The Role Of Mir-23b/27b/24-1 Cluster In Metabolic Abnormalities Associated With Polycystic Ovary Syndrome

Chapter Ⅰ:Expression and correlation of miR-23b/27b/24-1 cluster in women with PCOSObjectivePolycystic ovary syndrome(PCOS)is a complex disorder characterized by sporadic/anovulatory,hyperandrogenic and polycystic ovarian changes,involving multiple endocrine and metabolic changes.Typical clinical manifestations include menstrual sparseness or amenorrhea,infertility,hyperandrogenemia,hirsutism and obesity;and long-term complications include insulin resistance,type 2 diabetes,hypertension and cardiovascular disease.PCOS is a complex disease with significant genetic heterogeneity and phenotypic complexity,which does not follow the Mendelian inheritance and is influenced by multiple micro-effect genes and environmental factors.Our group has identified the 9q22.32 region as a PCOS susceptibility region,which contains several microRNAs(miRNAs).miR-23b,miR-27b and miR-24-1 are three very important ones that occur in clusters in close proximity to each other in the genome(～1 Kb).MiRNAs are involved in a range of important processes,including early embryonic development,cell proliferation,apoptosis,glycolipid metabolism,and tumorigenesis.However,the role and mechanism of miR-23b/27b/24-1 cluster in PCOS has not been reported.Therefore,the aim of this study was to investigate the role of miR-23b/27b/24-1 cluster in the pathogenesis of PCOS.MethodsIn this chapter,we collected peripheral blood from 22 healthy women and 47 women with PCOS and examined the expression of miR-23b,miR-27b and miR-24-1 in the cohort by qRT-PCR.The inclusion criteria were the Rotterdam diagnostic criteria and the control population were healthy women who presented to our hospital for male factors during the same period.After that,the PCOS group was further divided into PCOS normal weight group and PCOS overweight group according to BMI(24 kg/m2)level;PCOS non-hyperandrogenism group and PCOS hyperandrogenism group according to testosterone(60 ng/dL)level respectively.Then the differences between miRNAs in the subgroups and the control population were compared.Finally,Spearman correlation analysis was used to compare the correlation between miR-23b,miR-27b and miR-24-1 and their respective correlation with clinical indicators.ResultsⅠ.miR-23b/27b/24-1 cluster is reduced in the PCOS populationThe PCOS group was age-matched to the control group,while BMI(p=0.001),LH(p<0.001),T(p<0.001)and E2(p=0.007)were higher than the control group.miR-23b was significantly lower(p=0.020),miR-27b tended to be lower(p=0.200)and miR-241 was significantly lower(p=0.009)in PCOS.There were no significant differences in miR-23b,miR-27b and miR-24-1 expression in subgroups differentiated either by BMI or by testosterone levels.Ⅱ.Correlation between miR-23b/27b/24-1 clustersmiR-23b,miR-27b and miR-24-1 occur in clusters in the genome,and the same expression pattern exists for all three of them in the present study population.There was a strong correlation between miR-23b,miR-27b and miR-24-1 in the control population,the PCOS population and the total population(r=0.8-0.97).Ⅲ.Correlation of miR-23b/27b/24-1 cluster with clinical indicatorsmiR-23b was positively correlated with TC in the control population(r=0.481,p=0.043).miR-23b was not found to be correlated with clinical indicators in the PCOS population,but miR-23b was significantly negatively correlated with T(r=-0.292,p=0.015)and E2(r=-0.262,p=0.03)in the total population.miR-27b was not found to correlate with clinical indicators in either the PCOS population or the total population,but miR-27b was significantly positively correlated with TC in the control population(r=0.614,p=0.007)and also significantly negatively correlated with fasting glucose(r=0.489,p=0.046).miR-24-1 correlated with clinical indicators in combination with miR23b and miR-27b patterns.miR-24-1 was positively correlated with TC in the control population(r=0.484,p=0.042)and also negatively correlated with fasting glucose(r=0.546,p=0.023),while in the total population miR-24-1 was negatively correlated with T(r=-0.305,p=0.011)and E2(r=-0.316,p 0.008).ConclusionThe expression of miR-23b/27b/24-1 cluster was reduced in women with PCOS,but the difference was not significant in subgroups.Further correlation analysis showed that this cluster correlated with TC,FBG,T and E2.Based on this,we speculate that the miR-23b/27b/24-1 cluster may be involved in the regulation of glucolipid metabolism and hormone metabolism,and therefore we explore it’s in vivo function by constructing knockout mice.Chapter Ⅱ:Exploring the reproductive and metabolic phenotypes of miR-23b/27b/24-1 cluster