| Primordial Germ Cells（PGCs）are the progenitor cells of germ cells and are vital in the process of life.Studying the occurrence of PGCs is not only meaningful for understanding the mechanism of spermatogenesis,but also has a reference value for understanding the reproductive process of humans and animals.Poultry PGCs have a unique migration path,which can provide effective tool cells for genetic engineering of poultry,reprogramming of somatic cells and protection of precious germplasm resources.However,the number of PGCs isolated from chicken embryos is relatively small,and the number of in vitro culture induction cannot meet the needs of research and production.The key problem is that the mechanism of regulating the development of PGCs is still unclear,and the key regulatory factors affecting its occurrence have not been found Therefore,it is urgent to deeply analyze the regulatory factors in the process of PGCs.The occurrence of PGCs is a complex and precise regulation process,which is affected by many factors.At present,researchers have found that some genes,signaling pathways.growth factors and epigenetic modification factors play important roles in regulating the occurrence of PGCs,but the efficiency of inducing the generation of PGCs in vitro has not reached the expected requirements.Therefore,it is urgent to conduct innovative research on the generation mechanism of PGCs from a new field.With the development of sequencing technology and biological information analysis technology,the role of long non-coding RNA（lncRNA）in life activities has gradually received attention,and research has confirmed that it plays an important role in the process of germ cell differentiation.But the function and mechanism have not been reported.Based on the high-throughput sequencing results of the research group’s early ESCs（Embryonic Stem Cells,ESCs）and PGCs transcriptomes.lncRNA（TCONS 00874170）.which is only highly expressed in PGCs,was screened for bone formation proteins 4 gene（Bone Morphogenetic Protein 4,BMP4）,therefore named lncRNA-BMP4.This study takes the key lncRNA-BMP4 that regulates the formation of PGCs as an entry point,and explores in detail how lncRNA regulates the target gene BMP4 to promote the formation of PGCs.In order to reveal the role and mechanism of key influencing factors in the process of early chicken PGCs.improve the regulatory network of PGCs development,further improve the induction efficiency of PGCs.and provide a theoretical basis for the application of PGCs.The research results are as follows:（1）Screening,cloning and bioinformatics analysis of lncRNA-BMP4.Transcriptome sequencing analysis of chicken ESCs and PGCs revealed that lncRNA（TCONS00874170）was specifically expressed in PGCs.and it was predicted that the IncRN A targeted BMP4.so it was named lncRNA-BMP4;Northern Blot results showed that lncRNA-BMP4 was in cells At the same time,the cloned lncRNA-BMP4 transcript is 1244bp;qRT-PCR detection results show that the expression pattern of lncRNA-BMP4 in three cells（ESCs:1;PGCs:3.738±0.237;SSCs:0.323 ± 0.087）and sequencing results Consistent,expression profile detection also found that lncRNA-BMAP4 is highly expressed in the gonads;nucleocytoplasmic separation results show that IncRNA-BMP4 is distributed in the nucleus and cytoplasm,but its（4.952 ± 0.251）distribution in the nucleus is higher than that of the cytoplasm（2.808±0.083.P<0.01）:Bioinformatics predicts that lncRNA-BMP4 has an ORF of 231 bp.The constructed ORF prokaryotic fusion expression vector can be induced to express in BL E.coli in vitro.Encode a small peptide of 12.4kD（2）Verify the function of lncRNA-BMP4 in the process of PGCs formation in vitro and in vivo.The lncRNA-BMP4 overexpression vector oelncRNA-BMP4 and the interfering vectors shlncRNA-BMP4-1,shlncRNA-BMP4-2 and shlncRNA-BMP4-3 were constructed respectively,and the activity of the interfering vector transfected with DF-1 was compared The results showed that the shlncRNA-BMP4-2 vector had the best interference effect（0.349±0.028,P<0.01）.which was used in subsequent experimental studies.The functions of lncRNA-BMP4 in the formation of chicken PGCs were verified in vitro and in vivo.respectively.In vitro tests,morphological results showed that a large number of clustered embryoid bodies appeared on the 4th day of the overexpression group,and the number reached the peak on the 6th day.Only a small amount of embryoid bodies appeared on the 6th day;qRT-PCR detected that the overexpression group Cvh（39.733 ± 3.003）,C-kit（29.687 ± 2.107）and BMP4（24.68 7± 2.936）increased significantly on the 6th day.