Discovery And Mechanism Investigation Of MiR-552-3p In Improving Glycolipid Metabolism Disorders

Glycolipid metabolism disorders are a type of diseases that can cause systemic organs damage and dysfunction due to abnormal metabolism of glucose and lipids.Hyperglycemia,hyperlipidemia,liver lipid accumulation and insulin resistance,etc are the main clinical symptoms of these diseases.With increasing incidence of glycolipid metabolism disorders,it has become a serious threat to people.However,their pathogenesis is still unclear,resulting in the lack of effective treatment drugs.As important regulatory molecules in many physiological processes,microRNAs have been increasingly discovered for their role in glycolipid metabolism disorders.To study the roles of microRNAs will be helpful to explore the pathogenesis and drug development of glycolipid metabolism disorders.In our previous studies,we found miR-552-3p regulated the expression of CYP2E1 by dual mechanism.Further in-depth sequence analysis of miR-552-3p revealed that there was a similar sequence with the response element of nuclear receptor superfamily1(NR1)in miR-552-3p.Based on the function of NR1 in regulating metabolism related genes,we raised our scientific question--The relationship between miR-552-3p and glycolipid metabolism and its role in glycolipid metabolism disorders.At present,the biological function of miR-552-3p is mostly concentrated in tumor,and its regulatory effect on glucose and lipid metabolism has not been touched.On these bases,we conducted a series of in vivo and in vitro experiments to investigate the function and mechanism of miR-552-3p in regulating glycolipid metabolism.The discovery of miR-552-3p regulating metabolism-related gene expression: Using RNA-seq and KEGG analysis,we found 1,529 genes were regulated by miR-552-3p,in which down-regulated genes were more closely related to metabolism.At the same time,we reduced the content of endogenous miR-552-3p in HepG2 cells using miR-552-3p inhibitor and detected the metabolism-related genes expression.The results showed that endogenous miR-552-3p had the ability to regulate the expression of metabolism-related genes.The discovery of miR-552-3p improving glycolipid metabolism disorders: We established the mouse model of glycolipid metabolism disorders with high-fat and high-fructose diet,and overexpressed miR-552-3p in the liver of model mice or control mice(normal diet)by liver-specific adeno-associated virus to evaluate the function of miR-552-3p in vivo.Subsequently,we confirmed its ability to regulate glucose metabolism in 8-week-old db/db mice.(1)In terms of dyslipidemia,we found that miR-552-3p reduced the content of serum TC,TG and LDL-c in HFHFr mice,whereas had no effect on the content of HDL-c.In control mice,the miR-552-3p group had a litter higher LDL-c level and had no effect on TC,TG and HDL-c.Meanwhile,the results of the OLTT assay showed that the miR-552-3p group can enhance lipid metabolism in both HFHFr and control mice.(2)In terms of hepatic lipid,we found that miR-552-3p overexpression could significantly reduce the vacuoles in liver through H&E staining assay and the areas stained by oil red O staining,indicating that miR-552-3p inhibited liver lipid accumulation caused by high fat and fructose diet.Additionally,the concentration of TG and TC in liver was significantly reduced in miR-552-3p overexpression mice.To further confirm the effect of miR-552-3p on lipid metabolism,the primary hepatocytes was used and the results showed that primary hepatocytes from the mice overexpressed miR-552-3p had stronger resistance to lipid accumulation caused by FFA.(3)In terms of glucose metabolism disorders,overexpression miR-552-3p averted the increase of fasting blood glucose in HFHFr mice and db/db mice.OGTT assay showed that miR-552-3p could improve glucose tolerance in HFHFr mice and db/db mice.Further,we found that miR-552-3p alleviated insulin resistance and repaired insulin signaling pathway in liver through the detection of HOMA-IR score,ITT and in vivo insulin signaling assay.The results from in vivo PTT assay and in vitro glucose output assay showed that miR-552-3p could inhibit hepatic gluconeogenesis.(4)Finally,the effect of miR-552-3p on whole-body metabolic rate and energy balance was detected through metabolic chambers.The experimental results showed that miR-552-3p increased the oxygen and heat consumption,voluntary activity in HFHFr mice and had no influence on control mice.In summary,miR-552-3p regulated glycolipid metabolism and metabolic rate in mice with glucose and lipid metabolism disorders caused by many different inducements.The discovery of miR-552-3p regulating FXR and LXR? transcriptional activity: As reported,miR-552-3p regulated gene expression both in the cytoplasm and nucleus.Based on the function of NR1 in transcriptional regulation,we speculated that miR-552-3p plays a role in regulating metabolic gene expression in the nucleus.We used siRNA to knock down the expression of importin8,which is in charge of the transport of mature microRNA into the nucleus,to reduce the content of intranuclear miR-552-3p,and found that miR-552-3p regulated metabolic genes expression mostly in the nucleus.Next,we found LXR/RXR and FXR/RXR pathways might be the major contributors to the function of miR-552-3p in metabolic regulation via IPA analysis of differentially expressed genes in RNA-seq and protein mass spectrum.Furthermore,we demonstrated that miR-552-3p could dose-dependently inhibit LXR? transcriptional activity and activate FXR transcriptional activity through nuclear receptor transcription activation experiments.Finally,using Stigmasterol(FXR antagonist)and GW3965(LXR agonist)could reverse the regulatory function of miR-552-3p in FXR and LXR? transcriptional activities.The mechanism of miR-552-3p regulating the transcriptional activity of FXR and LXR?: Given the ability of miR-552-3p in binding to DNA,we tested the interaction between miR-552-3p and FXR or LXR? response elements(IR1,DR4).With DNA Pull Down and FRET experiments,we proved that miR-552-3p could bind to the antisense sequence of DR4 and the sense or antisense sequences of IR1.According to sequence analysis,we designed the mutations of miR-552-3p and performed related experiments to demonstrate that the AGGTCA-like sequence of miR-552-3p was important to its role in regulating the transcriptional activity of FXR and LXR?.Then using EMSA and ChIP assay,we confirmed that the combination of miR-552-3p and NR1 response elements affected the binding affinity between transcription factors and their response elements,thereby regulating the transcriptional initiation of downstream genes.In addition,we demonstrated that miR-552-3p regulated gene expression at the transcription level in vivo by detecting gene expression in liver and ex vivo ChIP assay.Finally,Stigmasterol and GW3965 were used to prove miR-552-3p restored glycolipid disorders by influencing FXR and LXR?.In conclusion,we found that miR-552-3p modulated FXR and LXR? transcriptional activities by binding to their response elements,thereby regulating the expression of downstream genes and improving the glycolipid metabolism disorders through in vivo and in vitro experiments.In my thesis,we first discovered the function of miR-552-3p in improving glucose and lipid metabolism disorders.At the same time,we first proposed a new mechanism for microRNA to regulate gene expression through nuclear receptor.These discoveries provide a novel mechanism of microRNAs regulating gene expression,supplies a new evidence for microRNAs participating in glucose and lipid metabolism,and gives a probable direction for the development of drugs against glycolipid metabolism disorders.