knockout miceObjectivePolycystic ovary syndrome(PCOS)is a heterogeneous disorder involving multiple alterations in reproduction and metabolism.Both ovarian and extra-ovarian factors have been implicated in the pathogenesis of PCOS,but which are the initiating factors and which are secondary alterations remain to be determined.The previous chapter found that miR-23b/27b/24-1 cluster expression was reduced in women with PCOS,correlating with TC,FBG,T and E2.This suggests that this cluster may play a role in the pathogenesis of PCOS.Therefore,in this chapter we explore the reproductive and metabolic effects of miR-23b/27b/24-1 cluster in knockout mice.MethodsKnockout mice were constructed using CRISPR-Cas9 technology and knockout efficiency was verified by detection of mature miRNAs as well as genome sequencing.Phenotypes were explored by both normal chow diet(NCD)and high-fat diet(HFD).In terms of reproduction,the effect of miR-23b/27b/24-1 cluster elimination on puberty initiation was investigated by observing vaginal opening time;the effect of miR23b/27b/24-1 cluster deletion on ovaries was investigated by H&E staining of ovaries in different states and superovulation assays.In terms of metabolism,the effects of miR23b/27b/24-1 cluster deletion on glucose metabolism were investigated by performing glucose tolerance tests(GTT),pyruvate tolerance tests(PTT)and insulin tolerance tests(ITT)on knockout mice in different states.The effects of miR-23b/27b/24-1 cluster ablation on lipid metabolism were investigated by enzymatic catabolism of triglyceride(TG)and total cholesterol(TC)levels in serum and liver tissues as well as by H&E and Oil-Red O staining.ResultsⅠ.miR-23b/27b/24-1 cluster knockout female mice showed no significant abnormalities in growth and developmentKnock-out mice(KO)showed no significant abnormalities in growth and development,and the proportion of offspring born was in accordance with Mendelian inheritance.Adult females were able to give birth normally,with no significant abnormalities in gestation frequency or number of pups per litter.No significant abnormalities were found in ovarian morphology at puberty(PD 4w),adulthood(PD 12w)and with HFD(12w).The ovulation test revealed no statistical difference in the number of eggs obtained as well as the rate of first polar body expulsion in KO females compared to wild-type(WT)controls.Ⅱ.miR-23b/27b/24-1 cluster affects glucolipid metabolism in female miceAdult KO female mice exhibit impaired glucose tolerance,a phenotype that becomes more pronounced with ageing and with the stress of HFD.Fasting glucose was significantly lower in KO females in the NCD state,whereas this change became non-significant in the HFD state.There was no significant difference in random blood glucose between the two groups of mice.Further PTT experiments showed that pyruvate utilization in KO females was reduced in both the NCD and HFD states,while insulin sensitivity was not significantly different.There were no significant differences in either blood or liver lipids under NCD status;however,the increase in liver lipids in KO mice under HFD status was significant,accompanied by significant alterations in key genes in the TC and TG metabolic pathways.Ⅲ.Impaired glucose tolerance in miR-23b/27b/24-1 cluster knockout male miceThere was no significant difference in body weight between WT and KO male mice.Under HFD status,there was a trend towards higher body weight and slightly higher subcutaneous and perigonadal fat weights in KO males.GTT results indicated that glucose tolerance was impaired in KO males and that this impairment was more severe after HFD stress.Random blood glucose levels were not significantly different between KO and WT mice,regardless of dietary conditions,and insulin sensitivity was not significantly different.In addition,insulin levels after glucose stimulation,and TG and TC levels were not significantly altered.Metabolic cage results showed no significant differences in VO2,VCO2,thermogenesis and XTOT,but the RER was significantly lower in KO male mice.In the HFD state,there was a trend towards increased liver TC and TG in KO males,and ORO staining also revealed increased hepatic steatosis in KO males,but not as pronounced as in females.ConclusionThe knockout mouse model revealed that the miR-23b/27b/24-1 cluster has less effect on reproduction and more effect on metabolism.miR-23b/27b/24-1 cluster knockout female and male mice exhibited different degrees of reduced glucose tolerance and hepatic steatosis.Chapter Ⅲ:Mechanistic studies on the role of miR-23b/27b/24-1 cluster in liver metabolismObjectiveMetabolic abnormalities are one of the important clinical features of polycystic