| Background and objectives Esophageal cancer(EC)is one of the most common digestive malignant tumors in China and other countries,it is characterized with high incidence and poor prognosis.The number of EC patients in China accounts for approximately half of the total number of global cases,and it results in a serious burden on the society.The occurrence of EC is a chronic process and influenced by multiple factors,such as environmental factors,dietary habits,and genetic factors.In recent years,the increasing number of research have already shown that the microbiota plays an unignorable role in the occurrence and development of EC.However,most of research on EC and microbiota are mainly concentrated on microbial analysis of oral or esophagus,the relationship of gut microbiota and the occurrence and development of EC are still poorly understand.Therefore,in this study,16 S r RNA gene sequencing technology was used to identify the characteristics of gut microbiota spectrums in EC patients,compare the difference of gut microbiota in the EC patients and healthy individuals,and explore the effect of bacterial endotoxin on the biological functions of EC cells and the mechanism that affects the invasion and migration of EC cells.In order to provide reasonable data reference and research support for further understanding of the internal relationship between the occurrence and development of EC and gut microbiota,and find new targets for the diagnosis and treatment of EC.Methods1.23 patients who have been diagnosed with EC according to the diagnostic criteria of EC were selected in Huai’an City,Jiangsu Province,23 age-and sex-matched healthy individuals were recruited as the control.The stool and blood samples of the EC patients and healthy individuals were collected,fecal genomic DNA extraction kit was used to extract microbial DNA,and the 16 S rDNA V4 region was specifically amplified,After completing the sequencing work on the Illumina-Hi Seq high-throughput sequencing platform,we analyzed the sequencing results in order to compare the differences between the composition and structure of the gut microbiota in two groups,and analyze the intestinal microbes with significant difference in relative abundance of EC patients and healthy individuals.2.Looking for representative bacterium in the sequencing result,designing specific primer according to its 16 S rDNA sequence,real-time fluorescent quantitative PCR was used for absolute quantitative analysis of bacteria in 46 samples.Stool samples from 100 EC patients and 50 healthy individuals were collected and DNA was extracted,to verify the absolute abundance of the above bacteria.3.ELISA was used to analyze serum level of bacterial endotoxin in EC patients and healthy individuals,and q RT-PCR was used to detect the expression of receptor TLR4 in EC tissues and cancer-adjacent normal tissues.4.The effect of bacterial endotoxin on EC cell proliferation was detected by CCK8 assay,transwell assays were used to observe the role of bacterial endotoxin on cell migration and invasion.5.The protein expression of TLR4/ NF-κB were measured by Western Blot,and used the TLR4 specific pathway inhibitor TAK242 and the NF-κB specific pathway inhibitor PDTC to treat EC cells respectively,then observed the changes of EC cells in invasion and migration capabilities.6.ELISA was used to detected the level of IL-6 and TGF-β1 in EC cells after treated with bacterial endotoxin,which EC cells were pretreated with or without TAK242 and PDTC.7.Western Blot was used to detect the expression of key proteins E-cadherin,N-cadherin and Vimentin in the EMT pathway.ResultsⅠ.Identification of the gut microbiota profile of patients with EC1.Alpha(α)diversity analysis of gut microbiota in EC patients and healthy controls(1)The result showed that the age,smoking rate,drinking rate and dietary habits of EC patients were not significantly different from those of healthy controls.(2)A total of 1831 OTUs were obtained from 46 samples,and the average number of OTUs per sample was 392,of which the two groups had a total of 609 OTUs,the number of OTUs unique to the EC group was 878,and the number of OTUs unique to the control group was 304,the rarefaction curve showed that the depth of the sample sequencing has been basically covered,and the sequencing data was reliable.(3)α diversity index indicated that the Ace and Chao1 indexes of the EC group were higher than control group(p <0.05).However,Shannon and Simpson indexes were not significantly different between the two groups(p > 0.05).It indicated that compared with healthy individuals,the species richness of the gut microbiota of EC patients has increased significantly,but the evenness has not changed.2.Beta diversity analysis of gut microbiota in EC patients and healthy controls(1)According to the results of PCo A analysis and UPGMA cluster tree analysis,the samples between the same group were obviously clustered,and the samples between different groups were obviously separated,indicating that the microbial species composition of EC patients was significantly different from that of the healthy controls.