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。
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 expre
作者
付强
岳巧娴
锡建中
宋鹏琰
陈晓勇
周荣艳
FU Qiang;YUE Qiaoxian;XI Jianzhong;SONG Pengyan;CHEN Xiaoyong;ZHOU Rongyan(College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第1期66-75,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
河北省自然科学基金(C2019204039)
河北农业大学精准畜牧学科群建设项目(1090064)。
