Research On The Role Of LncRNA-CEPT8 On The Formation Of Chicken Primordial Germ Cells

Primordial germ cells belong to upstream cells in germ cells and have the potential to differentiate into spermatogonia or oogonia.Primordial germ cells have always been the focus of researchers.Through the gradual exploration of primordial germ cells from shallow to deep,revealing the key genes in the process of germ cell formation and development and elucidating their mechanism of action have become a hot spot in reproductive biology.Research on primordial germ cells is of great significance in many aspects:(1)Research on primordial germ cells is conducive to the production of genetically modified animals and related biological drugs;(2)Nowadays,a large number of reproductive diseases plague people,because of ethical issues,experiments on human embryos are not allowed,and Research on primordial germ cells is helpful to reveal the growth phase of the reproductive system.The mechanism of gene action and cell development provide theoretical basis for fundamentally solving human reproductive diseases;(3)Primordial germ cells are progenitor cells of gametes of sexual reproductive animals.Studying the formation and differentiation of primordial germ cells can help to protect germplasm resources and even make it possible to reproduce and revive extinct species across species.However,in the current study of primordial germ cells,the lack of access to primordial germ cells has been restricting the development of related research.Primordial germ cells induced in vitro have many advantages,such as low cost,easy access to experiments and so on.However,due to the lack of in-depth study on the regulation mechanism of primordial germ cell formation,it is difficult to obtain a large number of high-quality primordial germ cells in vitro,so the research on primordial germ cell formation becomes very important.Previously,high-throughput single-cell sequencing of chicken embryonic stem cells(ESC)and primordial germ cells(PGC)was carried out in vivo.According to the results,some lncRNAs with high specific expression in PGC were screened,and lncRNA-CEPT8 is one of the lncRNAs with high specific expression in chicken PGC.Because of its low expression in ESC,it is highly expressed in chicken PGC.It is speculated that the IncRNA is involved in the regulation of the formation of PGC.In order to study the function and regulation mechanism of lncRNA-CEPT8 in chicken PGC formation,the Rugao Yellow Chicken was used as the research object.The function and mechanism of lncRNA-CEPT8 in the formation of PGC were explored in vivo and in vitro by RNA interference technology,which provided a theoretical basis for improving the efficiency of PGC induction in vitro.The results are as follows:(1)lncRNA-CEPT8 full-length amplification,subcellular localization and coding ability identification:Based on high-throughput sequencing results,a specific high-expression lncRNA in chicken PGC was screened,named lncRNA-CEPT8.The full-length of lncRNA-CEPT8(1248 bp)was obtained by RACE assay,and its specific high-expression in chicken PGC cells was confirmed by qRT-PCR(ESC:2.562±0.285),PGC:6.447±0.837),SSC:3.852±0.261).Similar to the transcriptome sequencing results(ESC:0.074 ± 0.011,PGC:1.477 ± 0.235,SSC:0.100±0.003),subcellular localization revealed that lncRNA-CEPT8 was enriched in the cytoplasm(77.1%).A total of 246 BP ORF was obtained by predicting the sequence of lncRNA-CEPT8 through ORF Finder online software,encoding 81 amino acids,and constructing prokaryotic fusion expression vector.Western Blot was used to detect the coding ability of lncRNA-CEPT8.It was found that lncRNA-CEPT8 could encode a small peptide with a length of about 9KD,which proved that lncRNA-CEPT8 had coding ability(2)To study the function of lncRNA-CEPT8 in the process of PGC formation in vitro and in vivo:Three RNA interference sites were designed according to the full-length sequence of lncRNA-CEPT8.The interfering RNA sequences were linked to pGMLV-SC5 vector and transfected into DF-1 cells to detect the interference efficiency.The results showed that shRNA2-CEPT8 had the highest interference efficiency.Therefore,shRNA2-CEPT8 was used to encapsulate lentivirus.The full length of lncRNA-CEPT8 was cloned and pcDNA3.0-basic vector was connected to construct pcDNA3.0-CEPT8 vector.The expression of Cvh and C-kit marker genes in PGC cells isolated from genital crest was detected by qRT-PCR at 4.5 days after incubation.The expression of Cvh and C-kit in the interference group was significantly lower than that in the control group and the over-expression group.Flow cytometry analysis showed that the positive cell rate in the interference group was significantly lower than that in the control group and the over-expression group.Immunohistochemical staining of embryonic paraffin sections showed that the number of positive cells in the over-expression group was more than that in the control group and the interference group,and the number of positive cells in the interference group was still less than that in the control group.The interference and over-expression vectors were transfected into chicken ESC cells respectively,and the morphological changes of cells in each group were observed under BMP4-induced