Mechanism Study On SIDT1-dependent Food-derived Micro RNA Absorption SIDT1-dependent Absorption In The Stomach Mediates Host Uptake Of Dietary And Orally Administered Micro RNAs

In 2012,Zhang Lin and others discovered that exogenous plant mi RNAs exist in mammalian serum and other tissues.Furthermore,exogenous plant mi R168a can bind to human or mouse low-density lipoprotein receptor adapter protein 1(Ldlrap1)m RNA,inhibitting the expression of LDLRAP1 in the liver,thereby regulating the content of LDL cholesterol in the blood.More and more studies have shown that intact plant mi RNAs in food can be absorbed through the gastrointestinal tract(GI-tract)of mammals,cross kingdom regulating mammalian gene expression.However,the mechanism of how mi RNAs in food is completely absorbed through the mammalian GI-tract to enter the circulatory system and other tissues of the body remains unclear.In Caenorhabditis elegans,Systemic RNA Interference Deficiency(SID-1)has the function of transporting extracellular double-stranded RNA(ds RNA)into the cell.Studies have shown that SID-1 may have 11 transmembrane domains and function as a multimer.SID-1 can transport ds RNA of different lengths and molecules with partial double-stranded RNA structures(such as mi RNA precursors and hairpin RNA).SID-1transmembrane family member 1(SIDT1),as a homologous protein of SID-1 in nematodes in mammals,is also located on the cell membrane and mediates the transfer of extracellular small interfering RNA(si RNA)and mi RNAs between connected cells.Based on the above findings,we put forward the hypothesis:SIDT1 mediates the uptake of exogenous mi RNAs in mammals.In this study,we studied Sidt1^-/-mice.On Sidt1^-/-mice,the results of high-throughput sequencing and quantitative PCR showed that the background level of plant mi RNAs in their bodies was significantly reduced,and their ability to dynamically absorb plant mi RNAs in food was also significantly lower than that of wild-type mice.It indicates that the absorption of exogenous mi RNAs in food or oral administration in mice is SIDT1-dependent,that is,SIDT1 protein is necessary for the absorption of exogenous mi RNAs in the digestive tract of mammals.Furthermore,we found that SIDT1 protein is mainly expressed in the stomach of mice,and further experimental results show that SIDT1 protein is located on the cell membrane of the pit cells of the gastric mucosa.We performed pyloric ligation on mice,and found that after the operation,the absorption of gavage mi RNAs in wild-type mice was not significantly different from that in the unligated group,while SIDT1 knockout mice had no significant absorption.Pylor ligation experiments show that the stomach is the first and primary organ for mice to absorb mi RNA from food.In vitro,we cultured primary gastric mucosal epithelial cells and simulated the acidic environment of the stomach.We found that acidic stimulation significantly enhanced the absorption of mi RNA molecules in the culture medium by gastric mucosal epithelial cells,while Sidt1^-/-mice-derived gastric mucosal epithelium Cells have no obvious absorption of mi RNA molecules in the culture medium,and acidic stimulation cannot increase their absorption of mi RNA molecules in the culture medium.This suggests that in the digestive tract,gastric acidic environment is essential for SIDT1-dependent mi RNA absorption.Using in vitro cell model,we extracted exosomes and analyzed the plant mi RNA content,finding that the plant mi RNAs absorbed by primary gastric mucosal epithelial cells derived from wild-type mice under acidic conditions can be released into the culture medium through exosomes.in.The luciferase report experiment constructed in vitro proved that the plant mi RNAs in these exosomes also has biological functions.Finally,using bioinformatics methods,we found that mi R2911 may target human or mouse transforming growth factor?1(TGF-?1)gene.TGF is Considered to be the key molecule.Using mouse models of carbon tetrachloride(CCl₄)induced liver fibrosis,we tried to down-regulate the TGF-?1 gene by oral plant mi R2911 to remedy liver fibrosis.After 4 weeks of oral mi R2911 treatment,plant mi R2911 increased significantly in wild-type mouse liver tissues,and TGF-?1 content in liver tissues decreased significantly.Pathological studies have shown that several indicators of liver fibrosis,including hydroxyproline,?-SMA,Collegen-I and Sirius Red staining showed significant improvement.In Sidt1^-/-mice,after 4 weeks of oral mi R2911,plant mi R2911 did not increase significantly in mouse liver tissue,and the content of TGF-?1 in liver tissue did not decrease significantly.After intravenous injection of mi R2911,the plant mi R2911 bypassed the digestive tract and so was absorbed in the liver tissues.The content of mi R2911 in the Sidt1^-/-mouse liver was significantly increased,and the content of TGF-?1 was significantly reduced,as was in the wild-type mouse liver.Pathological studies showed that several indicators of liver fibrosis,including hydroxyproline,?-SMA,Collegen-1 and Sirius Red staining all showed significant improvement.The above in vivo experiments show that SIDT1 is necessary for the absorption of oral mi RNA.In summary,our research results clarify the possible molecular mechanism of mammalian absorption of exogenous mi RNA through the digestive tract for the first time,that is,SIDT1 protein mediates the absorption of exogenous mi RNA in mammals;in addition,we have discovered a new function of the stomach,the stomach Direct absorption of exogenous mi RNA in food;low p H promotes the absorption of exogenous micro RNAs mediated by SIDT1,which explains why mammals can completely absorb and utilize exogenous micro RNAs from the biochemical point of view;in addition,oral micro RNAs can be passed through SIDT1 mediated absorption provides a potential direction for small RNA-based drug development.