| Primordial germ cells(PGCs),the progenitor cells of gametes,are essential for germ cell development.The study of the occurrence and differentiation mechanism of PGCs has important theoretical and practical significance in the fields of animal germplasm conservation,innovation and utilization,and genetically modified chicken production.The occurrence of PGCs is affected by many exogenous factors.At present,researchers have found that some key genes,signaling pathways,growth factors,and epigenetic modifications play an important role in this process.Although these factors played a certain role in the formation of PGCs,they did not improve the efficiency of PGCs generation.Therefore,it is imperative to carry out an innovative exploration of the mechanism of generation of PGCs from new areas,and to understand and understand the process of production of PGCs in order to obtain a large number of PGCs cells that can meet the needs of production research.With the development of sequencing technology and biological information analysis technology,the role of long non-coding RNA(lncRNA)in the process of germ cell differentiation and embryonic development has gradually gained attention.The regulation of cell differentiation by most IncRNAs is in the upstream stage of the gene and is less interfered.Studies on the effects and mechanisms of IncRNAs on the occurrence of PGCs can provide new research strategies for improving the in vitro generation efficiency of PGCs.This study was based on the previous single-cell high-throughput sequencing results to screen chicken PGCs specific expression lncRNA-M1ANCR,using chicken as a research object to explore its function in the process of PGCs formation,using RNA interference technology to explore both in vivo and in vitro levels.The function and mechanism of lncRNA-M1ANCR in differentiation of embryonic stem cells into primordial germ cells provide a theoretical basis and reference for effectively improving the efficiency of differentiation of ESCs into PGCs.The results of the study are as follows:(1)Full-length lncRNA-M1ANCR amplification,identification of cell localization and coding capacityThe lncRNA-M1ANCR full-length was obtained by RACE based on previous high-throughput sequencing results.qRT-PCR demonstrated that the specific high expression of lncRNA in PGCs was consistent with the results of transcriptome sequencing and was expressed in the cytoplasm;according to bioinformatics prediction analysis lncRNA-There is an open reading frame sequence in full length of M1ANCR,and the construction of prokaryotic fusion expression vector for WB discovery does not have coding capacity.(2)The function of lncRNA-M1ANCR in the formation of PGCs in vitro and in vivoBased on the lncRNA-M1ANCR full-length sequence,three shRNA target sites were designed and ligated into the pGMLU-SC5 interfering vector.After transfection of DF-1 cells,the interference efficiency(67.16%±2.0%)of target site 3 was found to be the highest by quantitative detection.Significantly higher than the control group(P<0.01);simultaneously cloned the full length of lncRNA-M1ANCR,and successfully constructed the pcDNA-M1ANCR overexpression vector;through the in vitro and in vivo injection of two aspects of lncRNA-M1ANCR in the formation of PGCs function,The results showed that the lncRNA-M1ANCR lentivirus interfering vector and the overexpression vector were transfected into chicken ESCs cells in vitro under RA-induced conditions.Through cell morphology observation,it was found that the number of PGCs-like formation induced by IncRNA was higher than that of RA-induced group and overexpression.The group was significantly reduced;qRT-PCR was further used to detect the expression levels of Cvh and C-kit in specific markers of PGCs,and the expression level in the interference group(Cvh:1.029±0.062,C-kit:1.154±0.009)was significantly lower than that in the control group.The expression group(Cvh:1.672±0.123,C-kit:2.232±0.029)and the induction group(Cvh:1.232±0.234,C-kit:1.561±0.017)(P<0.01),and the expression of the totipotency gene Nanog(Nanog:1.029±0.024)was significantly higher than the overexpression group(Nanog:0.918±0.038)and the induction group(Nanog:0.984±0.033)(P<0.01).To further verify this result,indirect immunofluorescence was used to detect the fluorescence expression of Cvh.It was found that PGCs formation was significantly reduced by interference with IncRNA expression.Flow cytometry analysis showed that the number of PGCs-like cells(3.96%±0.32%)produced after 4 days of induction in the interference group was significantly less than that in the RA-induced group(9.67%±0.24%).)and overexpression group(12.1%±0.46%)(P<0.01);In vivo chicken embryo vascular injection experiment,the interference vector expression vector was integrated into the chicken embryo,and the expression of the reproductive marker gene in the PGCs was detected and found to be significant Decreased sex(Cvh:2.631±0.208,C-kit:3.028±0.23)(P<0.01).At the same time,the number of PGCs cells in the genital ridges was observed by paraffin sections.It was found that the formation of PGCs in the interference group was significantly reduced(15±0.57).To further illustrate the effect of lncRNAs on the formation of PGCs,flow cytometry experiments revealed that interference with IncRNAs does have an inhibitory effect on the differentiation of PGCs(4.0±1.1%),and in vitro induction Induced,it showed IncRNA can be positive regulation process for chicken ESCs cells into PGCs cells.