Mechanism Of Gga-miR-31-5p Regulating Meiosis And Spermatogenesis In Chicken

Spermatogenesis is a complex and elaborate regulatory process.At present,most of its research has focused on the level of genes and drugs,while the systematic study of its regulatory networks has rarely been involved.This study aims to explore a new regulatory network for chicken spermatogonial stem cells?SSC?differentiate into spermatozoa.By studying the microRNA?miRNA?upstream of Stra8,a key gene in meiosis,the Transcription factors?TF?miRNA-Stra8 regulation mechanism and long non-coding RNA lncRNA?lncRNA?-miRNA-Stra8 competitive regulation mechanism during meiosis has been explored.Thus to provide a theoretical and experimental basis for clarifying the mechanism of spermatogenesis and the establishment of a ESC-oriented differentiation system for sperm.Based on this goal,this article took chicken as the research object and applied miRNA-seq technology to study the miRNA in the key node cells during Embryonic stem cells?ESC?differentiation into male germ cells by Retinoic acid?RA?in vitro.The key miRNAs involved in this process which may regulate Stra8 were screened and verified both the in vitro and in vivo to confirm the relationship between these key miRNAs and Stra8 during meiosis.Finally,the transcriptional regulation mechanism?TF-miRNA-Stra8 and lncRNA-miRNA-Stra8?of miRNA was further explored with a view to providing a theoretical and experimental basis for elucidating the mechanism of spermatogenesis and the establishment of a system for the induction of ESC differentiation into sperm.The research results are as follows:?1?In order to explore the specific regulatory mechanism of RA-induced ESC differentiation and the reason why the expression of RA-regulated gene Stra8 was not significantly different expressed in this process,cells at critical time points during RA induction,self-differentiating ESC at the same time node and freshly isolated ESC were performed miRNAs sequencing to analyze the key miRNAs that may be involved in germ cell differentiation in chicken.qRT-PCR was used to detect expression of key miRNAs at Od ESC?c₀?,RA induced 4d/10d?R₄/R10?,and self-differentiation 4d/10d?z₄/z10?,Primordial germ cell?PGC?and SSC.It was found that the four miRNAs:gga-miR-146c-5p,gga-miR-148a-3p,gga-miR-21-5p and gga-miR-30d were highly expressed in c₀,R₄,R10,z₄ and z10.Their target genes are all related to cell differentiation,of which gga-miR-30d,gga-miR-21-5p and gga-miR-148a-3p target genes are related to male germ cell development and differentiation,and are enriched in Notch.GnRH.MAPK and TGF-? signaling pathways.Analysis of z₄ vs c 0 and R₄ vs c₀ revealed 55 different miRNAs including gga-miR-31-5p,compared with z10 vs c 0 and z10 vs c₀ found 77 miRNAs including gga-miR-148a-3p.Compared with R₄ vs c₀ and z10 vs c 0,there were only two common differential miRNAs,and these two miRNAs play an important role in the formation of germ cells.Comparing the sequencing results of miRNAs in PGC/SSC/Spermatogonia?SP?,it was found that during the RA induction process,the expression trends of the differential miRNAs including gga-miR-30d were basically consistent with the trend in the above three cells.miRNAs targeting Stra8,such as gga-miR-31-5p,were also up-regulated during RA induction.The above results indicated that miRNAs such as gga-miR-31-5p are very likely to participate in the regulation of ESC differentiate into SSC and affect Stra8 expression?2?In order to further explore the molecular regulatory mechanism regulating Stra8 gene expression,this study used 3'RACE technology to amplify Stra8 3'UTR.Using the bioinformatics online software Targetscan and miRBase to reverse predict the miRNAs targeting the chicken Stra8 gene,the overexpression vectors of each miRNA and the Stra83'UTR luciferase reporter vector were constructed respectively.The miRNA binding site identifies the key miRNAs targeting Stra8 and constructs its interference vector.Then anti-Stra8 serum was prepared by immunoassay.qRT-PCR was used to detect the expression profiles of key miRNAs and Stra8 in different cells.In vitro,SSC was treated by overexpression and interference with key miRNAs and combined with RA induction.Flow cytometry was used to detect cell phenotype and qRT-PCR was used to detect meiosis-related gene expression.Sex-mature cocks were treated by testicular injection in vivo,the