绵羊miR-127/FOXO4反馈环路及其对卵泡颗粒细胞凋亡相关基因表达的影响

doi:10.11843/j.issn.0366-6964.2022.01.007

Abstract

Abstract: The aim of this study was to investigate the feedback loop between oar-miR-127 and target gene FOXO4 and its effect on sheep follicular granulosa cells. The miR-127 promoter region of sheep was predicted through Promoter 2.0. The binding sites of transcription factor FOXO4 in oar-miR-127 promoter region were predicted with JASPAR database. The pGL3-basic-miR-127 luciferase reporter vector containing the predicted binding site of FOXO4 and pcDNA-FOXO4 overexpression vector were constructed. The binding sites of oar-miR-127 located in 3'-UTR region of predicted target gene FOXO4 were analyzed with TargetScan software. Recombinant luciferase reporter plasmids of pmir-GLO-FOXO4 with wild-type, mutant-type and deleted fragment containing predicted binding sites were constructed. The pGL3-basic-miR-127 luciferase reporter vector and pcDNA-FOXO4 overexpression vector were co-transfected (or separately transfected) into sheep follicular granulosa cells cultured in vitro. The wild-type, mutant-type, and deleted FOXO4 recombinant plasmids were co-transfected with miR-127 mimic/mimic NC into 293T cells cultured in vitro, and luciferase activity was detected after 48 h. The expression levels of miR-127 (FOXO4) and apoptosis genes (Casp3, Bax and BCL2) in granulosa cells were detected by qRT-PCR after the pcDNA-FOXO4 overexpression vector (miR-127 mimic/mimic NC) was transfected into sheep follicular granulosa cells for 48 h. Overexpression of transcription factor FOXO4 significantly decreased the luciferase activity of miR-127 promoter (P<0.05) and the expression level of miR-127 (P<0.05); The luciferase activity of granulosa cells co-transfected with miR-127 mimic and FOXO4 wild-type recombinant plasmid was significantly lower than that of miR-127 mimic and FOXO4 deletion/mutant recombinant plasmid (P<0.05). The expression of FOXO4 in granulosa cells transfected with miR-127 mimic was significantly decreased (P<0.05), while the expression of Casp3 and Bax genes was extremely significantly increased (P<0.001). There was no significant change in the expression of Casp3 and Bax after overexpression of transcription factor FOXO4 in granulosa cells(P>0.05). The relative expression of BCL2was significantly decreased with overexpression of FOXO4 (P<0.05). This study confirmed a feedback loop between oar-miR-127 and target gene FOXO4, and both could promote the apoptosis of granulosa cells. These results laid a foundation for the study of its mechanism in the development of sheep ovarian follicles.

Key words: miR-127, FOXO4, sheep granulosa cells, apoptosis

CLC Number:

S826.2

FU Qiang, YUE Qiaoxian, XI Jianzhong, SONG Pengyan, CHEN Xiaoyong, ZHOU Rongyan. Effect of Oar-miR-127/FOXO4 Feedback Loop on Genes Associated with Apoptosis of Sheep Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 66-75.

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Effect of Oar-miR-127/FOXO4 Feedback Loop on Genes Associated with Apoptosis of Sheep Granulosa Cells

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