Research On The Mechanism Of MiR-202-5p Regulating The Proliferation,apoptosis,lipid Deposition And Steroid Hormone Synthesis Of Goose Follicular Granulosa Cells

Follicular selection during follicular development is a critical molecular event that determines the egg production performance of birds.Granular cells（GCs）,as an important component of follicles,their physiological and functional changes are an important manifestation of follicular selection.MiR-202-5p is one of the most abundant mature miRNA in some vertebrate reproductive organs and inhibits the proliferation of goat follicular GCs.However,its effect on GCs during goose follicular selection and its regulatory mechanism are still completely unknown.The present study took Tianfu meat goose maternal line as the experimental object,and thoroughly investigated the effects of miR-202-5p expression changes on the proliferation,apoptosis,lipid deposition,and steroid hormone synthesis of pre-hierarchical follicular granulosa cells（phGCs）and hierarchical follicular granulosa cells（hGCs）using overexpression and interference techniques.Combining transcriptome sequencing and target gene interference/overexpression assays,this study elucidated the regulatory mechanism of miR-202-5p on GCs during follicular selection.The main research results are as follows:1.The expression pattern of miR-202-5p showed a trend of increasing and then decreasing with the development of follicles.It peaked in the GCs of 8-10mm follicles,and decreased obviously in the GCs of F2-F4 follicles.2.The detection results of proliferation,apoptosis,lipid deposition and steroid hormone synthesis showed that after overexpression of miR-202-5p,the expression of CCND1,CCND2,BCL2,cell activity,and proliferation rate of phGCs and hGCs decreased significantly,while their apoptosis rate increased significantly.In addition,the expression of PPARγand St AR,deposition level of intracellular lipid droplets,the content of intracellular triglyceride（TG）,and the level of extracellular progesterone（P₄）of hGCs were also significantly decreased;Meanwhile,after miR-202-5p interference,the expression of CCND1,CCND2,BCL2,cell activity,the proliferation rate of phGCs and hGCs were significantly increased,and their apoptosis rate decreased significantly.Moreover,the expression of PPARγand St AR,and the deposition level of intracellular lipid droplets of hGCs were significantly increased.These results suggested that miR-202-5p was involved in inhibiting the proliferation of phGCs and hGCs,promoting the apoptosis of phGCs and hGCs,inhibiting the lipid deposition of hGCs,and inhibiting the steroid hormones synthesis of hGCs.3.Transcriptome sequencing results showed that in phGCs,45 genes were differentially expressed after miR-202-5p overexpression,and 721 genes were differentially expressed after miR-202-5p interference.In hGCs,70 genes were differentially expressed after overexpression of miR-202-5p,and 53 genes were differentially expressed after the interference of miR-202-5p.Functional enrichment analysis and gene annotation results revealed that DEGs related to the PI3K/Akt signaling pathway were present in both phGCs and hGCs.It is suggested that PI3K/Akt signal pathway may be the key pathway for miR-202-5p to regulate the proliferation and apoptosis of goose phGCs and hGCs.In addition,the DEGs screened in hGCs contain ACSF2 and PLA2G4F,which were related to fatty acid synthesis,indicating that miR-202-5p may inhibit intracellular lipid deposition of hGCs by inhibiting the expression of ACSF2 and PLA2G4F.4.The combined analysis of transcriptome sequence results and target genes predicted by the database found that BTBD10 was identified as a potential critical gene targeted by miR-202-5p to modulate the proliferation and apoptosis of GCs.Subsequent target relationship validation tests confirmed that miR-202-5p downregulated the expression of BTBD10 by targeting its 3’UTR region.After overexpression of BTBD10,the expression of CCND1,CCND2,and BCL2,cell activity and cell proliferation rate of phGCs and hGCs increased significantly,while the apoptosis rate decreased significantly;After the interference of BTBD10,the expression of CCND1,CCND2,and BCL2,cell activity and cell proliferation rate of phGCs and hGCs decreased significantly,while the apoptosis rate increased significantly.And compared to the co-transfection group of miR-202-5p mimic and negative control for overexpression（ov NC）,the co-transfection of miR-202-5p mimic and BTBD10 overexpression plasmid（ov BTBD10）restored the proliferation rate and apoptosis rate of phGCs and hGCs to levels that were not significantly different from the control group（mimic-NC and ov NC co-transfection group）.These results showed that miR-202-5p could inhibit the proliferation of geese phGCs and hGCs,and promote their apoptosis by targeting BTBD10.5.By integrating the predicted target gene by miRDB and Target Scan databases,ACSL3has been predicted as a potential critical gene targeted by miR-202-5p to modulate the lipid depositon of GC cells.Subsequent target relationship validation tests confirmed that miR-202-5p downregulated the expression of ACSL3 by targeting its 3’UTR region.The results of ACSL3interference test showed that the interference of ACSL3 could significantly inhibit the expression of PPARγ,DGAT2,and St AR.Besides,a significant reduction was also observed in intracellular lipid droplet deposition,TG content,and extracellular estradiol（E₂）level.These results suggest that miR-202-5p inhibit lipid deposition and steroid hormone synthesis of hGCs by targeting ACSL3.In conclusion,this study analyzed the molecular mechanism of miR-202-5p regulating the proliferation,apoptosis,lipid deposition and steroid hormone synthesis of GCs during goose follicular selection,providing a new theoretical reference for exploring the molecular mechanism of goose follicular selection.