSO packages. To assess whether there is causal relationship between esophageal cancer and gut microbes, we applied multiple methods: Inverse variance weighted (IVW), MR Egger regression, Weighted median, Maximum likelihood, Weighted mode. The IVW method was predominate and the others was utilized as sensitivity analyses to further confirm the reliability of the IVW method. The IVW method calculated a weighted average of Wald ratio estimates of each SNP. If all of the SNPs are valid instrumental variables, there will be no bias in the results [ 24 ]. Besides, we used the Cochran Q test to quantify the heterogeneity of IVs. As the p value ≤ 0.05, which standing for the existence of heterogeneity, we carried out a random effects IVW MR analyses. Moreover, the Leave-one-out analysis demonstrated the meta effects of the remaining SNPs when a certain SNP was removed in turn. If the results changed greatly after the elimination, it indicated the heterogeneity of the removed SNP. We also performed the reverse MR analyses in the same methods of the forward MR analyses. Results Genetic Association Between Gut microbiota and EC A total of 208 SNPs were screened out to be valid instrumental variables for 211 bacterial genera according to the selection principles. Figure 2 and Figure S1 are the forest plot and the scatter plots of significant microbial taxa with risks of esophageal cancer. In the forward MR analyses, eight significant microbial taxa were found to be significantly associated with esophageal cancer: the genera Eubacterium , oxidoreducens , Coprobacter , Lachnospira , Romboutsia , Ruminococcus1 , Senegalimassilia , Turicibacter , and Veillonella . The analyses suggests that Eubacterium and oxidoreducens (OR 0.999, 95%CI 0.998-1.000), Lachnospira (OR 0.998, 95%CI 0.996-1.000), Romboutsia (OR 0.999, 95%CI 0.998-1.000) and Turicibacter (OR 0.999, 95%CI 0.998-1.000) played a protective role in esophageal cancer, whereas Coprobacter (OR 1.001, 95%CI 1.000-1.002), Ruminococcus1 (OR 1.001, 95%CI 1.000-1.002), Senegalimassilia (OR 1.002, 95%CI 1.000-1.003) and Veillonella (OR 1.001, 95%CI 1.000-1.002) were concerned with the increased risk of EC. The results of all five statistical methods applied in the forward MR analyses have been displayed in Table S1 . Figure S2 shows the results of MR leave-one-out sensitivity analyses, indicating the stability of the IVs. There is no presence of heterogeneity in Cochran Q test. Moreover, based on the results of MR-PRESSO test and MR Egger regression, no evidence of pleiotropy was identified. Genetic Association Between EC and Gut microbiota We extracted 14 strongly-associated and independent SNPs of esophageal cancer after excluding a palindromic SNP (rs3802909). The reverse MR analyses was carried out as prescribed. The forest plot (Fig. 3 ) and scatter plot (Figure S3 ) are clearly demonstrated the effect of esophageal cancer on gut microbes. We found esophageal cancer have causal relationship with the genera Intestinimonas , Eggerthella , Coprococcus2 and Christensenellaceae R.7 . The occurrence of Esophageal carcinoma is consistent with the increased enrichment of the genera Eggerthella (Beta 37.63, 95%CI 4.76–70.50), Coprococcus2 (Beta 23.90, 95%CI 1.65–46.15) and ChristensenellaceaeR.7 (Beta 22.75, 95%CI 4.22–41.28). Nevertheless, the abundance of the genera Intestinimonas (Beta − 33.24, 95%CI -54.90–11.58) tends to decrease under the influence of EC. The results of all five statistical methods applied in the forward MR analyses have been displayed in Table S2 . Moreover, the results of Cochran Q test verified no heterogeneity in the IVs. The MR Egger regression intercept which is close to zero also proved that there no pleiotropy existed (Figure S4 ). Discussion The Two-Sample MR study we conducted clearly showed the bidirectional causal relationship between esophageal cancer and gut microbes without the influence of confounding factors. The forward MR revealed that genera Eubacterium oxidoreducens , Lachnospira , Romboutsia and Turicibacter had a protective effect on esophageal cancer, while Coprobacter , Ruminococcus1 , Senegalimassilia and Veillonella might be the risk factors for the formation of esophageal malignant neoplasms. Meanwhile, in the reversed MR, Esophageal cancer enriched the abundance of the genera Eggerthella , Coprococcus2 and Christensenellaceae R.7 , and the abundance of the genera Intestinimonas was found to be decreased. In recent years, there has been an increasing focus on the correlation between microorganisms and digestive tract diseases in academic research. For example, Helicobacter pylori has been the well-recognized key causative factor for gastric cancer [ 25 ]. Previous researches have clarified that virus infection such as human papillomavirus (HPV) and Epstein-Barr virus, as well as the changes of gut microbial composition contribute to the carcinogenesis of the esophagus [ 26 ]. In a recent study, the tumor development in LFD-fed GF mice, which received the fecal microbiota from HFD-fed L2-IL1B mice, indicated the crucial role of intestinal microbes in esophageal cancer development and verified its linkage with the IL8/CXCL1 chemokine family [ 27 ]. In an observational study, which recruiting 23 EC patient and 23 health individuals from Huai'an First People's Hospital (Huai'an, China), the abundance of Lachnospira was observed to be reduced in the EC patients, and Lachnospira , of which the AUC = 1, showed a great discrimination ability between EC patients and the healthy [ 17 ]. Reinforcing our analyses, it is plausible that Lachnospira plays a protective role in the pathogenesis of esophageal cancer. The genera Lachnospira , belonging to the phylum Firmicutes and the family Lachnospiraceae , generates acetic acid and butyric acid. It has been recognized that SCFAs not only maintain the stability of Gastrointestinal homeostasis but also function through immunoregulation [ 28 ]. Studies have shown that Lachnospira might connect with nonalcoholic fatty liver disease [ 29 ], Asthma [ 30 ], gastric cancer [ 31 ], colorectal cancer [ 32 ], depression and anxiety in patients with active ulcerative colitis [ 