Effect And Mechanism Of MicroRNAs On Prenatal And Postnatal Rats From Maternal Thyroid Hormone Deficiency

Objective:Thyroid hormone（TH）plays a key role in the development of fetal brain.Clinical congenital hypothyroidism or maternal thyroid hormone insufficiency during pregnancy have an effect on fetal neural cell proliferation,differentiation,migration,synaptic development,myelin formation and improved function.Subclinical hypothyroidism（SH）is a common clinical problem.Man and suggested that maternal hypothyroidism was associated with lower intelligence quotients in the offspring.We had found at midgestation,maternal SH were statistically lower at motor and intellectual development.In animal studies,we had found early maternal SH alters cerebral cortex and cell architecture,the cell migration of cerebral cortex,and impairs LTP induction in hippocampus of rats and impairs spatial learning in the offspring.However,we still unkonw the mechanisms of SH during pregnancy influence the neurodevelopment of offspring.MicroRNAs（miRNAs）,endogenous short RNAs and closely related to gene expression,are important regulators of gene expression which were found in recent years.MiRNAs that play an important role in the regulation of the mammalian brain development,from spatial and temporal patterns,fine control the stage-specific brain development,which points out miRNAs as widespread fine-control moleculars of gene expression,regulate the activities of life by network.In this study,we determined the profile of microRNAs expressed in the fetal and neonatal brains from maternal rats with subclinical hypothyroidism,compared to normal rats.To study the molecular mechanism of rat fetus brain development influenced by subclinical hypothyroidism through rapid and efficient micorRNA Chip arrays.Methods:72 female Wistar rats which were kept in a specific pathogen-free condition（body weight from 200 to 220g）were divided into two groups.（1）control group（n=24）:sham operated as a negative control;（2）hypothyroidism group（n=24）:performed with complete thyroidectomy.All blood samples from female rats were collected by puncture of the retro-orbital venous plexa a month after thyroidectomy.Serum TSH and Total thtyroxine（T₄）were measured.The models of hypothyroid rats were established and thyroid surgery completely as serum TT₄ concentrations fell to<1μg/dL.（3）subclinical hypothyroidism group（n=24）:hypothyroidism rats were injected L-T₄ at a dose of 1μg per 100g body weight per day.It last for nine days.Blood samples were collected and sera were analyzed for thyroid function.The models of SH were established as elevated serum TSH level and normal total T₄（TT₄）level.These rats were mated with normal male Wistar rats,embronic day 0（E0）was identified as sperm were found in the vaginal smear by microscopy the next day.Cesarean sections were performed on E13 and E17.Brains of prenatal and postnatal rats at the following ages were collected and frozen in tube at-80℃:E13,E17,postnatal day（P）P0,and P7.The hippocampus and cortex were separated on P7.To reduce the influence of individual variance,three samples were analyzed at each time point.45 brain samples were sent to KangChen Bio-Tech Corporation.miRNA profiles were detected by Exqion miRCURY?LNA.Functional enrichment of Gene Ontology（GO）biological processes and the Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis were also conducted.We analyzed the expression of ten miRNAs（miR-20a,miR-106b,miR-32,miR-335,miR-16,miR-33,miR-124,miR-376a,miR-26a,miR-375）by quantitative reverse transcription-polymerase chain reaction（qRT-PCR）.Total RNAs were isolated from tissues with the kit.miRNAs stem-loop（Stem-loop）primers were designed for reverse transcription,determined quantitatiy by SYBR Green Real-time PCR and U6 snRNA were regarded as internal references.Potential target genes BCL-2,BCL-W were determined by quantitatiy PCR and GAPDH were regarded as internal references.According to the data of hypothyroidism maternal E13 offspring brain miRNA chip compared with control,we selected miR-125b-5p and potential target genes STAT3.We used real-time quantitative PCR to verify.Using target genes prediction software,we found that 3‘UTR of STAT3 had two binding sites with miR-125b-5p,we build rat STAT3 wild type,mutant 1 and mutant 2 luciferase reporter vector and transfected these vector with miR-125b-5p mimics into（HEK）293 cells,48 h later,collected cell lysis solution,detected luciferase activity to understand the inhibition of miR-125b-5p on exogenous target genes,to find the binding sites of miR-125b-5 p with STAT3.miRNA negative control was as negative control.Using anion exchange resin to deleted the thyroid hormones of fetal bovine serum,and cultured the C17.2 neural stem cells.transfected 50 nM、100 nM miR-125b-5p mimics,100 nM、150 nM miR-125b-5p inhibitor into C17.2 neural stem cells for 24h,48h,72h and 96h.Using CCK-8 assay kit to detect the proliferation of miR-125b-5p on C17.2 stem cells.Using real time PCR to detected the mRNA expression of TUBB3 and GFAP.Data Analysis for miRNAs include:low intensity filtering and data normalization,quality assessment of miRNA data after filtering,differentially expressed miRNAs screening and heat map and hierarchical clustering.When Fold change>1.5 or<0.67 and p<0.05,the miRNA was significant different expression miRNA.Other