Screening Of LncRNA Regulating Chicken Germline Stem Cell Differentiation And Functional Mechanism Of PGClnc1

Germ cells carry the important task of transmitting genetic information in generations in sexually reproducing organisms,and produce new individuals through sperm-egg binding.For germ cells with these special properties,scientists have devoted ten years ago to the study of in vitro culture and induction of differentiation into sperm and eggs,in an attempt to make the source of sperm and egg energy continuously produced by in vitro differentiation.Once this study has been Success will have far-reaching implications for understanding the occurrence of germ cells,regulatory mechanisms,genetic modification,and clinical treatment of infertility diseases.Over the years,the study of its mechanism has never stopped,including endogenous regulatory factors and exogenous active substances.It mainly involves the regulation of key genes,growth factors and other factors.With the deepening of research,lncRNA The important role played in cell differentiation has been gradually discovered.At present,the role of lncRNAs in the regulation of many physiological processes in epigenetic and transcriptional levels has been well understood.However,the regulation of germ cell differentiation is relatively rare.In this study,embryonic stem cells（ESCs）,chicken primordial germ cells（PGCs）,spermatogonial stem cells（SSCs）,and chicken fibroblasts were detected by single-cell high-throughput sequencing.The IncRNA expression pattern of the four cells（CEFs）,through the comparative analysis of the results,we found that the complex process of differentiation of embryonic stem cells into male germ cells was related to many lncRNAs and signaling pathways,and that they were specifically selected among PGCs.The highly expressed lncRNA,TCONS₀0948124,was lowly expressed in ESCs and SSCs.It was hypothesized that the IncRNA may be involved in the regulation of the development of PGCs,and it was named PGClnc1.In order to systematically study the function and mechanism of PGClnc1.We performed functional gains and deletions of PGClncl,explored its function in the process of PGCs formation,and studied the regulatory factors of its transcriptional level;using RNA-Pull Down,mass spectrometry analysis and other experimental techniques for its interaction mechanism and ceRNA competition mechanism A preliminary study was conducted to study the molecular regulation mechanism of PGClncl.Our findings can be summarized in the following points:（1）Single Nucleic Acid Sequencing Analysis of Differentiation of ESCs to PGCs and SSCs RNA-seq was performed at the single-cell level to screen for differential lncRNAs involved in the differentiation of embryonic stem cells（ESCs）into male germ cells（PGCs,SSCs）,of which 8 expressions of lncRNAs and ESCs varied by more than 10 in PGCs.At times,the four lncRNAs and PGCs in the ESCs and SSCs group were associated with one lncRNA in the SSCs group,including STRA8,WDR91,WNT7A,PTPDC1,and BARX1.The GO function annotation of DELs indicated that more than 75%of DELs were enriched in biological processes,including cytokine stimulation response and differentiation;more than 15%of DELs were enriched in molecular function and associated with miRNA and double-stranded DNA;over 8%of DELs are enriched in cellular components and are associated with the cell membrane surface and organelle compartments.Enriched by the KEGG pathway analysis of DELs,20 signal pathways were significantly enriched in the three groups,including the Jak-STAT signaling pathway and cytokine-cytokine receptor interactions,and folic acid biosynthesis（Folate Biosynthesis）,TGF-beta signaling pathway,Wnt signaling pathway and mTOR signaling pathway,endoplasmic reticulum processing,purine metabolism,insulin signaling pathway and Hedgehog signaling pathway,etc..Among these signal paths,DELs mainly include ENSGALG00000002005,ENSGALG00000005733,ENSGALG00000002403,ENSGALG00000001967,ENSGALG00000003339,ENSGALG00000006431,ENSGALG00000006794,and so on.Fluorescent quantitative PCR results showed that RNA-seq obtained lncRNA expression profile information is accurate and reliable.（2）lncRNA-specific analysis,PGClncl mining,and coding for small peptides There were 354,654 and 239 specific novell lncRNAs in the ESCs vs.PGCs,ESCs vs.SSCs and PGCs vs.SSCs groups,and 367 specifically expressed lncRANA were involved in germ cell differentiation,including the differentially selected lncRNAs.ENSGALG0000000038,ENSGALG0000015297,ENSGALG0000011415,etc.;KEGG pathway enrichment results indicate that lncRNAs such as XLOC₆12989（ACVR2A）,XLOC₄78357（Lef-1）,and XLOC₂25283（MAPK1）can be significantly enriched in 18 key signaling pathways such as TGF-(3,Notch and Jak-Signal pathways such as STAT.lncRNA specifically expressed in PGCs（TCONS₀0948124）was named PGClncl and was enriched in TGF-β signaling pathway.The expression pattern analysis showed that PGClncl was highly expressed in the gonads,and organelle localization revealed that PGClnc1 was mainly expressed in the cytoplasm.Four open reading frames for PGClnc1,ORF-2,ORF-3,ORF-6,and ORF-8,were cloned and ligated into the pET28a（+）vector,respectively,and were induced by a histidine-tagged prokaryotic fusion expression vector.Bacterial protein,Western Blot validation found that PGClncl ORF2 can induce the expression of his-tagged protein,and then determine that PGClncl has the ability to encode small peptides.