Roles Of MiRNA In The Treatment Of Lipid Metabolism Disorder By Polygonatum Kingianum

Object:Polygonatum kingianum Coll.et Hemsl is a Yunnan medicinal herb and plays an important role in the prevention and treatment of lipid metabolism disorder.Previous research found that polysaccharides from Polygonatum kingianum can enhance insulin sensitivity and reduce insulin resistance in rats;reduce serum TC and TG levels in type 2 diabetic rats induced by high-fat diet and streptozotocin;The expression level of muR-122 in mice;and it has a regulatory effect on intestinal microbes in rats with lipid metabolism disorder.In recent years,researchers discovered a complex and interesting association between gut microbes and miRNAs(microRNAs): miRNAs affect bacterial growth by entering bacteria and regulate bacterial gene expression;miRNAs are critical for maintaining gut microbiota.However,it is not known which specific miRNAs are involved in the treatment of lipid metabolism disorder by Polygonatum kingianum.Based on this,we administered the extract of Polygonatum kingianum in the lipid metabolism disorder rats,using high-throughput sequencing technology to identify differentially expressed miRNAs,predict new miRNAs and Target gene,enrichment analysis to find biological processes and signaling pathways related to lipid metabolism disorder-related target genes;correlation analysis to find correlation between differentially expressed key miRNAs and intestinal microbial abundance;intestine Caco-2 cell level to verify the regulation of some key miRNAs and their target genes.This study aim to elucidate the role of miRNAs in the improvement of lipid metabolism disorder by Polygonatum kingianum.Methods:A rat model of lipid metabolism disorder was constructed,and the total polysaccharide from Polygonatum Kingianum(PS),high molecular weight polysaccharides(>100KDa)from Polygonatum kingianum(PSF),and water extract from Polygonatum Kingianum(PWE)with different molecular weights were administered for intervention for 14 weeks.By the end of the experiment,the rat liver was subjected to high-throughput sequencing to identify changes in miRNA,predict new miRNAs,target genes,and enrich the biological processes and signaling pathways of target genes.The 16 S rDNA V4 hypervariable region assay was used to analyze the diversity index of fecal samples,and the correlation analysis was used to find the correlation between differentially expressed key miRNAs and intestinal microbial abundance.Intestinal Caco-2 cells were given water extract from Polygonatum Kingianum and Diosgenin to observe the expression changes of miRNA and its target genes and overexpression/inhibition of miRNA,and then further administrated water extract from Polygonatum Kingianum and Diosgenin to observe miRNA and its target gene Expression changes.Results:By comparing the expression levels of miRNAs in the model group and the normal group and each experimental group,613 known miRNA matures and 438 known miRNA precursors were identified;135 differentially expressed miRNAs were screened,70 of which were up-regulated 65 down-regulation,some miRNAs have been reported to play a key regulatory role in the pathogenesis of lipid metabolism disorder;137 new miRNA matures and 142 new miRNA precursors were predicted.11 miRNAs with high reproducibility and significant difference were selected for qRT-PCR validation,and the expression trend was consistent with high-throughputsequencing results,which proved that high-throughput sequencing results were reliable.Target gene prediction was performed on the known and new miRNAs obtained by the analysis,and a total of 16038 target genes were obtained.Further analysis of the biological role of miRNA target genes revealed that most of the target gene aggregation has a wide range of biological functions,but most of them are involved in metabolic pathways such as sugar,fat and nucleotide.At the same time,the enrichment map showed that the target genes of candidate miRNAs are mainly enriched in three types of signaling pathways: type 2 diabetes,insulin secretion(Insulin secretion)and Glycerolipid metabolism.We performed a correlation analysis between significantly altered miRNAs and intestinal microbes with significant changes in abundance.It was found that miR-484 has a correlation with many intestinal microbes such as Bacteroides,Roseburia,Ruminococcus,Blautia,Prevotella,Christensenellaceae,Coprococcus,etc,especially with Roseburia.At the same time,miR-204-5p was positively correlated with Helicobacter and negatively correlated with Lactobacillus and Psychrobacter.miR-15a-3p was positively correlated with Lactobacillus and Psychrobacter.On the other hand,the results also showed that changes in genus such as Lactobacillus were significantly associated with changes in multiple miRNAs.Lactobacillus was positively correlated with miR-15a-3p and negatively correlated with miR-148a-5p,miR-27b-5p,miR-204-5p and miR-6329.Functional verification of miR-122 and its target genes showed that both water extract from Polygonatum Kingianum and Diosgenin can reduce the expression of miR-122 and its target genes;overexpression/inhibition of miR-122,it was found that miR-122 and its target genes have a negative relationship between regulation and regulation;the administration of Polygonatum kingianum water extract and diosgenin can reduce the expression of miR-122 target gene.Polygonatum kingianum water extract and main component diosgenin candown-regulate the expression of miR-184 and its target gene,and up-regulate the expression of miR-378 b and miR-484.After the corresponding mimic/inhibitor was administered,the Polygonatum kingianum water extract and diosgenin were administered,and similar results to miR-122 were obtained.Conclusion:Polygonatum kingianum has a regulatory roles on miRNA and its target genes in lipid metabolism disorder rats,and regulates the body through miRNA-target gene mechanism to improve lipid metabolism disorder.It also regulates intestinal microbes in rats with lipid metabolism disorder.The interaction of Polygonatum kingianum on intestinal flora and miRNA will provide more research ideas for the treatment of lipid metabolism disorder.