Molecular Mechanism Of Small Intestinal MicroRNA-mediated Stachyose-induced Changes In Intestinal Microbiota Composition

Stachyose is a non-reducing functional oligosaccharide widely present in foods such as beans.Relevant studies have shown that stachyose can promote the proliferation of bifidobacteria with high selectivity and improve intestinal health in a targeted manner.In order to verify whether microRNA secreted by small intestinal epithelial cells is involved in the process of stachyose regulating gut microbiota,this study used 16S rDNA sequencing technology and Small RNA sequencing technology to systematically investigate the effects of stachyose on the structure of mouse gut microbiota and small intestine.The influence of microRNA expression,screening of intestinal bacteria with significantly changed abundance and microRNAs with significantly changed expression induced by the intervention of stachyose,and further constructing a PLSR partial least square regression model to correlate the selected microRNAs with intestinal bacteria Prediction,and finally use flow cytometry and microbial in vitro culture and fermentation technology to verify the prediction results.The main research contents and results are as follows:(1)After 10 weeks of stachyose intervention,the mice had normal body weight and stable food intake.After 16S rDNA sequencing of the cecal contents of normal mice and stachyose intervention mice,we found that stachyose intervention in the intestines of mice.The microbial diversity,species distribution and clustering methods of microbiota have changed significantly.The sequencing results were further screened to obtain 8 kinds of bacteria with significant changes in abundance,namely Lactobacillus gasseri,Bacteroides acidifaciens,Bacteroides vulgatus,Ruminococcus flavefaciens,Parabacteroides goldsteinii,Lactobacillus murinus,Lactobacillus alimentarius and Akkermansia muciniphila.On this basis,we prefer to select Lactobacillus gasseri,Lactobacillus alimentarius and Akkermansia muciniphila,which have been proven by multiple studies to have intestinal probiotic effects and are proliferated by stachyose,for follow-up confirmatory experiments.(2)Using Small RNA sequencing technology to analyze the small intestinal microRNA expression profiles of normal mice and stachyose intervention mice,and it was found that stachyose significantly changed the expression levels of 27 microRNAs in the small intestine tissues of mice.At the same time,qPCR technology was employed to further verify the levels of these 27 differentially expressed microRNAs in small intestine tissues and feces.On this basis,combined with TPM,P value and FDR values,9 microRNAs with the same expression trend in the results of Small RNA sequencing and qPCR verification were screened out,and most of the microRNAs showed a downward-regulation trend.(3)The PLSR partial least squares regression model was successfully constructed with the stachyose-induced significant change in abundance as the dependent variable and small intestinal microRNA as the independent variable.The results showed that:three intestinal probiotics Lactobacillus gasseri,Lactobacillus alimentarius and Akkermansia muciniphila have a significant correlation with a variety of differential microRNA levels.Integrating the relative abundance of these three microorganisms at the genus level and species level,as well as the expression of microRNA in the small intestine and feces,we found that miR-133b-3p and miR-3068-5p,which are downregulated by stachyose,and these three probiotics Its abundance is significantly related,and it is speculated that it may be involved in mediating the growth regulation of stachyose on Lactobacillus gasseri,Lactobacillus alimentarius and Akkermansia muciniphila in the intestine.(4)After co-cultivating the chemically synthesized fluorescent-labeled microRNA with intestinal bacteria,the fluorescence intensity in the bacteria was monitored by flow cytometry,and it was found that both fluorescent-labeled miR-133b-3p and miR-30685p can enter Lactobacillus gasseri,Lactobacillus alimentarius and Akkermansia muciniphila.In addition,the results of bacterial growth curve determination showed that miR-133b-3p can significantly inhibit the proliferation of Lactobacillus gasseri in vitro.In summary,the intervention of stachyose significantly increased the abundance of probiotics such as Lactobacillus gasseri in the intestine of mice,and at the same time changed the expression of miR-133b-3p and other microRNAs in the small intestine tissue.The PLSR prediction results combined with the co-culture verification results showed that miR-133b-3p significantly inhibits the proliferation of Lactobacillus gasseri.From this we have concluded that stachyose down-regulates the expression of miR-133b-3p,which targets the growth of Lactobacillus gasseri in the small intestine,and increases the abundance of the intestinal probiotic Lactobacillus gasseri,thereby improving the structure of the gut microbiota of mice.