33 ]. However, most of them are association studies, lacking in exploration of mechanisms. Veillonella , one of the major oral microorganisms, promoted the cancer development according to our analyses. The genera contain Veillonella parvum and Veillonella alkalinogens . Oral microbial colonization of the intestine is concerned with many diseases, such as inflammatory bowel disease (IBD) [ 34 ] and colorectal cancer [ 35 ]. A latest study illustrated that the Veillonella ectopically colonize the intestine by taking advantage of inflammatory nitrates derived from inducible nitric oxide synthase (iNOS) activity in host cells [ 36 ]. As a member of Type II bacteria (Gram-negative and anaerobic/microaerophilic bacteria) of the Esophageal Flora, the genera Veillonella may serve as collaborative mechanism to facilitate gastric reflux through activating the iNOS pathway. It is considered that the alteration of Veillonella promotes the progression of gastroesophageal reflux to Barrett's esophagus, which eventually leads to adenocarcinoma [ 37 ]. The studies mentioned above all further validated our analysis. Further studies are urgently needed to determine the mechanism of intestinal bacteria in the pathogenesis of esophageal cancer. In our reverse MR analyses, we revealed that the alteration of gut microbes after suffering from esophageal cancer. Eggerthella is among the top five most abundant genera of the phylum Actinobacteria in the human gut. A study found that the colonization of Eggerthella activates intestinal Th17 by removing from inhibition of the Th17 transcription factor Rorγt, exacerbating the severity of colitis [ 38 ]. Moreover, with proinflammatory properties, Eggerthella is involved in the synthesis of key neurotransmitters in depression, such as glutamate, butyrate, serotonin, and γ-aminobutyric acid (GABA) [ 39 ]. In another study, Eggerthella lenta was found to promotes the accumulation of uremic toxin in serum and exacerbate the progression of kidney disease in a CKD rat model [ 40 ]. However, there is little research on the association of Eggerthella and EC, let alone the mechanism. Our analyses showed the decreased abundance of Intestinimonas under the exposure of EC. Intestinimonas produces butyrate from both sugars and amino acids, protecting the host from harmful metabolites. The enrichment of the genus Intestinimonas in gastric intraepithelial neoplasia [ 41 ] and the reduced abundance of this genera in the obesity [ 42 ] has been observed. Besides, there's already evidence that Intestinimonas showed a good distinction ability between the patients with chronic renal diseases (CKD) and the healthy [ 43 ] and correlated with the total functional ability score and the IL-4 level in patients with Huntington's disease [ 44 ]. Currently, there is limited research on the intestinal microbiota identified in our reverse study. Further investigation is necessary to determine whether alterations in gut microbiota following esophageal cancer have a positive or negative impact, which could enhance the prognosis and adjuvant therapy of EC. Our study has several strengths. Previous studies are more focused on esophageal cancer and esophageal microbiota. Moreover, changes in the microbiota in observational studies are merely clinical manifestations. We analyzed GWAS data by MR to confirm the possible interaction and causal relationship between EC and intestinal microbiota. Bidirectional analyses guarantee causality inferred from both directions. The selected bacteria lay the foundation for further exploration in the future. It also has contributed to the screening of biomarkers for esophageal cancer and the improvement of prognosis. In the procedure of the MR analyses, we strictly screened for strong-associated SNPs as instrumental variables (F statistics > 10). MR-PRESSO test and MR Egger regression was conducted to exclude SNPS with pleiotropy. Despite our exploration is meaningful, there are several limitations. The population we obtained from the UK Biobank was restricted to the European, in which the most common type of esophageal cancer is adenocarcinoma. We did not get the original information on the patients with EC, making it difficult for us to perform subgroup analysis. Besides, genus was the lowest taxonomic level in the gut microbe datasets, further exploration at the species level was restricted. In conclusion, this two sample MR found that the bilateral causality between esophageal cancer and intestinal microbes. Several genera such as Lachnospira and Veillonella had a protective or carcinogenic effect on esophageal cancer. Moreover, the alteration of the genera Eggerthella and Intestinimonas demonstrated the influence of EC on the gut microbiome. Further RCT study and animal experiments are in an urgent need to figure out the mechanisms involved, thus making the candidate intestinal microbes useful for clinical screening and treatment. Declarations Ethics approval and consent to participate The present MR analysis was based on summary data from previous studies that had gained written informed consent and ethics approval. No ethical permit is required for the secondary analysis of summary data. Consent for publication This manuscript has not been previously published. All authors have consented to the publication of the manuscript in this journal. Competing interests The authors declare no conflict of interest exists. Funding This study was funded by the National Natural Science Foundation of China (No. 82100594), China Postdoctoral Science Foundation (No. 2023M731417), Spring Sunshine Program from the Ministry of Education of China (No.202201552) and Jiangsu province Hospital (the First Affiliated Hospital with Nanjing Medical University) Clinical Capacity Enhancement Project (JSPH-MC-2022-29). Authors' contributions Z.X.Y. and S.W. drafted the manuscript. C.H. and L.X.L were responsible for conception and design the study. Z.X.Y. and Y.B.X. performed data analysis and interpretation. S.X.M. and Z.W.F. reviewed the data analysis. Z.G.X. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Acknowledgements The authors thank all investigators and participants from GWAS databases for sharing these data. References Sung, H.; Ferlay, J.; Siegel, R. 