(3)Identification of core region of lncRNA-M1ANCR promoter combined with transcription factor analysisAccording to the results of RACE and bioinformatics analysis,the promoter region of lncRNA was determined.After constructing the eukaryotic expression vector p0-EGFP,the DF-1 cells transfected with DF-1 cells were found to be able to stably express green fluorescence,indicating that this region has promoter activity;After constructing the promoter deletion vectors pGL3-p1-783,pGL3-p2-530,pGL3-p3-269 and pGL3-p3x respectively,two-luciferase reporter assays were performed.The results showed that pGL3-p3-269 had the highest activation activity(4.39 ± 0.35),indicating that there is an important regulatory element in the range of-0 to-269 bp,which has an important effect on promoter activity.Further through the JASPAR analysis of the bioinformatics analysis site,there was a transcription factor p53 binding site in the promoter region-0--269bp,in order to investigate whether p53 has a regulatory effect on promoter activity.In this study,the p53 interference was constructed separately.Vectors and overexpression vectors.,Double-luciferase activity was detected again after transfection of DF-1 cells.The activity of the core region after interference with p53(1.65±0.53)was significantly lower than that of the normal group(4.06±0.41)and the overexpression group(4.55±0.66),In summary,the transcription factor p53 can regulate the activity of the core region of the lncRNA-M1ANCR promoter,thereby regulating IncRNA expression.(4)Preliminary investigation of inhibition mechanism of lncRNA-M1ANCR and gga-mir-1591 competitive bindinglncRNAs have potential ceRNA properties.In this study,bioinformatics analysis yielded miRNAs gga-mir-1591 and gga-mir-3539 that may compete with IncRNA-MlANCR to construct miRNA mimics and inhibitors.After in vitro transfection of chicken ESCs cells under RA-induced conditions,it was found that inhibition of the expression of gga-mir-1591 promoted the formation of PGCs,and that the expression of gga-mir-1591 interfered with the formation of PGCs.The gga-mir-3593 mimics and inhibitors had no effect on the formation of PGCs.The expression of lncRNA-M1ANCR,MAPK1,totipotent genes,and reproductive marker genes was detected by qRT-PCR.The transfection of gga-mir-1591 was found.After inhibitors,the expressions of MAPK1 and IncRNA were significantly up-regulated(MAPK1:12.293±2.012,lncRNA:2.137±0.324),and the expression level of the germline marker gene(Cvh:6.731±0.921,C-kit:6.631±0.918)was also significantly higher than In order to further verify the effect of gga-mir-1591 on the formation of chicken PGCs,the flow cytometric analysis showed that the positive rate of PGCs-like cells(3.13±0.22%)was significantly higher after gga-mir-1591 inhibition.Other groups(P<0.01),show miRNA gga-mir-1591 having a regulatory role in the differentiation of chicken PGCs ESCs,and lncRNA-Ml ANCR there may be competitive binding inhibition relationship.In order to further verify the relationship between gga-mir-1591 and lncRNA-M1ANCR,this study transfected the lncRNA-M1 ANCR overexpression vector and the interference vector on the basis of transfection of gga-mir-1591 mimics and inhibitors.Through the rescue experiment to verify the existence of competitive binding inhibition relationship between the two.The observation of cell morphology revealed that the inhibition of the formation of PGCs after gga-mir-1591 could be affected by lncRNA interference vectors,resulting in the reduction of PGCs-like formation and vice versa;meanwhile,the detection of lncRNA-M1ANCR,MAPK1,totipotency by qRT-PCR was performed.The expression of gene and reproductive marker genes revealed that the expression of MAPK1 was significantly decreased in the inhibition of gga-mir-1591 expression and the interference of IncRNA at the same time(p<0.01)compared with the inhibition of gga-mir-1591 group,and the expression of the reproductive marker gene was also significant.Decrease(P<0.05);In summary,there is a competitive binding relationship between gga-mir-1591 and lncRNA-M1ANCR,and lncRNA-M1ANCR binds to miRNA gga targeting MAPK1 via a "sponge-like" adsorption method.-mir-1591 regulates the expression of MAPK1 and regulates the formation of chicken PGCs.(5)Screening and identification of lncRNA-M1ANCR interaction proteinsBy analyzing the RNA pull down results of lncRNA-M1ANCR,the lncRNA-M1ANCR may interact with ILF3 protein during the differentiation of chicken ESCs into PGCs.To further verify the mechanism of interaction between the two,the present study was confirmed by Western Blot.The expression of ILF3 protein in chicken PGCs was found,and the interaction between ILF3 and lncRNA was found through RIP experiments.In order to further explore the interaction between the two,qRT-PCR was performed on the key genes of downstream signaling pathways related to ILF3 and lncRNA.The results showed that MAPK and WNT,the key downstream signaling genes,interfere with lncRNAs.MAPK:10.342±1.192,WNT:6.323±0.624)was significantly lower in the overexpression group(MAPK:14.432±1.394,WNT:10.960±0.934).In summary,during the formation of chicken PGCs,there is an interaction between lncRNA-M1ANCR and ILF3 proteins,and changes in lncRNA expression can affect the expression of key genes in downstream signaling pathways. |
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