semen concentration and volume were then measured.qRT-PCR was used to detect meiosis-related gene expression and HE staining was used to stain testicular tissue.The results showed that the overexpression vectors of each miRNA and the Stra8 3 'UTR dual luciferase reporter vector,as well as the Stra8 3 'UTR luciferase reporter vector of the gga-miR-31-5p mutation site were successfully constructed.Dual luciferase activity test showed that gga-miR-31-5p had the best inhibitory activity on Stra8.The detection results of dual luciferase activity after the mutation site showed that gga-miR-31-5p can directly target Stra8.After overexpressing or interfering with gga-miR-31-5p in cells,Stra8 and gga-miR-31-5p are expressed in the opposite state Elisa results showed that anti-Stra8 serum was successfully prepared.After overexpression of gga-miR-31-5p,haploid formation efficiency showed significantly decrease by flow cytometry assay in vitro?P<0.01?.qRT-PCR results showed that overexpression of gga-miR-31-5p can significantly reduce the expression of meiosis-related genes such as Stra8?P<0.01?.In vivo experiment has shown that overexpression of gga-miR-3 1-5p significantly reduced the semen concentration and volume in the rooster?P<0.01?.At the same time,qRT-PCR results showed that the expression of meiosis-related genes such as Stra8 was significantly down-regulated?P<0.05?.And result of WB showed that Stra8 protein expression was significantly suppressed.HE staining results showed that overexpression of gga-miR-31-5p loosened testicular tissue and reduced sperm content in the lumen.The above results indicated that gga-miR-31-5p targets Stra8 and regulates sperm formation by inhibiting Stra8 expression.?3?In order to systematically clarify the difference in the expression of gga-miR-31-5p after adding RA to different cells,this study combined UCSC and NCBI databases to query the pre-miRN A upstream sequence of gga-miR-31-5p about 2180bp.Full length was amplified,replaced the CMV promoter in pEGFP-N1 vector,constructed recombinant vector pEGFP-miR-31-2180,then transfected into DF-1 cells to determine whether the fragment has promoter activity.By constructing different deletion fragments of the promoter region of gga-miR-31-5p,pGL3-584,pGL3-1344 and pGL3-2180,after transfection with DF-1,the dual-luciferase reporter system was used to screen out important regulatory elements that may be present in the gga-miR-31-5p promoter region.The gga-miR-31-5p promoter and Stra8 promoter were transfected into DF-1/ESC/SSC cells respectively.After RA treatment,the dual luciferase reporter system was used to detect the effect of RA on each promoter.Then the gga-miR-31-5p promoter was co-transfected with the Stra8 promoter,and after RA treatment,the respective regulation by RA was detected by dual luciferase reporter system.Screening for transcription factors that act only on the gga-miR-31-5p promoter and differentially expressed in ESC and SSC.Then vectors with deletion of its binding site were constructed.Dual luciferase reporter system was used to detect overexpressing/interfering transcription factors/deletion of the binding site on the activity of the gga-miR-31-5p promoter.Finally,the DF-1 cells transfected with the gga-miR-31-5p promoter were treated with 10-6 M TSA and 10-5 M RA,respectively,and the dual luciferase activity reporting system was used to detect the activity of the gga-miR-31-5p promoter.qRT-PCR was used to detect the expression of gga-miR-31-5p in ESC/SSC treated with TSA and RA.Sequencing results showed that pEGFP-miR-31-2180 was successfully constructed,and the vector can express green fluorescence after transfecting into DF-1 cells,indicated that it has promoter activity.The sequencing results of the promoter deleted fragments also showed that the vectors were successfully constructed,and the dual luciferase activity test further confirmed that the gga-miR-31-5p promoter 2180bp has promoter activity.It was found that RARa,c-Jun,HNF-4a REV-ErbA,ER and HNF-1 binding sites exist only in the region-2180bp?