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B.; Hurley, E.; O'Riordain, M.; Shanahan, F.; O'Toole, P. W. The oral microbiota in colorectal cancer is distinctive and predictive. Gut 2018 , 67 (8), 1454-1463. DOI: 10.1136/gutjnl-2017-314814 PubMed. Rojas-Tapias, D. F.; Brown, E. M.; Temple, E. R.; Onyekaba, M. A.; Mohamed, A. M. T.; Duncan, K.; Schirmer, M.; Walker, R. L.; Mayassi, T.; Pierce, K. A.; et al. Inflammation-associated nitrate facilitates ectopic colonization of oral bacterium Veillonella parvula in the intestine. Nat Microbiol 2022 , 7 (10), 1673-1685. DOI: 10.1038/s41564-022-01224-7 PubMed. Henderson, G.; Yilmaz, P.; Kumar, S.; Forster, R. J.; Kelly, W. J.; Leahy, S. C.; Guan, L. L.; Janssen, P. H. Improved taxonomic assignment of rumen bacterial 16S rRNA sequences using a revised SILVA taxonomic framework. PeerJ 2019 , 7 , e6496. DOI: 10.7717/peerj.6496 PubMed. Alexander, M.; Ang, Q. Y.; Nayak, R. R.; Bustion, A. E.; Sandy, M.; Zhang, B.; Upadhyay, V.; Pollard, K. S.; Lynch, S. V.; Turnbaugh, P. J. Human gut bacterial metabolism drives Th17 activation and colitis. Cell Host Microbe 2022 , 30 (1). DOI: 10.1016/j.chom.2021.11.001 PubMed. Radjabzadeh, D.; Bosch, J. A.; Uitterlinden, A. G.; Zwinderman, A. H.; Ikram, M. A.; van Meurs, J. B. J.; Luik, A. I.; Nieuwdorp, M.; Lok, A.; van Duijn, C. M.; et al. Gut microbiome-wide association study of depressive symptoms. Nat Commun 2022 , 13 (1), 7128. DOI: 10.1038/s41467-022-34502-3 PubMed. Wang, X.; Yang, S.; Li, S.; Zhao, L.; Hao, Y.; Qin, J.; Zhang, L.; Zhang, C.; Bian, W.; Zuo, L.; et al. Aberrant gut microbiota alters host metabolome and impacts renal failure in humans and rodents. Gut 2020 , 69 (12), 2131-2142. DOI: 10.1136/gutjnl-2019-319766 PubMed. Zhang, X.; Li, C.; Cao, W.; Zhang, Z. Alterations of Gastric Microbiota in Gastric Cancer and Precancerous Stages. Frontiers In Cellular and Infection Microbiology 2021 , 11 , 559148. DOI: 10.3389/fcimb.2021.559148 PubMed. Thingholm, L. B.; Rühlemann, M. C.; Koch, M.; Fuqua, B.; Laucke, G.; Boehm, R.; Bang, C.; Franzosa, E. A.; Hübenthal, M.; Rahnavard, A.; et al. Obese Individuals with and without Type 2 Diabetes Show Different Gut Microbial Functional Capacity and Composition. Cell Host Microbe 2019 , 26 (2). DOI: 10.1016/j.chom.2019.07.004 PubMed. Hu, X.; Ouyang, S.; Xie, Y.; Gong, Z.; Du, J. Characterizing the gut microbiota in patients with chronic kidney disease. Postgrad Med 2020 , 132 (6), 495-505. DOI: 10.1080/00325481.2020.1744335 PubMed. Du, G.; Dong, W.; Yang, Q.; Yu, X.; Ma, J.; Gu, W.; Huang, Y. Altered Gut Microbiota Related to Inflammatory Responses in Patients With Huntington's Disease. Front Immunol 2020 , 11 , 603594. DOI: 10.3389/fimmu.2020.603594 PubMed. Supplementary Figure Supplementary Figure 4 is not available with this version. Supplementary Figure 4. Leave-one-out plots for the causal effects of esophageal cancer on gut microbiota. Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Supplementary Figure 1. Scatter plots for the causal effects of significant microbial taxa on esophageal cancer. FigureS2.tif Supplementary Figure 2. Leave-one-out plots for the causal effects of gut microbiota on esophageal cancer. FigureS3.tif Supplementary Figure 3. Scatter plots for the causal effects of esophageal cancer on significant microbial taxa. TableS1.xls Supplementary Table 1. The causal effects of gut microbiota on esophageal cancer. TableS2.xlsx Supplementary Table 2. The causal effects of esophageal cancer on gut microbiota. TableS3.STROBEMRchecklist.docx Supplementary Table 3. STROBE-MR checklist of recommended items to address in reports of Mendelian randomization studies. 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. 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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-3149703","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":216996465,"identity":"e0e95b36-28b3-4cab-9e02-6295ff66f4d3","order_by":0,"name":"Xiaoying Zhou","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Zhou","suffix":""},{"id":216996466,"identity":"aa45646a-2c68-4e38-a1f4-45c721c8642c","order_by":1,"name":"Wei Su","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Su","suffix":""},{"id":216996467,"identity":"f29bb8da-12ef-46be-ac4a-40d3c0e13ab4","order_by":2,"name":"Han Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Chen","suffix":""},{"id":216996468,"identity":"af88a2a7-c174-4a80-a08f-e08341337e90","order_by":3,"name":"Bixing Ye","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bixing","middleName":"","lastName":"Ye","suffix":""},{"id":216996469,"identity":"25902d9f-4feb-4a7d-a417-c558b41a6aca","order_by":4,"name":"Xinmin Si","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinmin","middleName":"","lastName":"Si","suffix":""},{"id":216996470,"identity":"6cbf048d-ada9-43ba-abd5-e1e9d9fd465b","order_by":5,"name":"Weifeng Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weifeng","middleName":"","lastName":"Zhang","suffix":""},{"id":216996471,"identity":"4cb3a100-4ecb-4a67-92c0-5c9fc7939460","order_by":6,"name":"Xueliang Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xueliang","middleName":"","lastName":"Li","suffix":""},{"id":216996472,"identity":"b445bbbf-5978-4330-a03b-2d4e2e541bda","order_by":7,"name":"Guoxin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYLCCBAYGOQaGAyAmM/FajEnUAgSJDRCaCC3yEcnHJB7uqEvfzng6TYKhwjqxgf3sAbxaDG+kJRsknmHL3dlwdpsEw5n0xAaevAT8WmbkGD5IbOPJ3XAAqIWx7XBigwSPASEtBgcS2yTSDcBa/hGhRV4CbItBAkRLAxFaDHieAf3SlmAIdNhmi4Rj6cZtPDkEbGlPPib5s61O3uDG2Y03PtRYy/aznyFgywEYS+IAOE4Z2PCqB9nSAGPxN+BWNQpGwSgYBSMbAACHFkgCgVX6nAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Guoxin","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-07-07 15:14:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3149703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3149703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":39999142,"identity":"40ea3a13-1e22-4aaf-955d-00dc0d6629c7","added_by":"auto","created_at":"2023-07-13 18:34:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":614600,"visible":true,"origin":"","legend":"\u003cp\u003eThe study design of the bidirectional MR study. Abbreviations: MR, Mendelian Randomization; SNP: Single nucleotide polymorphisms.