Whole Transcriptome Analysis On Chicken Small Yellow Follicles With Different FSHR Expression Levels

Follicle selection has become a hot topic in poultry reproductive biology in recent years.The study of molecular mechanism of follicle selection is required to fully explore follicle resources in hens and improve the laying and reproductive performance of hens.Follicle selection mainly depends on the regulation of FSH secreted by pituitary and the expression of FSHR.The follicle with high expression level of FSHR shows great responds to the stimulation of FSH.In previous studies,non-coding RNAs including lncRNA and miRNA play an important role in regulating biological processes such as cell differentiation,transcriptional activation and transcriptional interference,but little is known about how it affects the development of animal ovarian follicles,especially the effects on follicle development and selection of avian ovary.In this study,the whole transcriptome sequencing of small yellow follicles with different expression level of FSHR in Jining Bairi chicken was performed,and the expression of 30 randomly selected mRNAs,lncRNAs and miRNAs was validated by qPCR.Preliminary studies and bioinformatics analysis were performed on the selected mRNA,lncRNA,miRNA and their target genes.The results are as follows:1.Basic statistics of the whole transcriptome sequencing data.About 99,105,865 clean reads were obtained from SY1,accounting for 98.87% of the raw reads,and contained 42,794 mRNAs,8011 ncRNAs,and 200 miRNAs.About 10605080 clean reads were obtained from SY2,accounting for 98.83% of the raw reads,and contained 42850 mRNAs,8138 ncRNAs,and 229 miRNAs.2.Identification and functional prediction of differentially expressed genes.Totally 815 significant differentially expressed mRNAs were screened,of which 223 were significantly upregulated and 592 were significantly downregulated.The results showed that these differentially expressed genes were enriched in the pathways of cell process-related phagocytosis,environmental information processing related neuroactive ligand-receptor interaction and related pathways.The functional annotations showed that these genes are significantly enriched in the extracellular region,responding to external stimulus,etc.;A total of 134 significant differentially expressed lncRNAs were screened,of which 52 were significantly upregulated and 82 were significantly downregulated.Selection of 29 significantdifferential expression lncRNAs to predict the target gene,showed that the target genes were enriched in metabolic pathways,FoxO signaling pathways,and most of which were significantly enriched in binding,cell and cell structure.Thirty-four significant differentially expressed miRNAs were screened,including three upregulated miRNAs and 31 downregulated miRNAs;We selected 33 differentially expressed miRNAs to predict their target genes,the results showed that the target genes were enriched in AGE-RAGE signaling pathway in diabetic complications,neuroactive ligand-receptor interaction and other related pathways.Functional annotation indicated that most genes are significantly enriched in binding,single organism process,biological regulation,membrane and so on.3.Integration analysis results.In recent years,lncRNA was shown to interact with miRNAs to collectively regulate the expression of target genes.For example,lncRNA can increase the expression level of target gene by competitively binding with miRNA.By comparing the relationship of differentially expressed genes,differentially expressed lncRNA target genes and differentially expressed miRNA target genes,we found that the SowahA is the target gene of miRNA-6615-3p,miRNA-1560-3p,miRNA-3540 and lncRNA-210520.2.Analysis of the sequencing results showed that the expression levels of SowahA and lncRNA-210520.2 were significantly upregulated by FSHR,while the expression levels of the three miRNAs were downregulated.4.The function of lncRNA-210520.2.After designing and transfecting siRNA against lncRNA-210520.2 into granulosa cells of hierarchal follicle,quantitative analysis showed that with the decline of lncRNA-210520.2 expression,the expression of target gene SowahA was significantly upregulated,while no significant changes were detected in the expression of three miRNAs;the expression of CYP11A1 was significantly upregulated after siRNA with lncRNA-210520.2;the proliferation rate of granulosa cells of hierarchal follicle was significantly higher than control group at 24 h after transfection of siRNA.The results indicated that inhibition of lncRNA-210520.2 could increase the secretion level of steroid hormones,also could stimulate the proliferation of follicle granulosa cells.In summary,the whole transcriptome sequencing of small yellow follicles with different expression levels of FSHR was performed,and a large number of differentially expressed mRNAs,lncRNAs and miRNAs were identified.The results indicate that SawahA is a common target gene for miRNA-6615-3p,miRNA-1560-3p,miRNA-3540 and lncRNA-210520.2.During chicken follicular development,lncRNA-210520.2 can inhibit the secretion level of steroid hormones and the proliferation of follicular granulosa cells.These results could provide a reference for the elucidation of the molecular mechanism of follicleselection in hens.