Potential Role And Mechanism Of Liver MiR-361-5p/miR-125a-5p Dysregulation In LOH Induced By Testosterone Deficiency

Part ?:Correlation between testosterone and miR-361-5p/miR-125a-5p levelsObjective:To investigate the correlation between miR-361-5p/miR-125a-5p levels and total testosterone/free testosterone concentration in LOH population and animal model.Methods:In clinical trial,elderly men were recruited for a 6-month testosterone supplementary therapy with testosterone undecanoate.Fasting blood were collected before treatment(0 month)and at the end of 3th and 6th month after treatment(3,6 month).15 participants with increased total and/or free testosterone were included for analysis.In animal experiments,male C57/BL6 at 8-10 weeks of age were used for control(n=10),castration(n=10)and testosterone supplementary therapy(testosterone propionate was injected weekly at 50 mg/kg in 100 ?l seam oil for 4 weeks,n=10).At the end of each experiment,all plasma and serum samples were collected for assessment of testosterone concentration and miR-361-5p/miR-125a-5p levels.Finally,the correlation between miR-361-5p/miR-125a-5p levels and total testosterone/free testosterone concentration in LOH population and animal model were analyzed by appropriate statistical methods.Results:After six-month testosterone supplementary therapy,serum total testosterone at 3 th and 6th month were significantly higher than 0 month(p<0.05),serum miR-361-5p/125a-5p levels(2-?Ct)at 6th month also significantly higher than 0 month(p<0.05).Total and free testosterone concentrations were significant correlated with miR-361-5p/125a-5p levels(r=0.650,0.569 for total testosterone and miR-361-5p/125a-5p,r=0.578,0.552 for free testosterone and miR-361-5p/125a-5p respectively,p<0.01).Similarly,in animal experiments,testosterone concentration after castrated one month was significantly lower than normal and testosterone supplementary mice(p<0.05).Serum miR-361-5p/125a-5p levels(2-?Ct)in castrated mice were significantly lower than normal and testosterone supplementary mice(p<0.05),total and free testosterone concentration were significant correlated with miR-361-5p/125a-5p levels(r=0.459,0.0.683 for total testosterone and miR-361-5p/125a-5p,r=0.636.0.825 for free testosterone and miR-361-5p/125a-5p respectively.p<0.01).Conclusions:Serum miR-361-5p/125a-5p levels were positively correlated with total and free testosterone concentration.Expression of miR-361-5p/125a-5p might be regulated by testosterone.Part II:Expression profile of miR-361-5p/miR-125a-5p in mouse organsObjective:To screen and verify the expression profile of miR-361-5p/miR-125a-5p in mouse organs,aiming to lay the foundation for subsequent study.Methods:In order to screen the miR-361-5p/miR-125a-5p specific expression organ,three male C57/BL6 at 8-10 weeks of age were sacrificed,Pancreas,duodenum,jejunum,ileum,colon,liver,spleen,testis,arteries,myocardium,lung,stomach,skeletal muscle and brain were obtained for total RNA extraction and real-time PCR analysis.After the screening,liver was considered as the target organ for further study.Liver of control mice,castrated mice and testosterone supplementary mice obtained from part I study were used for analyzing the miR-361-5p/miR-125a-5p levels between three groups.Moreover,human normal liver cell line L02 was selected for routine incubation and specific incubation in androgen-exhausted medium,aiming to analyze the differences of miR-361-5p/miR-125a-5p between two groups.Finally,most effective DHT concentration and stimulate time up-regulating miR-361-5p/miR-125a-5p expression were explored.Results:Dysregulation of miR-361-5p is correlated with hematological system diseases and miR-125a-5p highly expressed in brain,spleen,epididymis and ovaries based on miRanda(http://www.microrna.org/).Of the organs obtained from C57/BL6 mice,brain and liver were the firstly and secondly miR-361-5p/miR-125a-5p enriched organs.Liver was selected as the target organ for further study because that levels of the two miRNAs in serum should be influenced by the expression in liver rather than the brain with the existence of the blood-brain barrier.miR-361-5p/miR-125a-5p in C57/BL mice liver were significantly down-regulated after castration while significantly up-regulated after testosterone supplementary therapy(p<0.05).Meanwhile,miR-361-5p/miR-125a-5p levels in L02 cell line incubated in androgen-exhausted medium were significantly lower than in routine medium(p<0.05).Moreover,miR-361-5p/miR-125a-5p levels in L02 cell line increased most obviously at 10nM DTH stimulating 24 