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/6b2e04f4b6aa09983b93d23c.png"},{"id":39998924,"identity":"5010d153-5de3-40f0-9f04-94c7ca8de7bf","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1695040,"visible":true,"origin":"","legend":"\u003cp\u003eAssociations of genetically predicted gut microbiota with risk of esophageal cancer.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/c9c67801dc1427eef108cf97.png"},{"id":39998926,"identity":"2c81ef14-4053-461d-88da-dedf37fbbca1","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":787498,"visible":true,"origin":"","legend":"\u003cp\u003eAssociations of genetically predicted esophageal cancer with gut microbiota dysbiosis.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/a76bc3e7ad20d2c280513533.png"},{"id":40052903,"identity":"9e5d1b6d-64ac-4c8d-b5b7-24697552febd","added_by":"auto","created_at":"2023-07-14 17:44:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1042130,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/d2b9541e-86b7-4d8f-95b1-be20b71f4322.pdf"},{"id":39998932,"identity":"6bd490ea-f420-4b24-9fd8-74325a20c584","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8402812,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1. Scatter plots for the causal effects of significant microbial taxa on esophageal cancer.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/f02fbc857c8874accf62e604.tif"},{"id":39998931,"identity":"42e4a086-e877-4eac-9dbb-6989cbf263da","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3166760,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2. Leave-one-out plots for the causal effects of gut microbiota on esophageal cancer.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/0eceefa9e7a46bba4f910871.tif"},{"id":39998930,"identity":"aee9b30d-5c82-4b76-a797-b24674fa9684","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1569112,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3. Scatter plots for the causal effects of esophageal cancer on significant microbial taxa.\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/d311998ddd554bed16c0f4b0.tif"},{"id":39999143,"identity":"fcc328ca-6d4b-446d-a88f-0c6229ae4eb8","added_by":"auto","created_at":"2023-07-13 18:34:57","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":219648,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. The causal effects of gut microbiota on esophageal cancer.\u003c/p\u003e","description":"","filename":"TableS1.xls","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/4f748b104c52239589395c54.xls"},{"id":39998927,"identity":"b67b148f-bf81-413b-8eaf-3d276a95bbb5","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":65420,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2. The causal effects of esophageal cancer on gut microbiota.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/26dbfd462b10b8081e4d09b2.xlsx"},{"id":39998928,"identity":"0b5e8bcf-a925-41ec-85a0-7a183c94530a","added_by":"auto","created_at":"2023-07-13 18:26:57","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":34769,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3. STROBE-MR checklist of recommended items to address in reports of Mendelian randomization studies.\u003c/p\u003e","description":"","filename":"TableS3.STROBEMRchecklist.docx","url":"https://assets-eu.researchsquare.com/files/rs-3149703/v1/aa462cc9aaf141deafee5b0a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Causal Role of Esophageal Cancer and Gut Microbiota: A Bidirectional Mendelian Randomization Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer (EC) is one of the most common fatal cancers, which imposing significant worldwide health burdens. The global incidence and mortality of EC ranked tenth and sixth, respectively, in 2020 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The majority of cases (85% [512,500 cases]) were Esophageal squamous cell carcinoma (ESCC) and Esophageal adenocarcinoma (EAC) accounted for 14% (85,700 cases). The distribution vary from regions and adenocarcinoma is the main type in in European and American countries. Moreover, the incidence of EAC has been rising at an alarming rate in Western world [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is well recognized that host and environmental risk factors, such as obesity, gastroesophageal reflux diseases and Barrett's esophagus, contribute to EAC [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The symptoms of early esophageal cancer are often atypical and easy to be ignored, thus primary prevention and early screening play key roles in improving clinical outcome and survival of patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite researchers have done a lot of exploring, the exact pathogenic mechanism of esophageal cancer remains unknown to date, making it difficult to find potential noninvasive biomarkers and predictors.\u003c/p\u003e \u003cp\u003eGastrointestinal tract (GIT) is colonized by trillions of microorganisms that comprise the gut microbiota. Gut microbiota not only takes part in protecting against pathogens and maintaining gut ecosystem homeostasis but also involves in several cancers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. According to a recent study, about 13% of all cancer cases in 2018 can be attributed to infectious etiology [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. During the past few years, much attention has been paid to the association between esophageal cancers and gut microbes. Gut microbiota has a crucial role in the onset, development, and treatment efficacy of gastrointestinal malignancies, which may be caused by inflammation and immune disorders [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Several researches have revealed the different esophageal bacterial strains, from normal esophagus to ESCC and EAC [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Ajayi et al [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] showed that normal esophagus is mainly colonized by