Effect Of TGIF1 On E2/P4 Secretion And Apoptosis In Ovarian Granulosa Cells Of Dairy Goats

TG interaction factor 1(TGIF1)belongs to TALE homologue domain protein family.TGF-?1 can regulate hormone secretion and apoptosis on ovarian granulosa cells.In addition,the activity of TGF-? signaling pathway that is impacted by histone acetylation can be inhibited by TGIF1.However,the molecular regulation mechanism of TGIF1 on hormone secretion and apoptosis in ovarian granulosa cells of dairy goats has not been studied.In this study,Guanzhong dairy goats were used as research subjects,and ovarian granulosa cells were transfected with TGIF1 overexpression vector and TGIF1-si RNA,respectively.This study explored the mechanism of TGIF1 on the secretion of E2/P4 in ovarian granulosa cells and analyzed the effect of TGIF1 on apoptosis of ovarian granulosa cells to elucidate the biological function of TGIF1 gene in follicular development and atresia.The main results are as follows:1.Constructed TGIF1 overexpression vector and synthesized TGIF1-si RNA successfullyThe full length of CDS region of TGIF1 gene was predicted by NCBI,and the full length of 819 bp CDS region was amplified.The TGIF1 overexpression vector containing Hind III and Xho I restriction sites was constructed named pc DNA3.1-TGIF1,and the TGIF1 interfering RNA named TGIF1-si RNA was synthesized.The overexpression and interference of TGIF1 were detected by RT-q PCR and Western blot assay.The results have been found that pc DNA3.1-TGIF1 could significantly increase the expression of TGIF1,while TGIF1-si RNA could significantly decrease the expression of TGIF1.2.TGIF1 regulated E2/P4 secretion of ovarian granulosa cellsThe ovarian granulosa cells were respectivelytransfected into pc DNA3.1-TGIF1 and TGIF1-si RNA.The results were shown that overexpression of TGIF1 could significantly decrease the secretion of E2 and P4 in ovarian granulosa cells and interference of TGIF1 significantly increased the secretion of E2 and P4 in ovarian granulosa cells.More study has been shown that TGIF1 can significantly reduce the expression of hormone synthesis-related gene CYP19A1,CYP11A1,STAR,3B-HSD.These results suggested that TGIF1 can regulate the secretion of E2 and P4 by regulating the expression of hormone synthesis related genes.3.TGIF1 regulated the secretion of E2/P4 by granulosa cells through the TGF-? signaling pathwayTo investigate whether TGIF1 inhibits TGF-? signaling pathway and regulates E2/P4 secretion,ovarian granulosa cells were treated with TGF-? signaling pathway activator KGN(Kartogenin)for 2 h,6 h,12 h.It was found that the secretion of E2 and P4 increased significantly at 6 h,and the phosphorylation level of SMAD2/3 protein in TGF-? signaling pathway was the highest.After 6 h of KGN treatment,pc DNA3.1-TGIF1 was transfected into ovarian granulosa cells.Compared with control group,the result showed that TGIF1 could inhibit the effect of KGN on E2 and P4 by using ELISA.Further studyshowed that TGIF1 could significantly decrease the expression of SMAD2/3 protein and the phosphorylation level of SMAD2,however,knockdown of TGIF1 increased the expression of SMAD2/3 protein and its effect on phosphorylation was not significant.The results indicate that TGIF1 regulates the secretion of E2 and P4 through the TGF-?-SMAD signaling pathway.4.TGIF1 regulates the secretion of E2/P4 by influncing the activity of transcriptionfactor SP1In this study,we found that TGIF1 could significantly decrease the expression of transcription factor SP1 by using Western blot.To explore the role of SP1 in regulating the secretion of hormone by TGIF1,we knocked down the expression of SP1 via si RNA successfully.Then we detected the expression of key enzyme genes CYP19A1,CYP11A1,STAR and 3B-HSD after co-transfection of pc-TGIF1+si-SP1 and si-TGIF1+si-SP1.It was found that TGIF1 inhibits the expression of key enzyme genes for hormone synthesis by affecting the activity of transcription factor SP1.5.TGIF1 regulated apoptosis of ovarian granulosa cells through BCL-2/BAX pathway(1)The effect of TGIF1 on apoptosis of ovarian granulosa cells was detected by flow cytometry.The results showed that overexpression of TGIF1 significantly inhibited the apoptotic rate of granulosa cells compared with the control group,and knockdown of TGIF1 significantly increased the apoptosis rate of granulosa cells.(2)The expression of apoptosis-related genes which consisted of BCL-2,BAX,P53,Caspase3 was detected by RT-q PCR and Western blot.The results demonstrated that over-expression of TGIF1 significantly decreased the expression of BAX,P53,Caspase3 genes compared with the control group,while it had no significant effect on the expression of BCL-2.Knockdown of TGIF1 could significantly increase the expression of BAX,P53 genes,while it had no significant effect on the expression of BCL-2,Caspase3 genes.More studies indicated that over-expression of TGIF1 could significantly increase the ratio of BCL-2/BAX,while knockdown of TGIF1 significantly decreased the value of BCL-2/BAX.In conclusion,TGIF1 regulated the secretion of E2 and P4 through TGF-?-SMAD signaling pathway in ovarian granulosa cells.On the one hand,TGIF1 could inhibit the expression of SMAD2/3 and its phosphorylation level.It resulted in the decline of the transcription of hormone-related genes.On the other hand,TGIF1 could directly inhibit the expression of transcription factor SP1.Both of TGIF1 and transcription factor SP1 could reduce the expression of hormone-related genes CYP19A1,CYP11A1,STAR,3B-HSD.Besides,TGIF1 could inhibit the apoptosis of ovarian granulosa cells through BCL-2/BAX pathway.The above studies provided a theoretical basis for further elucidating the mechanism of the effect of TGIF1 on the secretion and apoptosis of granulosa cells and the pathway of follicular development.