hours.Conclusions:miR-361-5p/miR-125a-5p enriched in liver and they can be regulated by testosterone.Moreover,miR-361-5p/miR-125a-5p levels in L02 cell line increased most obviously at 10nM DTH stimulating 24 hours.Part ?:Mechanism of androgen regulation of miR-361-5p/miR-125a-5p expressionObjective:To search androgen response elements(AREs)in miR-361-5p/miR-125a-5p gene and investigate the potential mechanism of androgen regulation of miR-361-5p/miR-125a-5p.Methods:Firstly,promoter sequence of miR-361-5p/miR-125a-5p gene were searched using UCSC(http/://genome.ucsc.edu/),obtained promoter sequences were then uploaded on JASPAR(http://jaspar.binf.ku.dk/)for miR-361-5p/miR-125a-5p AREs prediction.After that,L02 cell were divided into five groups:? positive control,L02 cells were incubated in routine medium:? androgen exhausted group,L02 cells were incubated in androgen exhausted medium for 24 hours;? DHT group,androgen exhausted L02 cells were incubated in androgen free medium with 10nM DHT for 24 hours;? DHT+ Casodex,androgen exhausted L02 cells were incubated in androgen free medium with lOnM DHT and 10?M androgen receptor antagonist casodex for 24 hours;? vehicle group,androgen exhausted L02 cells were incubated in androgen free medium with 10?l absolute ethyl alcohol for 24 hours.All cells were collected and chromatin immunoprecipitation(CHIP)was conducted followed by manufacturers' protocol,DNA fragments obtained from CHIP were analyzed using RT-PCR.Finally,L02 cell miR-361-5p/miR-125a-5p levels of aforementioned five groups were also analyzed.Results:Five and four AREs in miR-361-5p/miR-125a-5p gene were found and they were named as miR-361-*5p-ARE-1/2/3/4/5 and miR-125a-5p-ARE-1/2/3/4 according to relative locus from transcription start site.CHIP and RT-PCR suggested that miR-125a-5p-ARE-1 levels was significantly increased after DHT stimulation compared to androgen deficiency L02 cells(p<0.05),but there was no significant alteration neither in androgen deficiency L02 cells nor stimulation with DHT+ casodex or absolute ethyl alcohol(p>0.05).Differences of expression levels of other three miR-125a-5p AREs between five groups were not observed(p>0.05).Additionally,five miR-361-5p AREs were not amplificated successfully in our experiments.Finally,we verified the CHIP and the results suggested that miR-361-5p/miR-125a-5p levels was significantly increased after DHT stimulation compared to androgen deficiency L02 cells(p<0.05),but there was no significant alteration neither in androgen deficiency L02 cells nor stimulation with DHT+casodex or absolute ethyl alcohol(p>0.05).Conclusions:Androgen can regulate miR-125a-5p expression in miR-125a-5p gene transcriptional level via combination of ARE.Part ?:Potential effects of miR-361-5p/miR-125a-5p dysregulation on liver cells functionObjective:We analyzed the biological function of miR-361-5p/miR-125a-5p target genes,aiming to explore the role and the potential effects of miR-361-5p/miR-125a-5p dysregulation on liver cells function.Methods:miR-361-5p/miR-125a-5p target genes were predicted using Gen Trail(http://genetrail.bioinf.unisb.de/mina_geneset converter.php).GO and KEGG pathway enrichment were then analyzed subjected to miR-361-5p/miR-125a-5p target genes.GO and KEGG analysis suggested that miR-361-5p/miR-125a-5p target genes were enrich in lipid and glucose metabolism,so miR-361-5p/miR-125a-5p target genes related to lipid and glucose metabolism were then strictly screened based on miRTarBase(http://mirtarbase.mbc.nctu.edu.tw/),TarBase(http://diana.imis.athena-innovation.gr/DianaTools/index.php),UCSC(http://genome.ucsc.edu/)and PubMed.Finally,L02 cells were incubated in routine and androgen free medium,to assess the expression levels of selected miR-361-5p/miR-125a-5p target genes at different androgen conditions.Results:874 miR-361-5p/miR-125a-5p target genes were predicted and there are enriched in various lipid and glucose metabolic process,such as MAPK cascade,proteasome-mediated ubiquitin-dependent protein catabolic process,insulin receptor signaling pathway,regulation of cellular amino acid metabolic process.To further identify specific miR-361-5p/miR-125a-5p target genes involved in lipid and glucose metabolic process,3 miR-361-5p target genes(Ncehl,Rac1 and Srsf3)and 9 miR-125a-5p target genes(Ehdl,Hk2,Lin28a,Lyplal,Ogt,Sgpll,Pctp,Ppp2ca and Sema4c)were stricily selected.Of these genes,L02 cell Ncehl and Lyplal were up-regulated in androgen deficiency condition.Conclusions:Dysregulation of miR-361-5p/miR-125a-5p may disturb liver lipid and glucose metabolism,thus lead to metabolic syndrome in LOH.