Streptococcus while esophagitis and BE are associated with gram-negative bacteria. Furthermore, certain G-bacteria, including Escherichia coli and Fusobacterium nucleatum, are linked to EA. Yamamura et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] revealed that Fusobacterium nucleatum aggravate the survival of ESCC patients. The limitation is, most of the sample were obtained from esophagus by invasive examination. It has been proven that intestinal microbiome involves in inflammatory bowel diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e],diabetes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and so on. Recently, Deng et al [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] demonstrated increased richness of gut microbes and significant change of the distribution proportion of some bacteria in EC compared with health individuals through 16S rDNA gene sequencing of fresh stool. While it is still unclear of the causal relationship between gut microbiota and esophageal cancer.\u003c/p\u003e \u003cp\u003eMendelian randomization (MR) is an original approach to exploring the causality between exposure or risk factor and outcome, which has been widely applied to research in various fields. It screens for genetic variants that can represent exposure as an instrument to examine the relationship between instrument and outcome. Compared to traditional observational studies, which are prone to biases such as reverse causality and residual confounding, MR rules out these biases due to the law of independent assortment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this text, we utilize the genome-wide association study (GWAS) summary statistics from UK Biobank and MiBioGen consortium. A two-sample MR is carried out to evaluate the causal relationship between gut microbes and esophagus.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the flowchart of the MR analysis. A bidirectional two-sample MR study was designed to explore the causal effect of gut microbiota and esophageal cancer.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Sources\u003c/h2\u003e \u003cp\u003eWe obtained the genetic variants datasets from public-available GWAS. The summary statistics of esophageal cancer was provided by UK Biobank. The project is a population-based cohort study involving in 500,000 middle-aged participants. The study consisted of 740 esophageal cancers cases and 372,016 controls, and almost of them are European [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The summary statistics of gut microbes was acquired from the large-scale meta-analysis of 24 cohorts including 18,340 participants conducted by MiBioGen consortium. In this study, fecal microbial composition was analyzed targeting the V4, V3-V4, and V1-V2 variable regions of the 16S rRNA gene and classified by direct taxonomic classification. To identify host genetic variants that was mapped to the genetic loci impacting the relative abundance or presence of microbial taxa, microbiome quantitative trait loci (mbQTL) mapping analysis was carried out. A total of 211 taxa (131 genera, 35 families, 20 orders, 16 classes, and 9 phyla) were involved [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSelection of the instrumental variables\u003c/h2\u003e \u003cp\u003eWe set the threshold of genome-wide significance (p\u0026thinsp;\u0026lt;\u0026thinsp;5*10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) to filter the SNPs strongly associated with the exposure in the MR analysis. Then, on the basis of the European ancestral individuals from the 1000 Genomes Project, we assessed the linkage disequilibrium to ensure the independence ,and appropriate SNPs within 10,000kb clumping window with an r2\u0026thinsp;\u0026lt;\u0026thinsp;0.001 were kept [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) Test was implemented to test the presence of horizontal pleiotropy, the \u003cem\u003ep\u003c/em\u003e value in each MR analysis should be greater than 0.05 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Before harmonizing the exposure and outcome datasets, we excluded the palindromic sequence due to the uncertainty of the direction for exposure and outcome. Moreover, F-statistics was calculated to evaluate the strength of IVs by the formula: F\u0026thinsp;=\u0026thinsp;R2(n\u0026thinsp;\u0026minus;\u0026thinsp;1\u0026minus;k)/(1\u0026thinsp;\u0026minus;\u0026thinsp;R2) k, (R2 represents the proportion of variation explained by each IV, k represents the number of eligible SNPs, and n represents the sample size) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When F statistics\u0026thinsp;\u0026gt;\u0026thinsp;10, the IVs was considered as strongly-correlated instruments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn this study, we performed the analysis in R 4.1.0 using TwoSampleMR [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and MRPRESSO packages. To assess whether there is causal relationship between esophageal cancer and gut microbes, we applied multiple methods: Inverse variance weighted (IVW), MR Egger regression, Weighted median, Maximum likelihood, Weighted mode. The IVW method was predominate and the others was utilized as sensitivity analyses to further confirm the reliability of the IVW method. The IVW method calculated a weighted average of Wald ratio estimates of each SNP. If all of the SNPs are valid instrumental variables, there will be no bias in the results [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Besides, we used the Cochran Q test to quantify the heterogeneity of IVs. As the p value\u0026thinsp;\u0026le;\u0026thinsp;0.05, which standing for the existence of heterogeneity, we carried out a random effects IVW MR analyses. Moreover, the Leave-one-out analysis demonstrated the meta effects of the remaining SNPs when a certain SNP was removed in turn. If the results changed greatly after the elimination, it indicated the heterogeneity of the removed SNP. We also performed the reverse MR analyses in the same methods of the forward MR analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGenetic Association Between Gut microbiota and EC\u003c/h2\u003e \u003cp\u003eA total of 208 SNPs were screened out to be valid instrumental variables for 211 bacterial genera according to the selection principles. