| Background:Subclinical hypothyroidism(SCH)is a group of syndromes caused by multiple etiologies.It is characterized by serum free thyroxin(FT4)levels within the reference range,only serum thyroid-stimulating hormone(TSH)levels above the reference range.The epidemiological survey showed that the prevalence of SCH had been increasing year by year in recent years,with the prevalence ranging from 4-20%in western developed countries.While the prevalence is higher in Asian countries,reaching 17%to 20%.Although SCH has mild or no obvious clinical manifestations,SCH is often associated with dyslipidemia and it is an independent risk factor for coronary heart disease and non-alcoholic fatty liver disease(NAFLD).However,the molecular mechanism of SCH related lipid metabolism is unclear.Liver played a key role in the process of lipid metabolism and is in the center place of lipid metabolism,so the liver can be used as a target for the study of lipid metabolism dysregulation in SCH.MicroRNAs(miRNAs),are small(21-23 nucleotides in length)non-coding RNAs.They interact with 3 ’untranslated region(3’ UTR)of mRNA in incomplete or complete complementary way to inhibit translation or degrade mRNA.An increasing number of evidence supported that miRNAs regulated various hepatic diseases including NAFLD and affected hepatic pathophysiological environment.The species and expression level of miRNAs are tissue specific and specific miRNA reflects some specific pathophysiological processes.However,there is no study of miRNAs in the process of lipid metabolism disorders in SCH.Proteomics studies the systematic analysis and recording of proteins expressed in specific organisms or specific tissues within a given time period.Proteomics studys the composition and function of all proteins encoded by the genome in different time and space and reveals the basic rules of life activity at the overall protein level of cells.It can help us obtain a comprehensive and intensive understanding of regulation network and pathophysiology process.As the development of genomics research,proteomics has become an extremely important part of the life science field.Proteomics is also widely used in the study of lipid metabolism.Isobaric Tags For Relative And Absolute Quantitation(iTRAQ)analysis results are sensitive and reliable.Also,it can give the molecular weight and rich structural information of each protein at the same time.Meanwhile,LC-MS/MS detection has high sensitivity and low boundary.It is a very good research method of this subject.In recent years,more and more studies have demonstrated lipid metabolism regulation of miRNA can be realized through a large regulatory network.Proteomics has been widely used to identify and quantify proteins regulated by miRNA.However,the characteristic of lipid metabolism related miRNA expression profile and proteome has not yet been studied in SCH mice.Objective:This experiment is intended to explore the expression profile of hepatic lipid metabolism related miRNAs and proteome changes,establish a module between miRNAs and proteins using TargetScan and miRanda for prediction by in-depth bioinformatics analysis of the differentially expressed miRNAs and screened proteins in SCH mice and obtain a more comprehensive understanding of the development of liver lipid metabolism disorders in SCH mice.Methods:1.Construction of SCH mouse model;Male C57BL/6 mice(aged 7 weeks)were randomly allocated into two groups.SCH group and control group(CON)received methimazole(MMI,0.08 mg/kg BW/d)in water and the corresponding volume of water for 16 weeks,respectively.Serum TSH level in serum was detected by Elisa.Serum free thyroid hormone in mice(FT4)level was detected using the I125 labeling method of radioimmunoassay(RIA).The liver function indexes of Alanine Transaminase(ALT)and Aspartate Transaminase(AST)were determined to exclude the drug side effects on liver.2.Serum total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were detected by automatic biochemistry analyzer in Shandong Provincial Hospital endocrinology laboratory.The content of TG and TC in liver were assayed using assay kits following the manufacturer’s protocol(Applygen Technologies,Beijing,China).Oil red O staining was used to determine hepatic lipid