(2)Anosim analysis found that the difference between the EC group and the control group was greater than the difference within the group(p < 0.05),its indicating that the structure of the intestinal microbial community of EC patients was significantly different from the healthy controls.(3)The result of LEf Se analysis indicated that at the genus level,compared with the control group,the bacterium with a significant increase in relative abundance in the EC group were Klebsiella,Verrucobacterium,Enterobacter,Bifidobacterium,and Streptococcus,the bacterium with a significant decrease in relative abundance in the EC group were Lachnospira,Bacteroides,and Lachnoclostridium.3.Analysis of gut microbial difference between the EC patients and healthy individuals.(1)At the phylum level,19 phyla were detected in the EC group and 23 phyla were detected in the control group.The phyla with relative abundance greater than 1% in the EC group were Firmicutes,Proteobacteria,Bacteroides,Actinobacteria,and Verrucomicrobia.The phyla with relative abundance greater than 1% in the control group were Bacteroides,Firmicutes,and Proteobacteria.Welch’s t test showed that the relative abundance of Firmicutes(57.1% VS 38.73%)and Actinomycetes(8.5% VS 0.12%)in the EC group were increased significantly compared with the control group,but the relative abundance of Bacteroides(11.28% VS 49.02%)was significantly decreased(p <0.05).(2)At the genus level,there are 197 genera were identified in the EC group,and 235 genera were identified in the control group.The genera with relative abundance greater than1% in the EC group were Klebsiella,Bacteroides,Streptococcus,Bifidobacterium,Subdoligranulum,Faecalibacterium,and Agathobacter.The genera with relative abundance greater than 1% in the control group were Bacteroides,Lachnospira,Klebsiella,Agathobacter,Faecalibacterium,and Subdoligranulum.Based on Welch’s t-test,compared with the control group,there were 9 genera,including Streptococcus,Bifidobacterium,Subdoligranulum,Blautia,Romboutsia,Collinsella,Paeniclostridium,Dorea,and Atopobium were highly increased in the EC group,and there also 9 genera,including Lachnospira,Bacteroides,Agathobacter,Lachnoclostridium,Parabacteroides,Paraprevotella,Butyricicoccus,Tyzzerella,and Sutterella were significantly decreased in the EC group.(3)According to the contribution of different bacterial genera,the top three genera that contributed to the difference between the two groups were Bacteroides,Klebsiella,and Streptococcus.Ⅱ.Quantitative analysis of the difference in intestinal flora between EC patients and healthy individuals1.Real-time fluorescence quantitative PCR was performed on 23 pairs of samples.The results showed that compared with healthy controls,the absolute abundance of Bacteroides(3.85E+09 VS 9.06E+10),Lachnoclostridium(1.59E+08 VS 2.61E+09),and Lachnospira(4.96E+07 VS 3.08E+8)were significantly decreased in EC patients;the absolute abundance of Bifidobacterium(6.27E+08 VS 4.21E+07)and Streptococcus(1.50E+09 VS 3.39E+08)were significantly increased in EC patients;while the absolute abundance of Enterococcus(1.83E+07 VS 9.61E+06)and Klebsiella(7.91E+09 VS 1.78E+09)were no significant difference between the two groups.2.The real-time fluorescence quantitative PCR was performed on 100 EC patients and50 healthy individuals.We found that the overall trend was roughly the same as the 23 pairs samples,the absolute abundance of the Bacteroides(1.00E+09 VS 1.14E+10),Lachnoclostridium(1.21E+07 VS 1.76E+08),and Lachnospira(6.42E+06 VS 8.106E+07)were obviously decreased,the absolute abundance of the Bifidobacterium(1.08E+09 VS2.93E+08),Streptococcus(1.17E+08 VS 1.62E+07),Enterococcus(1.17E+07 VS 3.61E+06)and Klebsiella(1.60E+09 VS 2.87E+08)were increased significantly in the EC group,and the differences were statistically significant(p <0.05).According to the results of the two experiments,the quantitative result of the gut microbiota between two groups were reliable and representative.Ⅲ.The effect of bacterial endotoxin on the biological function of EC cells1.By analyzing the level of lipopolysaccharide in the serum samples of EC patients and healthy controls,we found that the level of lipopolysaccharide in serum samples of EC patients was significantly higher than healthy controls(p <0.05),the levels of lipopolysaccharide in the EC group was 1.61-fold higher than that in the control group(748ng/L VS 464 ng/L).It was indicated that EC patients may be exposed to higher levels of lipopolysaccharide in daily life.2.RT-q PCR was used to ananlyze the expression of lipopolysaccharide receptor TLR4 m RNA in EC tissues and para-tumor tissues,the results indicated that the m RNA expression levels of TLR4 in EC tissues was increased compared with adjacent non-tumor tissues(2-fold upregulation of the expression level,p <0.05).It was indicated that TLR4 was specifically and highly expressed in EC tissues.3.After treating EC cells with different concentrations of lipopolysaccharide(0,500ng/m L,1μg/m L,5μg/m L,10μg/m L)for 24 h,48h,and 72 h,respectively.We detected the proliferation ability of EC cells by the CCK-8 assay,and the result showed that with the increase of the exposure time and the exposure dose,the proliferation ability of