ovary syndrome(PCOS).Studies of male relatives of women with PCOS have introduced the concept of male PCOS,suggesting that PCOS is not always a primary disease of female reproduction.Our study in the previous chapter found that the effects of miR-23b/27b/24-1 cluster knockout mice were not significant in terms of reproduction,but rather altered significantly in terms of glucolipid metabolism.Metabolic studies usually use males as subjects,and females have been discarded in large part because of the instability of the results.The liver is the main metabolic organ of the body and transcriptome sequencing has identified up to 72%of sex-dependent differential genes in the liver.Therefore,in this chapter we expect to explore the causes of impaired glucose tolerance due to deletion of the miR-23b/27b/24-1 cluster by combined multi-omics analysis and to explore its effects on liver metabolism in female and male mice respectively.MethodsChanges in mRNA levels and protein levels of key genes were detected by qRTPCR and Western blot.Non-targeted metabolomics and transcriptome sequencing were used to detect the effects of miR-23b/27b/24-1 cluster on metabolites and mRNAs in the liver.Steroid hormones and sulphated steroid hormones were measured by mass spectrometry.MetaboAnalyst 5.0(https://www.metaboanalyst.ca/)was used for metabolomics enrichment analysis and transcriptomic conjoint analysis.Transcriptomics enrichment analyses were performed using the Reactome online platform(https://reactome.org).Images were drawn using R or Hiplot(https://hiplot.com.cn/)or GraphPad Prism 7 software.ResultsⅠ.Impaired glycolysis and reduced PKLR in miR-23b/27b/24-1 cluster knockout miceG6PC and PCK1 are key enzymes involved in gluconeogenesis,while GCK and PKLR are key enzymes involved in glycolysis.G6pc,Pckl and Gck showed a decreasing trend in KO female mice,while Pklr was significantly decreased.With the stress of HFD,G6pc,Pck1 and Pklr all showed a significant decrease.Protein levels of PKLR,which is responsible for catalyzing phosphoenolpyruvate(PEP),an irreversible rate-limiting step in glycolysis,were also significantly reduced in knockout males.Differential metabolite-based enrichment analysis showed that Glycolysis,Warburg Effect and Pyruvate Metabolism pathways were significantly enriched and levels of reduced nicotinamide adenine dinucleotide(NADH),nicotinamide adenine dinucleotide(NAD),phosphate and the intermediate product PEP were all reduced to varying degrees in the glycolysis pathway.Ⅱ.Male mice with miR-23b/27b/24-1 cluster ablation had elevated retinoic acid and cortisolThe results of the joint transcriptomic and metabolomic analysis of male livers showed that Retinol metabolism and Steroid hormone biosynthesis ranked first and second in the enrichment results,with the main differential metabolites being retinoic acid(RA,log2 FC=0.34),cortisol(log2 FC=1.16)and desoxycortone(log2 FC=0.94).Elevated cortisol may be involved in the phenotype of impaired glucose tolerance in KO male mice by decreasing glucose utilization.Ⅲ.miR-23b/27b/24-1 cluster affects the sulphation of steroid hormones in female miceDifferential metabolite enrichment analysis in the liver of female mice revealed significant alterations in the steroid hormone anabolic pathway,with significant reductions in androstenedione(Δ4A),dehydroepiandrosterone(DHEA),and testosterone(T).Further joint transcriptomic and metabolomic analyses also revealed a significant enrichment of the steroid synthesis pathway.Both the liver proteome and transcriptome revealed a significant increase in SULT2A1(encoded by Sult2a1,which mediates the conversion of DHEA to DHEA-S),a result also supported by qRT-PCR of expanded samples.This implies that the reduced DHEA in KO female mice may be due to enhanced sulphation.Validation by targeted mass spectrometry of serum revealed that DHEA-S was indeed elevated in serum,in addition to an increase in Epitestosterone-S and Testosterone-S.Finally,analysis of an expanded sample revealed a significant increase in serum DHEA-S in patients with PCOS and a significant negative correlation with miR-23b.ConclusionPKLR,a key enzyme in the glycolytic pathway,was reduced with miR23b/27b/24-1 cluster ablation.The miR-23b/27b/24-1 cluster affects glucose homeostasis through the glycolytic pathway.Combined multi-omics analysis showed that Retinol metabolism and Steroid hormone biosynthesis were significantly altered.However,in male mice,the miR-23b/27b/24-1 cluster mainly showed an increase in RA and cortisol,whereas in females,the miR-23b/27b/24-1 cluster may regulate the sulfation of DHEA through SULT2A1,which in turn affects the synthesis and metabolism of steroid hormones.