environment The results showed that the number of PGC-like cells formed after interfering with lncRNA was significantly less.The expression of Cvh and C-kit in the interference group was significantly lower than that in the control group and the over-expression group by qRT-PCR.Flow cytometry analysis showed that the positive cell rate in the interference group was significantly lower than that in the control group and the over-expression group.Indirect immunofluorescence showed that the expression of Cvh and C-kit in the over-expression group was significantly higher than that in the control group.Group B and interference group.The results of in vivo experiment were consistent with those of in vitro experiment.It was concluded that IncRNA-CPET8 played an important role in the formation of chicken PGC.(3)lncRNA-CEPT8 targeting gene Trim8 and activating JAK/STAT signaling pathway in the process of PGC formation:According to bioinformatics prediction,lncRNA-CEPT8 targeting Trim8 gene was inferred,Trim8 gene sequence was obtained on NCBL and Trim8 gene primers were designed.The expression of Trim8 gene in PGC was detected by qRT-PCR after interference and overexpression of lncRNA-CEPT8.The results showed that the expression of Trim8 gene had the same trend as that of lncRNA-CEPT8.Three RNA interference sites were constructed according to Trim8 sequence,and three RNA interference sites were designed to connect the interfering RNA sequence to pGMLV-SC5 vector.The interfering efficiency was detected by transfection into DF-1 cells.The results showed that shRNA3-Trim8 had the highest interference efficiency.Therefore,lentiviral encapsulation experiments were carried out using shRNA3-Trim8.The full length of Trim8 was cloned and pcDNA3.0-basic vector was connected to complete the experiment.Overexpression of pcDNA3.0-Trim8 vector was constructed.The expression of Cvh and C-kit marker genes in PGC cells isolated from genital crest was detected by qRT-PCR at 4.5 days after incubation.The expression of Cvh and C-kit in the interference group was significantly lower than that in the control group and the over-expression group.Flow cytometry analysis showed that the positive cell rate in the interference group was significantly lower than that in the control group and the over-expression group.After PAS staining of embryonic paraffin sections,the number of positive cells in the over-expression group was more than that in the control group and the interference group,and the number of positive cells in the interference group was still less than that in the control group.The interference and over-expression vectors were transfected into chicken ESC cells,respectively.The morphological changes of cells in each group were observed under BMP4-induced environment.The results showed that the number of PGC-like cells in the interference Trim8 group was significantly less than that in the control group and the interference group.Over-expression group;qRT-PCR detection of Cvh and C-kit of PGC cell marker gene showed that the expression level of interference group was significantly lower than that of control group and over-expression group;flow cytometry analysis showed that the positive cell rate of interference group was significantly lower than that of control group and over-expression group;after interference and over-expression of Trim8 gene,qRT-PCR detection of JAK/STAT signaling pathway marker genes JAK2 and STAT3 showed that JAK2 and STAT3 were significantly lower than that of control group and over-expression group.JAK/STAT signaling pathway was activated.It is concluded that the target gene Trim8 of lncRNA-CEPT8 activates JAK/STAT signaling pathway by up-regulating Trim8 gene.(4)Identification and transcription factor analysis of core region of lncRNA-CEPT8 promoter:Bioinformatics predicted the promoter region of lncRNA-CEPT8,designed primer PCR to amplify 1170 bp upstream segment of IncRNA,constructed eukaryotic expression vector pEGFP-CEPT8,and transfected DF-1 cells,found that the cloned fragment could express fluorescence,proved its promoter activity;constructed promoter deletion vector p1-248,respectively.P2-564,p3-763,p4-946,p5-1170.After transfection into DF-1 cells,the relative fluorescence expression was detected by double luciferase reporting system.The results showed that p3-763 had the highest activity,so it was the core region of lncRNA-CEPT8 promoter.Predicting the transcription factor binding sites of p3-763,we found that there might be multiple transcription factor binding sites,and selected transcription factors PAX2,PAX5 and Klf4 to construct point mutations.The results showed that PAX2 and PAX5 negatively regulated the expression of lncRNA-CEPT8,Klf4 positively regulated the expression of lncRNA-CEPT8,and methylation inhibitor 5'Azadc and acetylation inhibitor TSA were added to the culture medium of DF-1 cells transfected with pGL3-p3(564bp-763bp core region of promoter region).The activity decreased significantly.In conclusion,the core region of lncRNA-CEPT8 promoter is 564 bp-763 BP upstream.There is a transcription factor PAX2.PAX5 negatively regulates the expression of lncRNA-CEPT8.Klf4 positively regulates the expression of lncRNA-CEPT8.Methylation and acetylation all affect the activity of lncRNA-CEPT8 promoter.