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e are the forest plot and the scatter plots of significant microbial taxa with risks of esophageal cancer. In the forward MR analyses, eight significant microbial taxa were found to be significantly associated with esophageal cancer: the genera \u003cem\u003eEubacterium\u003c/em\u003e, \u003cem\u003eoxidoreducens\u003c/em\u003e, \u003cem\u003eCoprobacter\u003c/em\u003e, \u003cem\u003eLachnospira\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eRuminococcus1\u003c/em\u003e, \u003cem\u003eSenegalimassilia\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, and \u003cem\u003eVeillonella\u003c/em\u003e. The analyses suggests that \u003cem\u003eEubacterium\u003c/em\u003e and \u003cem\u003eoxidoreducens\u003c/em\u003e (OR 0.999, 95%CI 0.998-1.000), \u003cem\u003eLachnospira\u003c/em\u003e (OR 0.998, 95%CI 0.996-1.000), \u003cem\u003eRomboutsia\u003c/em\u003e (OR 0.999, 95%CI 0.998-1.000) and \u003cem\u003eTuricibacter\u003c/em\u003e (OR 0.999, 95%CI 0.998-1.000) played a protective role in esophageal cancer, whereas \u003cem\u003eCoprobacter\u003c/em\u003e (OR 1.001, 95%CI 1.000-1.002), \u003cem\u003eRuminococcus1\u003c/em\u003e (OR 1.001, 95%CI 1.000-1.002), \u003cem\u003eSenegalimassilia\u003c/em\u003e (OR 1.002, 95%CI 1.000-1.003) and \u003cem\u003eVeillonella\u003c/em\u003e (OR 1.001, 95%CI 1.000-1.002) were concerned with the increased risk of EC. The results of all five statistical methods applied in the forward MR analyses have been displayed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e shows the results of MR leave-one-out sensitivity analyses, indicating the stability of the IVs. There is no presence of heterogeneity in Cochran Q test. Moreover, based on the results of MR-PRESSO test and MR Egger regression, no evidence of pleiotropy was identified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetic Association Between EC and Gut microbiota\u003c/h2\u003e \u003cp\u003eWe extracted 14 strongly-associated and independent SNPs of esophageal cancer after excluding a palindromic SNP (rs3802909). The reverse MR analyses was carried out as prescribed. The forest plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and scatter plot (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) are clearly demonstrated the effect of esophageal cancer on gut microbes. We found esophageal cancer have causal relationship with the genera \u003cem\u003eIntestinimonas\u003c/em\u003e, \u003cem\u003eEggerthella\u003c/em\u003e, \u003cem\u003eCoprococcus2\u003c/em\u003e and \u003cem\u003eChristensenellaceae R.7\u003c/em\u003e. The occurrence of Esophageal carcinoma is consistent with the increased enrichment of the genera \u003cem\u003eEggerthella\u003c/em\u003e (Beta 37.63, 95%CI 4.76\u0026ndash;70.50), \u003cem\u003eCoprococcus2\u003c/em\u003e (Beta 23.90, 95%CI 1.65\u0026ndash;46.15) and \u003cem\u003eChristensenellaceaeR.7\u003c/em\u003e (Beta 22.75, 95%CI 4.22\u0026ndash;41.28). Nevertheless, the abundance of the genera \u003cem\u003eIntestinimonas\u003c/em\u003e (Beta \u0026minus;\u0026thinsp;33.24, 95%CI -54.90\u0026ndash;11.58) tends to decrease under the influence of EC. The results of all five statistical methods applied in the forward MR analyses have been displayed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Moreover, the results of Cochran Q test verified no heterogeneity in the IVs. The MR Egger regression intercept which is close to zero also proved that there no pleiotropy existed (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Two-Sample MR study we conducted clearly showed the bidirectional causal relationship between esophageal cancer and gut microbes without the influence of confounding factors. The forward MR revealed that genera \u003cem\u003eEubacterium oxidoreducens\u003c/em\u003e, \u003cem\u003eLachnospira\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e and \u003cem\u003eTuricibacter\u003c/em\u003e had a protective effect on esophageal cancer, while \u003cem\u003eCoprobacter\u003c/em\u003e, \u003cem\u003eRuminococcus1\u003c/em\u003e, \u003cem\u003eSenegalimassilia\u003c/em\u003e and \u003cem\u003eVeillonella\u003c/em\u003e might be the risk factors for the formation of esophageal malignant neoplasms. Meanwhile, in the reversed MR, Esophageal cancer enriched the abundance of the genera \u003cem\u003eEggerthella\u003c/em\u003e, \u003cem\u003eCoprococcus2\u003c/em\u003e and \u003cem\u003eChristensenellaceae R.7\u003c/em\u003e, and the abundance of the genera \u003cem\u003eIntestinimonas\u003c/em\u003e was found to be decreased.\u003c/p\u003e \u003cp\u003eIn recent years, there has been an increasing focus on the correlation between microorganisms and digestive tract diseases in academic research. For example, Helicobacter \u003cem\u003epylori\u003c/em\u003e has been the well-recognized key causative factor for gastric cancer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous researches have clarified that virus infection such as human papillomavirus (HPV) and Epstein-Barr virus, as well as the changes of gut microbial composition contribute to the carcinogenesis of the esophagus [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In a recent study, the tumor development in LFD-fed GF mice, which received the fecal microbiota from HFD-fed L2-IL1B mice, indicated the crucial role of intestinal microbes in esophageal cancer development and verified its linkage with the IL8/CXCL1 chemokine family [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In an observational study, which recruiting 23 EC patient and 23 health individuals from Huai'an First People's Hospital (Huai'an, China), the abundance of Lachnospira was observed to be reduced in the EC patients, and \u003cem\u003eLachnospira\u003c/em\u003e, of which the AUC\u0026thinsp;=\u0026thinsp;1, showed a great discrimination ability between EC patients and the healthy [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Reinforcing our analyses, it is plausible that \u003cem\u003eLachnospira\u003c/em\u003e