EC cells was enhanced(the relative cell proliferation rate of the lowest dose group was about 1.1 times,and the highest dose group was about 1.3 times),the difference was statistically significant(p<0.05).It was indicated that lipopolysaccharide treatment can promote the proliferation of EC cells,but limited physiological significance.4.In order to analyze the effect of lipopolysaccharide on the invasion and migration of EC cells,we treated EC cells EC109 with different concentrations of lipopolysaccharide,and detected the invasion and migration ability of EC cells,respectively.The results demonstrated that lipopolysaccharide treatment can significantly increase the number of invasion and migration of EC cells compared with the control group(p < 0.05),and it was dose-dependent(the number of cells penetrating the membrane in the lowest dose group and the highest dose group were about 1.5 times and 3 times that of the control group,respectively).The higher the lipopolysaccharide concentration,the more the number of cells invasion and migration.This indicated that lipopolysaccharide can significantly promote the invasion and migration of EC cells.Ⅳ.The mechanism of bacterial endotoxin promoting the migration and invasion of EC cells1.In order to explore whether the TLR4/NF-κB signaling pathway can be specifically activated by lipopolysaccharide,we selected different concentration of lipopolysaccharide(0,500ng/m L,1μg/m L,5μg/m L,10μg/m L)to treat EC cells,the results indicated that with the increased concentration of lipopolysaccharide,the expression of TLR4 protein and NF-κB p65 protein increased in a dose-dependent manner(p < 0.05).2.In order to investigate whether the invasion and migration promotion effect of lipopolysaccharide on EC cells was related to the TLR4/NF-κB signaling pathway,we used the TLR4 signaling pathway inhibitor TAK242 and the NF-κB signaling pathway inhibitor PDTC to pretreate the cells,and then observed the effect of lipopolysaccharide on the invasion and migration ability of EC cells,we found that after treating EC cells with TAK242 or PDTC,the LPS-mediated promotion of EC cell invasion and migration was abolished(p<0.05).3.In order to explore the effect of TLR4/NF-κB signaling pathway on the secretion of IL-6 and TGF-β1 by EC cells,we treated EC cells with TAK242 and PDTC respectively,and detected the level of IL-6 and TGF-β1 in the cell supernatants.The results showed that compared with the control group,the level of IL-6 and TGF-β1 in the lipopolysaccharide treatment group was significantly increased,while after treatment with inhibitors,the level of IL-6 and TGF-β1 were significantly decreased(p <0.05).The results indicated that lipopolysaccharide can affect the secretion of IL-6 and TGF-β1 by EC cells through the TLR4/NF-κB signaling pathway.4.In order to verify whether IL-6 and TGF-β1 exert their biological effects on EC cells through the EMT signaling pathway,we further observed the expression of proteins related to the EMT signaling pathway.The results showed that compared with the control group,the expression level of E-cadherin protein in the lipopolysaccharide treatment group was significantly down-regulated,and the expression levels of N-cadherin protein and Vimentin protein were significantly up-regulated(p <0.05),while the expression level of E-cadherin protein was significantly up-regulated and the expression levels of N-cadherin protein and Vimentin protein were significantly down-regulated in treatment group after treated with inhibitor(p <0.05).The results indicated that lipopolysaccharide may promote the secretion of IL-6 and TGF-β1 of EC cells by activating the TLR4/NF-κB signaling pathway,then affected the EMT process of EC cells and promoted the invasion and migration of EC cells.Conclusions:1.Compared with healthy individuals,an obvious dysbiosis of the gut microbiota in the EC patients was observed,the composition of gut microbiota was significantly different between the two groups,the richness of the gut microbiota was significantly increased,while the difference in the evenness was not statistically significant.The differential abundance analysis of the genus found that there were 18 genera with different relative abundance of intestinal flora between EC patients and healthy individuals,9 bacterial genera with increased and decreased relative abundance,respectively.2.The absolute abundance of Streptococcus,Enterococcus,Klebsiella,and Bifidobacterium were significantly higher in the EC group,while the absolute abundance of Lachnospira,Bacteroides,and Lachnoclostrium were significantly lower in the EC group compared with healthy group.3.The level of bacterial endotoxin was higher in the serum of EC patients,and its specific receptor TLR4 was specifically and highly expressed in cancer tissues too.Cell experiments indicated that bacterial endotoxin can promote the proliferation,invasion and migration of EC cells.4.Bacterial endotoxin can induce the secretion of IL-6 and TGF-β1 by activating the TLR4/NF-κB signaling pathway to promote the EMT process of EC cells and affect the invasion and migration of EC cells. |
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