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

摘要/Abstract

摘要： 旨在研究oar-miR-127/FOXO4反馈环路及其对绵羊卵泡颗粒细胞的作用。本研究利用Promoter 2.0预测绵羊miR-127启动子，利用JASPAR数据库预测转录因子FOXO4与oar-miR-127启动子区的结合位点，构建包含预测结合位点的pGL3-basic-miR-127荧光素酶报告载体和pcDNA-FOXO4过表达载体；利用TargetScan软件在线预测oar-miR-127与FOXO4基因3'-UTR区结合位点，构建包含预测结合位点的pmir-GLO-FOXO4野生型、突变型和缺失型荧光素酶报告基因重组质粒；将pGL3-basic-miR-127荧光素酶报告载体和pcDNA-FOXO4过表达载体共（或单独）转染体外培养的绵羊卵泡颗粒细胞，FOXO4突变型、野生型和缺失型重组质粒与miR-127 mimic/mimic NC共转染体外培养的293T细胞，48 h后检测荧光素酶活性；pcDNA-FOXO4过表达载体（miR-127 mimic/mimic NC）转染绵羊卵泡颗粒细胞48 h，利用qRT-PCR检测miR-127（FOXO4）及凋亡基因（Casp3、Bax及BCL2）的表达水平。过表达转录因子FOXO4显著降低了oar-miR-127启动子相对荧光素酶活性（P<0.05）和oar-miR-127表达水平（P<0.05）；miR-127 mimic和FOXO4野生型重组质粒共转染的颗粒细胞荧光素酶活性显著低于共转染miR-127 mimic和FOXO4缺失/突变型重组质粒的细胞（P<0.05）。转染miR-127 mimic的颗粒细胞中FOXO4表达量显著降低（P<0.05），Casp3和Bax基因表达水平极显著升高（P<0.001）；过表达转录因子FOXO4的颗粒细胞中Casp3和Bax的表达水平无显著变化（P>0.05），但BCL2相对表达量显著降低（P<0.05）。本研究证实了oar-miR-127与靶基因FOXO4间存在反馈环路，并促进绵羊卵泡颗粒细胞的凋亡，为研究其在绵羊卵泡发育中的作用机制奠定了基础。

关键词: miR-127, FOXO4, 绵羊颗粒细胞, 细胞凋亡

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

中图分类号:

S826.2

付强, 岳巧娴, 锡建中, 宋鹏琰, 陈晓勇, 周荣艳. 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[J]. 畜牧兽医学报, 2022, 53(1): 66-75.

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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