accumulation.3.Detection of:miRNA expression in the liver of mice by quantitive PCR(qPCR).4.ITRAQ reagent was used to mark the enzyme hydrolyzed protein combined with LC-MS/MS technique to isolate liver protein.The software ProteinPilot 4.5 was used to obtain quantitative protein.The fold-change of SCH/CON protein expression above 1.80 or below 0.56 were deemed up-regulated or down-regulated threshold.5.The biological functions and the signaling pathways of different expression of liver proteins were analyzed by PANTHER and DAVID classification system.6.The agglomerative hierarchical clustering analysis of miRNA profile and differentially expressed proteins were performed by Cluster version 3 and Java Treeview 1.6.7.TargetScan and miRanda were used to find the targets of miRNA in the different expression of liver proteins.The results were overlapped and established miRNA-protein regulatory module.Results:1.SCH mouse model was successfully constructed.The clinical characteristic of SCH is elevated serum TSH levels while normal serum FT4 levels.When using MMI(0.08 mg/kg/d)for 16 weeks,the SCH mice exhibited normal serum FT4 levels(P>0.05)and higher TSH levels(P<0.01)compared with control mice.The above changes accord with the clinical features and diagnostic criteria of SCH.2.Dyslipidemia and Hepatic lipid metabolic disorders in SCH mice.Serum lipid profiles(TG,TC and LDL-C)in SCH mice increased significantly relative to controls(P<0.05),but there was no di口erence in serum HDL-C between two groups(P>0.05).Hepatic TG and TC contents of SCH mice also increased notably than control mice(P<0.05).Simultaneously,more lipid droplet accumulation was observed in the liver of SCH mice by oil red 0 staining.3.17 selected microRNAs expression profile.MiR-10b-5p,miR-24-3p,miR-29a-3p,miR-30b-5p,miR-34a-5p,miR-125b-5p,miR-130a-5p and miR-199a-5p were significantly down-regulated in SCH mouse livers(P<0.05).Hierarchical clustering analysis based on the 17 miRNAs could separate a majority of subjects of each group.Eight of 9 SCH mice were clustered together;Six of 7 CON mice were clustered together.4.Functional analysis of proteins identified by proteome.ITRAQ labelled quantitative proteomics study showed that a total of 1969 proteins were quantified in SCH and control group.The fold-change of SCH/CON protein expression above 1.80 or below 0.56 were deemed significant.36 proteins displayed significant differences in SCH mice compared to the controls,22 of these proteins were up-regulated while 14 proteins displayed reduced expression.The Gene Ontology(GO)analysis shows that most of the proteins are associated with“metabolic”and“cellular processes”and are binding proteins in cells and organelles or proteins with catalytic activity.KEGG analysis showed that most of the identified proteins were involved in the regulation of multiple pathways,such as Alzheimer’s disease,oxidative phosphorylation,Parkinson’s disease and NAFLD.Based on differentially expressed proteins hierarchical clustering showed the two groups had remarkable separation,indicating that liver proteins expressed in SCH mice are distinct from those in CON mice.5.MiRNA-protein regulatory analysis.In order to explore the interrelation between altered miRNAs and proteins in the liver of SCH mice,we established a module between miRNAs and proteins using TargetScan and miRanda for prediction.The regulatory module contains 3 miRNAs,5 proteins and 5 miRNA-protein connections.Aldh3a2 and Txn are targets of miR-34a-5p;Eeflb and Psap are targets of miR-130a-3p;MiR-24-5p targets Selenbp2.The results suggest that these miRNAAs and proteins may participate in the progression of lipid metabolism disorders in SCH mice.Summary and conclusions:1.SCH mice displayed dyslipidemia and liver lipid metabolism disorders.2.MiRNA profile in SCH mice liver changed compared to controls.Down regulation of miR-10b-5p,miR-24-3p,miR-29a-3p,miR-30b-5p,miR-34a-5p,miR-125b-5p,miR-130a-5p and miR-199a-5p may be a manifestation of lipid metabolism disorders.3.MiR-34a-5p/Aldh3a2,miR-34a-5p/Txn,miR-130a-3p/Eeflb,miR-130a-3p/Psap and miR-24-5p/Selenbp2 regulation provides the molecular basis for the pathogenesis of lipid metabolism disorders in SCH. |
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