（P<0.01）.the interference group Cvh（10.951 ± 1.637）and BMP4（24.687±2.936）decreased significantly（P<0.01）,C-kit（13.874 ± 1.783）decreased significantly flow cytometric analysis The results showed that the positive cell rate of CVH in the overexpression group was significantly increased（27.793%±1.942.P<0.01）,and the opposite result appeared after interference（4.196%±0.843.P<0.01）;indirect immunofluorescence results and flow cytometry results Consistent.In the in vivo test,the expression levels of Cvh（2.986 ± 0.146）,C-kit（2.374 ±0.276）and BMP4（2.637 ± 0.228）at the 4.5th day in the overexpression group were significantly increased（P<0.01）.and Cvh（0.344）in the interference group ± 0.020)and BMP4（0.436±0.029）decreased significantly（P<0.05）,C-kit（0.285 ± 0.036）decreased significantly（P<0.01）;the results of flow cytometry analysis showed that after overexpressing lncRNA-BMP4,CVH positive cell rate of protein was significantly increased（25.103%± 1.825.P<0.01）.and the opposite result appeared after interference（2.861%±0.849,P<0.01）;the immunohistochemical results were consistent with the flow cytometry results.Both in vitro and in vivo functional verification results show that lncRNA-BMP4 can promote the formation of chicken PGCs.（3）Explore the elements that regulate the differential expression of LncRNA-BMP4.The lncRNA-BMP4 promoter was amplified with a total length of 1270bp,and the recombinant vector plncBMP4-N1 was constructed.After transiently transfected with DF-1.it showed green fluorescence,indicating that the amplified promoter has promoter activity;further cloned by the piecewise truncation method lncRNA-BMP4 promoter series deletion fragments were connected to pGL3-basic to construct pGL3-1270,pGL3-1086,pGL3-929,pGL3-832.pGL3-651,pGL3-473,pGL3-310,pGL3-148 different The recombinant vector of large and small size was determined by the dual luciferase reporter system to determine that-833～-651bp is the active core region of the lncRNA-BMP4 promoter.Bioinformatics predicted the presence of SOX 17,CREB1,and STAT1 transcription factor binding sites in the promoter active region.Based on this,a dual-luciferase reporter recombinant vector that lacked three binding sites was constructed,and after transfection with DF-1,the detection of Cis regulation found that only the deletion of the STAT1 binding site can significantly reduce the activity of the lncRNA-BMP4 promoter（5.333 ± 0.418,P<0.01）;Then the Stat1 interference vectors shStat1-1,shStat1-2,shStat1-3 were constructed respectively,and the transfection of DF-1 showed comparative activity.The results of qRT-PCR showed that the interference effect of shStat1-1 was the best（0.368 ± 0.02,P<0.01）,can be used in subsequent experiments.Trans regulation test found that interfering with Statl can significantly reduce the activity of lncRNA-BMP4 promoter（10.847 ± 1.001,P<0.01）.The above results show that Statl can positively regulate the activity of lncRNA-BMP4 promoter.The DNA methylation inhibitor 5’Azacd and histone deacetylation inhibitor TSA were added on the basis of the recombinant vector transfected with the active core region of the promoter in DF-1.The detection results of the dual luciferase reporter system showed that lncRNA-The activity of BMP4 promoter was not affected by DNA methylation,but compared with the control group（17.283 ± 1.028）,the addition of TSA significantly increased the activity of IncRNA-BMP4 promoter（37.183 ± 1.176,P<0.01）.The results show that both transcription factor STAT1 and histone acetylation can activate lncRNA-BMP4 initiation activity,but DNA methylation has no effect on its initiation activity.