Effect Of Knocking Down Chicken FOXL2 On Gene Expression In Granulosa Cells

The egg laying performance of hens,which is closely associated with their ovarian development,is an important trait in poultry production.However,the hens' ovarian development is a dynamic and complex process and the specific factors and mechanisms related to this process regulation are still unclear.In mammals,FOXL2 has been reported to be a key gene keep the normal reproductive physiology of females,and it was also considered as an important gene function on ovarian development.The mutates of FOXL2 cause failure of ovarian premature,infertility,even cancer.In this study,we analized the gene expression changes with FOXL2 knockdown in chicken ovary poGC(Pre-ovulatory follicle)and phGC(Pre-hierarchical follicle),aimed to reveal the pathway regulated by FOXL2,thus indicate the related mechanisms might involve in follicular selection and follicular development in chickens.The main results are as follows:1.Three siRNAs which specific targeting on FOXL2 gene were designed and synthesized.These siRNAs and NC-siRNA was transfected by liposomes into primary cultured poGC cells for 24 hours.The knockdown efficiency of FOXL2 gene at the level of mRNA was detected by qPCR,and results show that all the three siRNAs poccess significant knockdown effects which ranged from 55% to 80%,and the siRNA with the highest knockdown efficiency was selected and named as FOXL2-siRNA for subsequent experiments.2.After transfecting the FOXL2-siRNA(knockdown group)and NC-siRNA(control group)into primary cultured poGC and phGC cells,The mRNA expression levels of FOXL2 gene was detected using qPCR at three time intervals(24h,36 h and 48h).Results showed that the expression level of FOXL2 gene in the poGC and phGC groups were significantly reduced at 48 h post-transfection.3.After transfecting the FOXL2-siRNA(knockdown group)and NC-siRNA(control group)into primary cultured poGC and phGC cells,the expression of FOXL2 gene at protein level was also detected using Westrn Blot at different time intervals(24h,36 h and 48h).The results showed that relative expression of FOXL2 at protein level in the was not significantly changed at 24 h and 36 h post-transfection,but significantly decreased at 48 h post-transfection(P < 0.05).Combined with the above results(2),the FOXL2 gene was significantly decreased at both mRNA and protein levels after transfected FOXL2-siRNA 48 hours.4.Thus,four groups of cells(named as poGC-KD,poGC-CT,phGC-KD and phGC-CT respectively)after 48 h post-transfection were collected for RNA sequencing.Total RNA and protein were from each sample for detecting the relative expression of FOXL2 gene by qPCR Westrn Blot.The results showed that FOXL2 gene was significantly decreased in both poGC and phGC groups at the levels of protein and mRNA.The batch samples(4 replicates in each group,16 samples in total)were selected to be qualified and sequenced by library and transcriptome.5.Transcriptome sequencing was performed to annotate the expressed genes and calculate their expression levels.Then the samples were grouped and compared as follows: poGC-CT vs.poGC-KD(named Comp.po),phGC-CT vs.phGC-KD(named Comp.ph),respectively.The differentially expressed genes(DEGs)of knockdown FOXL2 in poGC and phGC were obtained.The results showed that there were 1309 DEGs in Comp.Po group,it contains 611 up-regulated genes and 698 down-regulated genes;and 775 DEGs were obtained in Comp.ph group,it contains 368 up-regulated genes and 407 down-regulated genes.6.GO annotation analysis showed that the DEGs in Comp.po mainly acted on the nucleus,which was closely related to the regulation of cell division,proliferation and chromosome changes during cell cycle.The DEGs in Comp.ph were mainly involved in intracellular signaling pathways and immune-related processes.7.KEGG pathway enrichment analysis showed that the DEGs in Comp.po were mainly concentrated in DNA replication,ECM-receptor interaction,p53 signaling pathway and cell cycle pathway;the DEGs in Comp.ph were mainly concentrated in ECM-receptor interaction,cytokine-cytokine receptor interaction and quarterly adhesion plaques.Combining the results of transcriptome sequencing(6)it revealed that FOXL2 has different roles in poGC and phGC,and affects cell proliferation,cell cycle and DNA replication in poGC.8.CCK-8,EdU and flow cytometry were used to detect the effects of FOXL2 knockdown on cell proliferation,DNA replication,apoptosis and cell cycle in granulosa cells.The results showed that knocking down FOXL2 gene in poGC could promote cell proliferation,promote DNA replication,inhibit cell apoptosis and promote cell cycle process,but had no significant effect on the characteristics of these biological processes in phGC.