（3）Study on the Regulation Mechanism of Transcription Level of PGClncl Combine NCBI and UCSC database to query the sequence of about 1500 bp upstream promoter of PGClncl transcription initiation site to amplify the PGClncl promoter fragment,clone into pEGFP-N1 vector,replace the CMV promoter,and construct recombinant expression vector pPGClncl-EGFP,the green fluorescence of EGFP was observed after transient transfection of DF-1,indicating that the promoter region of the cloned PGClncl had promoter activity;by the deletion fragment cloning technique,different deletion fragment vectors of the PGClncl promoter were constructed,PGL3-P1～PGL3-.P6,and through the dual luciferase reporter system,the core region of the PGClncl promoter is found to be-1033 to-661 bp;the transcription factor binding site in the core region of the promoter is predicted to find STAT1,TCF7L2,and Sox2 transcription factor binding sites for these sites.The point mutation experiment was carried out to construct the corresponding transcription factor point mutation vector.The dual fluorescence reporter system detected that these transcription factors had a certain positive regulation on the activity of the promoter,of which TCF7L2 role is extremely significant;the last 10μmol/L,1μmol/L 5-Aza-2’-deoxycytidine（5-Azadc）,Trichostatin A（TSA）treatment,detection The effects of DNA methylation and histone acetylation on promoter activity of PGClnc1 revealed that DNA methylation inhibition of 5-Azadc and histone acetylation inhibitor TSA treatment resulted in 8.36±3.33 promoter activity increase to 68.99±2.29.141.7 ± 3.12.（4）In vitro and in vivo study of the function of PGClncl in the formation of PGCs We designed three shRNA target sites for the PGClncl sequence,constructed a lentivirus interfering vector,and performed lentivirus packaging to detect the interference efficiency of lentivirus interfering vectors by qRT-PCR.The interference vectors PGClncl-shl,PGClnc1-sh2 and PGClncl-sh3 were successfully constructed with interference efficiencies of 29%,77%and 47%,respectively.The function of PGClncl in the formation of PGCs was studied both in vitro and in vivo.The results showed that:During the experiment,reproductive-like cells could appear after 4 days of overexpression in the overexpression group,and there was a significant up-regulated expression of the reproductive marker gene（P<0.01）.The knockout group could not be in the same situation.Flow cytometry results showed that on the 4th day,the proportion of PGCs-like cells in normal-differentiated cells was 4.5%,and after overexpression of PGClncl,the proportion increased to 5.9%（P<0.01）.After knocking down PGClncl,the proportion dropped significantly to 1.1%（P<0.01）.The results of fluorescence quantitative experiments also showed that the expression of the marker gene cvh（0.3412±0.03）of PGCs after PGClncl knockdown was significantly down-regulated compared with the normal group（1.02454±0.03）（P<0.01）;the results of indirect immunofluorescence showed that relative In the RA-induced group,the expression of cvh protein increased after overexpression of PGClncl,but after knocking down PGClncl,the expression of cvh protein was significantly decreased.In vivo,the chicken embryo was injected with 10 μL of 106 TU/mL +90 μL of DMEM,which was able to be injected.EGFP is stably expressed and chicken embryos can excite green fluorescence.The results of qRT-PCR showed that after knocking down PGClnc1,cvh and c-kit genes related to reproduction significantly decreased to 0.245±0.199 and 0.17±0.0458.Overexpression of PGClncl resulted in opposite changes in the expression of genes in the body,including cvh,The c-kit significantly increased to 0.671±0.02,0.85±0.01（P<0.05）;PAS showed that knocking down PGClncl could significantly reduce the production of PGCs.Flow cytometry results showed that the proportion of cvh-positive cells in the Control group was 4.2%,and after overexpression of PGClncl,the proportion of cvh-positive cells increased to 5.8%（P<0.01）.After low PGClncl,the proportion of cvh-positive cells decreased to 1%（P<0.01）.（5）Study on the Molecular Regulation Mechanism of PGCsl Regulating the Formation of PGCs The sequence of PGClncl was analyzed by RNA-pull down and mass spectrometry.23 proteins interacting with PGClncl were successfully obtained,including known proteins such as GAPDH,KRT5,KRT19,LOC776816 and other unknown proteins;GAPDH was involved in the regulation of TGF-(3 signaling pathways.The signal transduction,combined with the results of the previous laboratory on the effect of TGF-β signaling pathway on germ cell differentiation,and after interference or overexpression of PGClncl,found that the Smad3 gene showed a significant reverse trend,both exist A negative regulation relationship（rises to 3.52±0.089 and falls to 0.50±0.026,respectively）,Smad3 protein expression trends and mRNA levels consistent with the same time,combined with RIP experiments we found that interference with PGClncl after the level of phosphorylation of GAPDH Protein expression affects,and then functions at the protein level;target genes predicted by the combination of PGClncl and its cis have miRNA analysis results for target binding,resulting in miRNAs with a common target,gga-mir-6557-5p,by constructing miRNAs The simulants and inhibitors were co-transfected with PGClncl interference overexpression vector into DF-1 cells and found to interfere with PGClncl,target gene expression.Was adjusted to 0.257 ± 0.020;overexpression of miR,BTRC downregulated to 0.566 ± 0.019.When BTRC was interfered,the expression of PGClncl was down-regulated to 0.788±0.031.When over-expressing miR,the expression of PGClnc1 was down-regulated to 0.574±0.025.While over-expressing PGClnc1,over-expressing miR,the expression of BTRC was up-regulated to 1.257±0.028.