（4）The dose-dependent effect of N6-methyladenine（m6A）on IncRNA-BMP4 as a sponge is involved in the study of ceRNA.Bioinformatics predicts that only miRNA-12211,miRNA-12258 and lncRNA-BMP4 and BMP4 have a common binding sequence on chickens.Therefore,we first verified whether these two miRNAs were involved in the formation of PGCs,and constructed miRNA-12211 and miRNA-12258 overexpression and interference vectors respectively.The in vitro morphological observation results showed that there was no embryoid bodies on the 2 d and 4 d after overexpression of miRNA-12211 A small number of embryoid bodies appeared on the 6th day of the embryo body.On the contrary,a large number of clustered embryoid bodies appeared on the 4th day after the interference,and the number reached the peak on the 6th day.The results of qRT-PCR showed that after overexpression of miRNA-12211,Cvh（5.837 ± 1.002）,C-kit（5.844 ± 1.004）,BMP4（5.394 ± 0.420）and lncRNA-BMP4（3.063 ± 0.055）all decreased significantly（P<0.01）,while the interference group Cvh（18.935± 1.071）increased significantly（P<0.05）.C-kit（18.247 ± 1.096）.BMP4（17.880 ±1.203）and lncRNA-BMP4（12.981 ± 0.972）all increased significantly（P<0.01）;Flow analysis results showed that overexpression of miRNA-12211 significantly reduced the positive cell rate of CVH protein（19.703%±0.981,P<0.01）.and the interference group significantly increased the positive cell rate of CVH（30.952%±1.252,P<0.01）.and the indirect immunofluorescence results are consistent with the flow cytometry results.The above results indicate that miRNA-12211 and lncRNA-BMP4 have opposite functions,can inhibit the formation of chicken PGCs,and inhibit the expression of BMP4 and lncRNA-BMP4.However,there was no significant difference between the over-expression and interference of miRNA-12258 and the control group,proving that miRNA-12258 had no effect on the formation of PGCs.Therefore,we chose miRNA-12211 to further verify its relationship with lncRNA-BMP4 and BMP4,and constructed a BMP4 3’UTR luciferase reporter vector,co-transfected with miRNA-12211 overexpression vector DF-1.luciferase report The activity of BMP4 3’UTR detected by the system was significantly reduced（3309 7.67 ± 3691.37.P<0.01）.On this basis,overexpression of lncRNA-BMP4 could significantly save the activity of BMP4 3’ UTR（50612.10±3939.78,P<0.01）.The results show that lncRNA-BMP4 can compete with miRNA-12211 to bind BMP4 3’UTR,and rescue BMP4 expression.Analyzing the sequence of lncRNA-BMP4,it was found that there are 13 m6A occurrence sites（RRACH）.Through RIP,m6A can be enriched on lncRNA-BMP4 in ESCs and PGCs.In order to detect the effect of m6A on lncRNA-BMP4 binding to miRNA-12211.this study constructed the interference vectors of m6A methyltransferase Mettl3 and Mettl14.transfected with DF-1 to detect activity,and qRT-PCR to detect shMettl3-1（0.483 ± 0.012,P<0.01）and shMettl14-3（0.355 ± 0.049,P<0.01）have the best interference effect and can be used in subsequent experiments.On the basis of overexpressing lncRNA-BMP4,transfect shMettl3-1 and shMettl14-3,respectively,or add m6A activator MA,m6A inhibitor NPC.qRT-PCR results show that the change of m6A level does not affect the transcription of lncRNA-BMP4;Next,a Luc-lncRNA-BMP4 luciferase reporter vector was constructed,and DF-1 was co-transfected with oemiRNA-12211,and its fluorescence activity decreased significantly（P<0.01）.On this basis,shMettl3-1 and shMettl14 were transfected respectively.-3,or the addition of NPC,the fluorescence activity of Luc-lncRNA-BMP4 increased significantly（P<0.05）.and the fluorescence activity decreased significantly after the addition of MA（P<0.05）,indicating that high m6A levels can promote the binding of lncRNA-BMP4 to miRNA-12211.In order to further explore the mode in which m6A affects the binding of lncRNA-BMP4,progressive accumulation mutations were constructed for 16 m6A occurrence sites,and 16 recombinant lncRNA-BMP4 luciferase recombinant vectors were constructed.RIP results showed that when progressively cumulative mutations reached positions 6,7,nd 8,the level of m6A enrichment on lncRNA-BMP4 showed a significant decrease（P<0.01）,uciferase reporter system detected Luc-lncRNA-BMP4 fluorescence activity showed a very significant increase（P<0.01）.The above results prove that lncRNA-BMP4 adsorbs miRNA-12211 and rescues BMP4 expression in a dose-dependent manner for the number of m6A modification sites.