Effect Of FOXL2 On The Expression Of COL4A1 Collagen Gene In Chicken Granulosa Cells

FOXL2(Forkhead box L2)is a member of the forkhead box(FOX)transcription factor superfamily.It is specifically expressed in the ovarian granulosa cell and is essential for the development of ovarian follicles.In the RNA sequencing of FOXL2 in chicken ovarian granulosa cells of our laboratory,we found that FOXL2 not only regulated the transcription of steroid hormone synthase and estrogen receptor,etc,but also was closely related to the expression of collagen genes in granulosa cells.This study explored the effect of FOXL2 on collagen gene expression in granulosa cells by overexpressing FOXL2 in precursor granulosa cells(pre-GC),pre-hierarchical follicle granulosa cells(ph-GC)and pre-ovulation follicle granulosa cells(po-GC).At the same time,the dual luciferase reporter system and chromatin immunoprecipitation(Ch IP)were performed to study how FOXL2 regulated the expression of collagen genes.The main results are as follows:(1)After Transfection with pc DNA3.1-FOXL2 48 h,the expression of total collagen in the cell supernatant of granulosa cells and chicken fibroblasts(DF-1)was detected by ELISA.The results showed that the expression level of total collagen in pre-GC was significantly reduced(P<0.05),and the expression level of total collagen in po-GC was significantly increased(P<0.05),but there was no significant change in ph-GC and DF-1,indicating that FOXL2 can affect the expression of collagen in a part of chicken granulosa cells;(2)To further explore the effects of FOXL2 on specific collagen genes in granulosa cells,the pc DNA3.1-FOXL2 was transfected into granulosa cells and DF-1,and the total RNA was collected after 24 h.The m RNA expression levels of FOXL2,COL1A1,COL1A2,COL2A1,COL3A1,COL4A1,COL4A4,COL5A2,COL6A3 and COL8A1 were detected by q PCR.As a result,it was found that FOXL2 was overexpressed 5-60 times in various cells,and the expression level of COL4A1 in each cell decreased significantly with the overexpression of FOXL2(P<0.05);(3)It is known that FOXL2 significantly reduced the expression of COL4A1 at them RNA level in various granulosa cells.In order to further explore whether FOXL2 affects the expression of COL4A1 protein,pc DNA3.1-FOXL2 were transfected into various granulosa cells and DF-1 of chicken,and total protein was collected after 48 h.Western Blot(WB)was used to verify that overexpression of FOXL2 in granulosa cells and DF-1 would significantly reduce the protein expression of COL4A1(P<0.05);(4)The COL4A1 promoter dual luciferase reporter vectors: PGL3-COL4A1-WT(normal sequence vector)and PGL3-COL4A1-MUT(mutant sequence vector)were successfully constructed,and they were co-transfected with pc DNA3.1-FOXL2 into DF-1respectively.Fluorescence detection was performed after 48 h.The results showed that,compared with the control group,the fluorescence value of PGL3-COL4A1-WT and pc DNA3.1-FOXL2 co-transfected group was significantly reduced(P<0.05),while the fluorescence value of PGL3-COL4A1-MUT and pc DNA3.1-FOXL2 co-transfected group was not significantly different from that of the control group(P>0.05).It indicated that FOXL2 directly regulated the transcription of COL4A1,and the binding site was located between-640 bp and-625 bp upstream of the coding region of COL4A1,and the sequence was CAGCAGCACCAGCAG;(5)The FOXL2 overexpression vector(PCMV-FOXL2-C-HA)containing HA tag protein was constructed and transfected into DF-1.The overexpression of FOXL2 was detected by q PCR after 48h(P<0.001).In order to further verify the specificity of vector,FOXL2 protein bands were detected clearly by Ch IP-WB using anti-HA antibody;(6)After transfection with PCMV-FOXL2-C-HA vector into DF-1 48 h,the enriched promoter sequence with anti-HA antibody was detected by Ch IP-q PCR,which was twice as much as the enriched sequence with anti-Ig G antibody(P<0.05),the direct regulation of FOXL2 on COL4A1 was verified once again;In conclusion,this study found that FOXL2 can regulate the expression of different genes encoding collagen in chicken granulosa cells,and FOXL2 can directly regulate the expression of COL4A1 by binding to the CAGCAGCACCAGCAG sequence between-640 bp and-625 bp upstream of the coding region of COL4A1.The biological significance ofthis regulation still needs further study.