plays a protective role in the pathogenesis of esophageal cancer. The genera \u003cem\u003eLachnospira\u003c/em\u003e, belonging to the phylum \u003cem\u003eFirmicutes\u003c/em\u003e and the family \u003cem\u003eLachnospiraceae\u003c/em\u003e, generates acetic acid and butyric acid. It has been recognized that SCFAs not only maintain the stability of Gastrointestinal homeostasis but also function through immunoregulation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Studies have shown that \u003cem\u003eLachnospira\u003c/em\u003e might connect with nonalcoholic fatty liver disease [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Asthma [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], gastric cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], depression and anxiety in patients with active ulcerative colitis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, most of them are association studies, lacking in exploration of mechanisms. \u003cem\u003eVeillonella\u003c/em\u003e, one of the major oral microorganisms, promoted the cancer development according to our analyses. The genera contain \u003cem\u003eVeillonella parvum\u003c/em\u003e and \u003cem\u003eVeillonella alkalinogens\u003c/em\u003e. Oral microbial colonization of the intestine is concerned with many diseases, such as inflammatory bowel disease (IBD) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and colorectal cancer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A latest study illustrated that the \u003cem\u003eVeillonella\u003c/em\u003e ectopically colonize the intestine by taking advantage of inflammatory nitrates derived from inducible nitric oxide synthase (iNOS) activity in host cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. As a member of Type II bacteria (Gram-negative and anaerobic/microaerophilic bacteria) of the Esophageal Flora, the genera \u003cem\u003eVeillonella\u003c/em\u003e may serve as collaborative mechanism to facilitate gastric reflux through activating the iNOS pathway. It is considered that the alteration of \u003cem\u003eVeillonella\u003c/em\u003e promotes the progression of gastroesophageal reflux to Barrett's esophagus, which eventually leads to adenocarcinoma [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The studies mentioned above all further validated our analysis. Further studies are urgently needed to determine the mechanism of intestinal bacteria in the pathogenesis of esophageal cancer.\u003c/p\u003e \u003cp\u003eIn our reverse MR analyses, we revealed that the alteration of gut microbes after suffering from esophageal cancer. \u003cem\u003eEggerthella\u003c/em\u003e is among the top five most abundant genera of the phylum \u003cem\u003eActinobacteria\u003c/em\u003e in the human gut. A study found that the colonization of \u003cem\u003eEggerthella\u003c/em\u003e activates intestinal Th17 by removing from inhibition of the Th17 transcription factor Rorγt, exacerbating the severity of colitis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, with proinflammatory properties, \u003cem\u003eEggerthella\u003c/em\u003e is involved in the synthesis of key neurotransmitters in depression, such as glutamate, butyrate, serotonin, and γ-aminobutyric acid (GABA) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In another study, \u003cem\u003eEggerthella lenta\u003c/em\u003e was found to promotes the accumulation of uremic toxin in serum and exacerbate the progression of kidney disease in a CKD rat model [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, there is little research on the association of \u003cem\u003eEggerthella\u003c/em\u003e and EC, let alone the mechanism. Our analyses showed the decreased abundance of \u003cem\u003eIntestinimonas\u003c/em\u003e under the exposure of EC. \u003cem\u003eIntestinimonas\u003c/em\u003e produces butyrate from both sugars and amino acids, protecting the host from harmful metabolites. The enrichment of the genus \u003cem\u003eIntestinimonas\u003c/em\u003e in gastric intraepithelial neoplasia [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and the reduced abundance of this genera in the obesity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] has been observed. Besides, there's already evidence that \u003cem\u003eIntestinimonas\u003c/em\u003e showed a good distinction ability between the patients with chronic renal diseases (CKD) and the healthy [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and correlated with the total functional ability score and the IL-4 level in patients with Huntington's disease [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Currently, there is limited research on the intestinal microbiota identified in our reverse study. Further investigation is necessary to determine whether alterations in gut microbiota following esophageal cancer have a positive or negative impact, which could enhance the prognosis and adjuvant therapy of EC.\u003c/p\u003e \u003cp\u003eOur study has several strengths. Previous studies are more focused on esophageal cancer and esophageal microbiota. Moreover, changes in the microbiota in observational studies are merely clinical manifestations. We analyzed GWAS data by MR to confirm the possible interaction and causal relationship between EC and intestinal microbiota. Bidirectional analyses guarantee causality inferred from both directions. The selected bacteria lay the foundation for further exploration in the future. It also has contributed to the screening of biomarkers for esophageal cancer and the improvement of prognosis. In the procedure of the MR analyses, we strictly screened for strong-associated SNPs as instrumental variables (F statistics\u0026thinsp;\u0026gt;\u0026thinsp;10). MR-PRESSO test and MR Egger regression was conducted to exclude SNPS with pleiotropy. Despite our exploration is meaningful, there are several limitations. The population we obtained from the UK Biobank was restricted to the European, in which the most common type of esophageal cancer is adenocarcinoma. We did not get the original information on the patients with EC, making it difficult for us to perform subgroup analysis. Besides, genus was the lowest taxonomic level in the gut microbe datasets, further exploration at the species level was restricted.