（5）The study that m6A affects lncRNA-BMP4 encoded protein to promote the formation of PGCs.Earlier it was confirmed that lncRNA-BMP4 can encode a12.4kD small peptide.In order to investigate whether the functioning body of lncRNA-BMP4 is the RNA chain or the encoded small peptide,the RNA chain overexpression vector oelnc of the mutant ORF start codon was constructed separately-ORF mut and small peptide overexpression vector oeORF.wild type lncRNA-BMP4 overexpression vector was used as a positive control.In vitro morphological observation results showed that a small number of embryoid bodies appeared on the 4th day of the blank control group,and the number of embryoid bodies increased and the body type became larger on the 6th day:a large number of embryoid bodies appeared on the 4th day of the oelnc-ORFmut and oeORF groups.The number was more in 6 d:the positive control group had the largest number of embryoid bodies.qRT-PCR detected the expression of PGCs marker genes on the 6th day.The Cvh of the oelnc-ORFmut group rose to 29.759 ±2.827（P<0.05）:the Cvh of the oeORF group rose to 30.294 ± 3.735（P<0.01）,and C-kit rose to 25.283 ± 1.073（P<0.05）,positive control group Cvh rose to 39.733±3.003（P<0.01）.C-kit rose to 29.687 ± 2.107（P<0.01）:the results of flow analysis showed that compared with the blank control group,The positive cell rate of CVH protein in oelnc-ORFmut group（16.747%±0.379）and oeORF group（17.739%± 0.491）both increased significantly（P<0.01）,but both were less than the positive control group（29.298%± 2.073）.The indirect immunofluorescence results are consistent with the flow cytometry results.The above results all show that not only the RNA chain of lncRNA-BMP4 can promote the formation of PGCs,but also that the small peptides encoded by it have the same function.In order to explore the mechanism of the function of small peptides,a small peptide fusion expression vector with a His tag was constructed.ChIP detection results showed that the encoded small peptides were combined as transcription factors in the BMP4 promoter region.Further construct a wild-type BMP4 promoter vector and a BMP4 promoter vector lacking a small peptide binding site,and co-transfect DF-1 with a small peptide overexpression vector,respectively.The detection results of the dual luciferase reporter system showed that overexpression of small peptides significantly increased the activity of the BMP4 promoter compared with the control group（329.938 ± 28.938,P<0.01）,but after the mutation of the binding site,the activity of the BMP4 promoter was extremely significant Decreased（119.398± 12.874.P<0.01）.and overexpression of small peptides on this basis could not affect the activity of BMP4 promoter.The results indicate that small peptides can bind to the BMP4 promoter region to promote its transcription.In order to investigate whether N6-methyladenine affects the ability of lncRNA-BMP4 to encode small peptides,the His fusion expression vector of small peptides was transfected into PGCs,on the basis of which were added m6A activator MA and inhibitor NPC,Western Blot results showed,When the m6A level increases,the expression of small peptides decreases,while the m6A level decreases,the expression of small peptides increases,indicating that low m6A levels can promote lncRNA-BMP4 encoding small peptides,the encoded small peptides bind to the BMP4 promoter region to activate BMP4 expression.His pull-down combined mass spectrometry analysis was performed on the small peptide proteins induced and purified in vitro,and 236 interaction proteins were obtained:Co-IP results showed that the small peptides combined with DDX5,indicating that DDX5 may be the interaction target protein of small peptides.However,its mechanism needs further study and verification. |
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