data were presented as mean±SEM and were evaluated by t-test.P<0.05 was considered as statistical significance.Results:Serum thyroid hormone levels in pregnant rats:TT₄ and TSH of Wistar rats in hypothyroidism group,respectively,were significantly lower and higher than the control group at E13,E17,P0 and P7;TSH of rat in subclinical hypothyroidism group were significantly higher than control group and TT₄ of rat in subclinical hypothyroidism had no significant difference with control group.2.miRNA microarray:compared with control group,1.5-fold increased miRNAs from subclincal hypothyroidism fetal/neonatal rat brain tissue were seen in E13:miR-26a、miR-16、miR-130b、miR-335、miR-93;E17:miR-32、miR-17-5p、miR-150、miR-30e;P0:miR-335、miR-376a、miR-181d、miR-30e;P7C:miR-20a、miR-410、miR-376a、miR-101a、miR-124;P7H:miR-33、miR-140.Reduced by 67%or more were seen in E13:miR-375、miR-294;E17:miR-675、let-7b、mir-181a;P0:miR-195*、miR-375、miR-200c*;P7C:miR-200c*、miR-291a-3p、miR-665;P7H:miR-24-1*.Compared with subclincal hypothyroidism group,1.5-fold increased miRNAs from hypothyroidism fetal/neonatal rat brain tissue were seen in E13:let-7e、miR-199a-3p、miR-181a、miR-30e、miR-199a-5p、miR-136、miR-341、miR-124;E17:let-7c、miR-376a;P0:miR-375、miR-24-1*;P7H:miR-483、let-7f-1*.Reduced by 67%or more were seen in E13:miR-125b-5p、miR-451、miR-503;E17:miR-493*;P0:miR-301a、miR-296;P7H:miR-204*、miR-542-3p.3.The validation of ten miRNAs and potential target genes by real time PCR:compared with control group,the expression of modulated miRNA up-regulated were as follows:E13:miR-335（fold change1.5,p<0.05）;E17:miR-106b（fold change 1.25,p<0.05）;P7C:miR-376a（fold change 1.23,p<0.05）;P7H:miR-33（fold change 1.29,p<0.05）;The expression of modulated miRNA down-regulated were as follows:P0:miR-33（fold change 0.67,p<0.05）、miR-124（fold change 0.41,p<0.01）、miR-376a（fold change 0.57,p<0.01）、miR-375（fold change 0.48,p<0.05）.Compared with control group BCL-2 of subclinical hypothyroidism maternal offspring brain was significantly lower in E17（fold change 0.68,p<0.05）,BCL-W was significantly lower in P0（fold change 0.77,p<0.05）.4.The GO analysis results illustrated a broad range of biological processes associated with the predicted target genes.The majority of the predicted target genes were potentially associated with binding(including protein binding and nucleotide binding,localization,positive regulation of biological process,etc.These KEGG data revealed involvement of the brain development-related signaling pathway,such as long-term depression（LTD）,axon guidance,and neurotrophin,as well as other signaling pathways related to the adherens junction,suggesting that these involved signaling pathways may be altered by the regulation of SDE miRNAs in M-SH-related offspring brains.5.Compared with control group,miR-125b-5p in hypothyroidism rat group were significantly lower in E13,the expression was down to 16%;miR-125b-5p expression was down to 68%、56.8%、42.9%by real-time PCR（p<0.05）at E13、P0 and P7C compared with control,respectively.Expression of STAT3 mRNA had no significant difference compared with control group（p>0.05）.The total protein of STAT3 were significantly higher compared with control at E13 and P7C,the fold change were1.5 and 1.33 respectively.6.Dual-luciferase reporter assay showed luciferase activities of wild-type and mutant 2 were reduced in HEK293 cells when co-transfected with miR-125b-5p mimics compared with miRNA negative control（p<0.05）.Mutant 1 co-transfected with miRNA mimics had no significant difference compared with negative control（p>0.05）.7.The expression of GFAP and TUBB3 in thyroid hormone depletion medium were lower than the nomal group.When the C17.2cells were treated with miR-125b-5p mimics,the expression of TUBB3 was up-regulated and GFAP was down-regulated（p<0.05）.8.The total protein expression of STAT3 had no significant difference compared with control group,and the phosphorylation of the protein were significantly higer than normal control group（p<0.05）.Overexpression of miR-125b-5p repressed the phosphorylation of STAT3.9.The data of CCK8 study showed miR-125b-5p had no proliferation effect on C17.2 cells.Overespression miR-125b-5p had no effect on the proliferation.Conclusion:1.We have found the microRNA expression profile of rat offspring brains associated with maternal subclinical hypothyroidism druing pregnancy.We found miR-335 in E13、miR-106b in E17、miR-376a in P7C、miR-33 in P7H、miR-124 and miR-375 in P0 were significant different compared with control group,and they may modulate the process invovled with BCL-2 and BCL-W.2.GO analysis indicates most of the altered miRNA-regulated genes were associated with binding,location and positive biological process regulation.KEGG pathway analysis indicates several important cellular signaling pathways in neurodevelopment,including long-term depression（LTD）,axon guidance,and neurotrophins,were regulated by the altered expression of miRNAs.3.The expression of miR-125b-5p was lower in hypothyroidism maternal offspring brain,the protein of STAT3 were higher in hypothyroidism group.4.miR-125b-5p can bind with the first binding site of STAT3 3’UTR in HEK293 cells.5.miR-125b-5p had no proliferation effect on C17.2 cells and it had a higher expression of TUBB3 and lower expression of GFAP.Overexpression of miR-125b-5p could inhibit STAT3 protein phosphorylation.