\u003c/p\u003e \u003cp\u003eIn conclusion, this two sample MR found that the bilateral causality between esophageal cancer and intestinal microbes. Several genera such as \u003cem\u003eLachnospira\u003c/em\u003e and \u003cem\u003eVeillonella\u003c/em\u003e had a protective or carcinogenic effect on esophageal cancer. Moreover, the alteration of the genera \u003cem\u003eEggerthella\u003c/em\u003e and \u003cem\u003eIntestinimonas\u003c/em\u003e demonstrated the influence of EC on the gut microbiome. Further RCT study and animal experiments are in an urgent need to figure out the mechanisms involved, thus making the candidate intestinal microbes useful for clinical screening and treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present MR analysis was based on summary data from previous studies that had gained written informed consent and ethics approval. No ethical permit is required for the secondary analysis of summary data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript has not been previously published. All authors have consented to the publication of the manuscript in this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (No. 82100594), China Postdoctoral Science Foundation (No. 2023M731417), Spring Sunshine Program from the Ministry of Education of China (No.202201552) and Jiangsu province Hospital (the First Affiliated Hospital with Nanjing Medical University) Clinical Capacity Enhancement Project (JSPH-MC-2022-29).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.X.Y. and S.W. drafted the manuscript. C.H. and L.X.L were responsible for conception and design the study. Z.X.Y. and Y.B.X. performed data analysis and interpretation. S.X.M. and Z.W.F. reviewed the data analysis. Z.G.X. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all investigators and participants from GWAS databases for sharing these data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung, H.; Ferlay, J.; Siegel, R. L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA Cancer J Clin \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e71\u003c/em\u003e (3), 209-249. 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Altered Gut Microbiota Related to Inflammatory Responses in Patients With Huntington\u0026apos;s Disease. \u003cem\u003eFront Immunol \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e11\u003c/em\u003e, 603594. DOI: 10.3389/fimmu.2020.603594 PubMed.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Figure","content":"\u003cp\u003eSupplementary Figure 4 is not available with this version.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 4. Leave-one-out plots for the causal effects of esophageal cancer on gut microbiota.\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, Bidirectional","lastPublishedDoi":"10.21203/rs.3.rs-3149703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3149703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eEsophageal cancer (EC) and gut microbiota are reported to be clinically relevant but their genetic association is unclear. We carried out a bidirectional MR analyses to assess the casual relationship between EC and gut microbiota from fecal samples.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe microbiome genome-wide association studies (GWAS) data of 18,340 individuals provided by MiBioGen consortium and the EC GWAS data (740 esophageal cancers cases and 372,016 controls) provided by UK Biobank were respectively utilized as exposure and/or outcome data. Reliable single nucleotide polymorphisms (SNPs) were obtained after rigorous screening. A bidirectional Mendelian randomization (MR) analysis was conducted using the inverse-variance weighted (IVW) method. The sensitivity analyses including the MR-Egger method, weighted median, MR-PRESSO and mode methods were performed to examine the stability, heterogeneity and pleiotropy of the results.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eForward MR analysis revealed genetic liability to gut microbiota was associated with a higher risk of EC (\u003cem\u003eCoprobacter\u003c/em\u003e (OR 1.001, 95%CI 1.000-1.002); \u003cem\u003eRuminococcus1\u003c/em\u003e (OR 1.001, 95%CI 1.000-1.002); \u003cem\u003eTuricibacter\u003c/em\u003e (OR 0.999, 95%CI 0.998-1.000); \u003cem\u003eSenegalimassilia\u003c/em\u003e (OR 1.002, 95%CI 1.000-1.003); \u003cem\u003eVeillonella\u003c/em\u003e (OR 1.001, 95%CI 1.000-1.002)) or a significantly lower risk of EC (\u003cem\u003eEubacterium oxidoreducens\u003c/em\u003e (OR 0.999, 95%CI 0.998-1.000); \u003cem\u003eLachnospira\u003c/em\u003e (OR 0.998, 95%CI 0.996-1.000); Romboutsia (OR 0.999, 95%CI 0.998-1.000)). Reverse MR analysis showed that genetic liability to EC was also causally linked to increased susceptibility of gut microbiota dysbiosis (genera \u003cem\u003eEggerthella\u003c/em\u003e (Beta 37.63, 95%CI 4.76\u0026ndash;70.50); \u003cem\u003eCoprococcus2\u003c/em\u003e (Beta 23.90, 95%CI 1.65\u0026ndash;46.15); \u003cem\u003eChristensenellaceaeR.7\u003c/em\u003e (Beta 22.75, 95%CI 4.22\u0026ndash;41.28); genera \u003cem\u003eIntestinimonas\u003c/em\u003e (OR -33.24, 95%CI-54.90\u0026ndash;11.58)).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings supported a bidirectionally causal relationship between gut microbiota and EC, implying the potential of gut microbiota as novel biomarkers and therapeutic drugs for EC.\u003c/p\u003e","manuscriptTitle":"The Causal Role of Esophageal Cancer and Gut Microbiota: A Bidirectional Mendelian Randomization Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-13 18:26:52","doi":"10.21203/rs.3.rs-3149703/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":"fd79c862-b301-4fde-a208-9a5a7b0c3845","owner":[],"postedDate":"July 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-14T17:44:21+00:00","versionOfRecord":[],"versionCreatedAt":"2023-07-13 18:26:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3149703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3149703","identity":"rs-3149703","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}