-1344bp of the gga-miR-31-5p promoter,in which RA can enhance gga-miR-31-5p promoter activity,and the gga-miR-31-5p promoter competitively binds RA with the Stra8 promoter.Moreover,it was also found that the transcription factor c-jun inhibits the activation activity of gga-miR-31-5p.When c-jun is present in large amounts,RA has no obvious effect on the activation activity of gga-miR-31-5p.In addition,it was also found that when TSA was added,the activity of the gga-miR-31-5p promoter was significantly inhibited?P<0.01?.The expression of gga-miR-31-5p in ESC/S S C after TSA or TSA and RA combined treatment was significantly reduced?P<0.01?,only in RA treatment in ESC can upregulate the expression of gga-miR-31-5p.The above results indicated that c-jun's inhibitory effect on the promoter of gga-miR-31-5p is stronger than that of RA on the promoter activation of gga-miR-31-5p.When histone acetylation occurs,it inhibits the transcription of gga-miR-31-5p.?4?In order to explore the existence of the lncRNA-gga-miR-31-5p-Stra8 ceRNA mechanism,this study combined lncRNA sequencing during RA-induced ESC differentiation to conduct a joint analysis of lncRNA-miRNA and screened lncRNAs which interact with gga-miR-31-5p.Then their overexpression vectors were contructed.LncRNA,gga-miR-31-5p and Stra8 3'UTR luciferase reporter vector were co-transfected into DF-1,and the dual luciferase reporter system was used to detect lncRNA that competed with Stra8 for binding to gga-miR-31-5p.The IncRNA gga-miR-31-5p binding site mutation vector was constructed to further confirm its competitive relationship.Four interference targets were designed for key lncRNA,and their interference vectors were constructed.The best interference active vectors were determined by qRT-PCR and dual luciferase activity detection.In vitro,lncRNA-IMS was overexpressed and interfered separately,and a co-transfection group of lncRNA and gga-miR-31-5p was set up,combined with RA induction,using flow cytometry to analyze cell ploidy,qRT-PCR to detect meiosis-related genes.In vivo,sex-mature cocks were treated by testicular injection.Then the semen concentration and volume were measured.qRT-PCR was used to detect meiosis-related gene expression and HE staining was used to stain testicular tissue.The results showed that 3 lncRNA overexpression vectors,lncRNA-IMS gga-miR-31-5p binding site mutation vector and 4 IncRNA-IMS interference vectors were successfully constructed.The dual luciferase report test showed that lncRNA-IMS is the key lncRNA,and it competitively binds to gtra-miR-31-5p with Stra8.Through qRT-PCR and dual luciferase activity reporting system,the best interference vector was determined as shIMS-2.In vitro experiments showed that overexpression of lncRNA-IMS significantly increased the haploid formation efficiency?P<0.01?,the expression of meiosis-related genes such as Stra8 was signficantly down-regulated?P<0.01?,and the interference showed completely opposite results.The expression level of gga-miR-31-5p did not change.At the same time,overexpression of lncRNA-IMS and gga-miR-31-5p,no matter the cell ploidy or meiosis-related genes have not changed,only gga-miR-31-5p expression is up-regulated.In vivo experiments showed that overexpression of LncRNA-IMS significantly increased the semen concentration?P<0.01?,and the expression of meiosis-related genes such as Stra8 were significantly increased.HE staining results showed that after overexpression of LncRNA-IMS,the tissue structure of the testis was complete,the sperm cells at all levels were arranged densely,and the sperm content in the lumen was high.After the interference,the semen concentration and volume were significantly reduced?P<0.01?,and the expression of meiosis-related genes such as Stra8 were down-regulated significantly?P<0.01?.The structure of the testis was loose.There were fewer sperm cells at all levels,a larger lumen,and less sperm content in the cavity.At the same time,overexpression of LncRNA-IMS and gga-miR-31-5p,the semen concentration and volume,meiosis-related genes have not changed?P>0.05?,the testicular tissue is intact,and there is more sperm in the lumen.It showed that LncRNA-IMS can compete with Stra8 to bind gga-miR-31-5p,reduce the inhibitory effect of gga-miR-31-5p on Stra8,and promote spermatogenesis.The above research shows that histone acetylation,c-jun and RA jointly regulate the expression of gga-miR-31-5p during the regulation of meiosis in chicken,which affects the expression of Stra8.At the same time,the LncRNA-IMS present in the cell competitively binds to gga-miR-31-5p,which in turn prevents the inhibitory effect of gga-miR-31-5p on Stra8,promotes meiosis in male germ cell,and increases the number of sperm production.