Cloning and Identification of Differentially Expressed lncRNAs in Follicles of Meishan Pigs and Duroc Pigs with Their Correlation Analysis with miRNAs
[Objective]The objective of this study was to clone and identify the differentially expressed lncRNA-ALDBSSCT0000005583 in M2 follicles on the fourth day of follicular stage in Meishan pigs and Duroc pigs,and to analyze the correlation between the expression of miRNAs in porcine granulosa cells,so as to provide a theoretical basis for exploring the role of lncRNAs in the development of follicles in sows by regulating miRNAs.[Method]Based on the differentially expressed lncRNA-ALDBSSCT0000005583 screened in Meishan and Duroc M2 follicles in our early research,the full-length sequence of ALDBSSCT0000005583 was verified by RT-qPCR and cloned by RACE;the coding ability of this lncRNA was predicted by the coding potential assessment tool of CAPT and CPC,which was further identified by the primary expression test;the coding ability of this lncRNA was identified by the primary expression test;the subcellular coding ability of NA-ALDBSSCT0000005583 was identified by the nucleoplasmic separation experiment and tested to identify its coding ability;the subcellular localization of lncRNA-ALDBSSCT0000005583 by nucleoplasmic isolation assay and its expression level in various tissues were detected by RT-qPCR;miRBase website was used to locate the miRNA database of pigs,and the combination of RNAhybrid and miRanda online software was used to predicte the relationship with the lncRNA-ALDBSSCT0000005583.The inter-species conserved miRNAs that interacted with lncRNA-ALDBSSCT0000005583 were predicted by TargetScan and miRanda,the target genes that interacted with lncRNA-ALDBSSCT0000005583 were predicted by TargetScan and miRanda,and their target genes were subjected to GO enrichment and KEGG signaling pathway analyses;the effects of target genes on miRNA expression were verified by overexpression as well as interference with lncRNA.[Result]The expression level of lncRNA-ALDBSSCT0000005583 in M2 follicles of Duroc pigs was significantly higher than that in Meishan pigs,and the size of lncRNA 5′RACE and 3′RACE fragments was 569 bp and 546 bp,respectively,and the sequencing analysis showed that the size of lncRNA-ALDBSSCT0000005583 was 588 bp.Bioinformatics predicted that the encoding potential was low,and the results of prokaryotic expression assay further proved that it did not code for proteins.Tissue expression profiling showed that lncRNA-ALDBSSCT0000005583 was expressed in the adrenal gland,spleen,liver and ovaries,and low in the hypothalamus and heart,while the subcellular localization results showed that the lncRNA was mainly present in the cytoplasm.After bioinformatics analysis,a total of 9 conserved miRNAs were screened for potential interaction with lncRNA-ALDBSSCT0000005583,including two miRNAs related to ovarian development:miR-193a-5p and miR-361-3p.KEGG and GO enrichment analysis showed that the target genes of miR-193a-5p and miR-361-3p were related to phylogenetic processes and biological processes such as cell-to-cell signaling.It was also significantly involved in oxytocin,Ras,NF-κB gonadotropin-releasing hormone secretion and other pathways.Subsequently,lncRNA-ALDBSSCT0000005583 was overexpressed in granulosa cells,and the expressions of miR-193a-5p and miR-361-3p were significantly down-regulated by RT-qPCR(P＜0.05),but there was no significant effect after interference with lncRNA-ALDBSSCT0000005583.[Conclusion]lncRNA-ALDBSSCT0000005583 was a lncRNA that did not have the ability to code for proteins.There was a significant difference in the expression level between the medium follicles of Meishan and Duroc pigs,and the expression level was higher in the adrenal glands,spleen,liver and ovaries,which is mainly found in the cytoplasm of granulosa cells,and might be involved in the development of porcine ovarian granulosa cells by interacting with miR-193a-5p and miR-361-3p.
国家科技期刊平台
NSTL
万方数据
维普
