miR-221-3p靶向BCL2L11调控小尾寒羊卵泡颗粒细胞凋亡

doi:10.3864/j.issn.0578-1752.2022.09.015

摘要/Abstract

摘要：

【目的】BCL2L11在哺乳动物中能够促进多种细胞凋亡,同时参与繁殖性状相关组织器官的发育及疾病治疗,文章利用分子生物学方法探究miR-221-3p靶向调控BCL2L11对小尾寒羊卵泡颗粒细胞凋亡的影响,为进一步研究BCL2L11在卵泡颗粒细胞凋亡和卵泡闭锁过程中的调控作用提供依据。【方法】在前期课题组卵巢组织全转录组测序分析的基础上,获得了候选基因BCL2L11及其调控元件miR-221-3p,利用半定量和组织荧光定量（RT-qPCR）分析BCL2L11在小尾寒羊不同组织中的表达情况;通过RT-qPCR定量试验在小尾寒羊卵泡期和黄体期卵巢组织中鉴定了BCL2L11及miRNA-221-3p的表达情况;构建BCL2L11 3’UTR野生型和突变型载体,在HEK293T细胞中共转染miR-221-3p mimic和BCL2L11野生型和突变型及阴性对照,采用双荧光素酶报告基因检测系统确定miR-221-3p与BCL2L11靶向性关系;在绵羊卵巢原代颗粒细胞中转染miR-221-3p mimic及阴性对照实现miR-221-3p过表达,使用RT-qPCR技术在mRNA水平上检测miR-221-3p对BCL2L11以及卵巢颗粒细胞凋亡标志基因XIAP和Fas表达水平的影响;同时利用EdU试验分析miR-221-3p过表达和阴性对照组中颗粒细胞的增殖变化。【结果】半定量和组织RT-qPCR分析均表明BCL2L11在卵巢组织中表达量高于其他组织;RT-qPCR定量结果显示miR-221-3p和 BCL2L11在小尾寒羊卵泡期和黄体期卵巢组织中差异表达,miR-221-3p在卵泡期卵巢中的表达量高于黄体期,而BCL2L11在卵泡期卵巢中的表达量低于黄体期,表现出负调控的现象;双荧光素酶报告基因验证分析显示,过表达miR-221-3p mimic显著抑制了BCL2L11 3’UTR荧光素酶的活性（P<0.05）,阴性对照组则没有显著影响;过表达miR-221-3p,靶基因BCL2L11 mRNA表达水平显著降低,同时,卵泡颗粒细胞凋亡标志基因XIAP和Fas的表达量也显著降低（P<0.05）;EdU试验分析显示,过表达miR-221-3p的颗粒细胞增殖率为18.9%,极显著高于阴性对照组的10.43%（P<0.01）。【结论】BCL2L11和miR-221-3p是调控绵羊卵巢发育的重要基因及调控元件,BCL2L11是miR-221-3p的靶基因之一,miR-221-3p过表达可抑制颗粒细胞凋亡,该作用结果可能通过抑制靶基因BCL2L11的表达进而影响了绵羊卵巢颗粒细胞的凋亡。

关键词: miR-221-3p, 小尾寒羊, 卵巢颗粒细胞凋亡, BCL2L11

Abstract:

【Objective】BCL2L11 could promote apoptosis in mammals and is involved in the development of tissues and organs related to reproductive traits and in disease treatment. The aim of this study was to explore the effect of miR-221-3p on the regulation of granulosa cell apoptosis in Small-Tail Han sheep by targeting BCL2L11, so as to provide evidence for further study of the regulation of BCL2L11 in granulosa cell apoptosis and atresia of follicles.【Method】Based on the whole transcriptome sequencing analysis of the ovarian tissue of the previous study in our group, the differentially expressed gene BCL2L11 and its regulatory element miR-221-3p were obtained in this study. Analysis of the expression of BCL2L11 in different tissues of the Small-Tail Han sheep by using semi-quantitative and tissue fluorescence quantification (RT-qPCR). The expression of BCL2L11 and miRNA-221-3p was identified in the ovarian tissues of the Small-Tail Han sheep in the follicular and luteal phase by RT-qPCR. The miR-221-3p mimic, BCL2L11 wild-type and BCL2L11 mutant-type were co-transfected in HEK293T cells with negative control, the dual luciferase reporter gene detection system was used to determine the targeting relationship between miR-221-3p and BCL2L11. The miR-221-3p mimic and negative control were transfected into ovarian granulosa cells to achieve the overexpression of miR-221-3p. RT-qPCR was used to detect the effect of miR-221-3p on the expression levels of BCL2L11 and the marker genes of the apoptosis of ovarian granulosa cell gene XIAP and Fas at the mRNA level. At the same time, the changes in proliferation of granulocytes in the miR-221-3p overexpression and negative control groups were also analyzed using the EdU assay. 【Result】The results showed that the expression of BCL2L11 in ovarian tissue was the highest, followed by spleen and lung tissues. RT-qPCR results showed that the expression of miR-221-3p and BCL2L11 was significantly different in the ovarian tissues of Small-Tail Han sheep between follicular and luteal phases. The expression of miR-221-3p was higher in follicular than that in luteal phase ovaries, whereas BCL2L11 was less expressed in follicular than that in luteal phase ovaries, which showed the phenomenon of a negative regulation. Dual luciferase reporter analysis showed that overexpression of miR-221-3p significantly inhibited the activity of BCL2L11 3’UTR vector (P<0.05). The overexpression of miR-221-3p significantly reduced the mRNA level expression of target gene BCL2L11, while follicular granulosa cell apoptosis expression of marker genes XIAP and Fas were also significantly reduced (P<0.05). Analysis of the EdU assay showed that the proliferation rate of granulosa cells overexpressing miR-221-3p was 18.9%, which was significantly higher than that of the negative control group at 10.43% (P<0.01). 【Conclusion】BCL2L11 and miR-221-3p were important genes and regulatory elements that regulate ovarian development in Small-Tail Han sheep. BCL2L11 was one of the target genes of miR-221-3p, and overexpression of miR-221-3p could inhibit granulosa cell apoptosis by target BCL2L11.

Key words: miR-221-3p, Small-Tail Han Sheep, granulosa cells apoptosis, BCL2L11

刘玉芳,陈玉林,周祖阳,储明星. miR-221-3p靶向BCL2L11调控小尾寒羊卵泡颗粒细胞凋亡[J]. 中国农业科学, 2022, 55(9): 1868-1876.

LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing. miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep[J]. Scientia Agricultura Sinica, 2022, 55(9): 1868-1876.

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基因名称 Name	引物序列 Primer sequence	片段大小 Length (bp)	退火温度 Tm (℃)
BCL2L11	F: 5′ ACCATTATCTCAGTGCAATGGC 3′	130	60.0
BCL2L11	R: 5′ CCAAAGGGTTGTAACCCTTCTT 3′	130	60.0
XIAP	F: 5′ CCAGCATCGTGAGGGTCAAT 3′	156	59.5
XIAP	R: 5′ TTCAGAGCAAGGCACACACT 3′	156	59.5
Fas	F: 5′ ATGCCAGCCTGGTGAACG 3′	109	59.5
Fas	R: 5′ ACTTCTAAACCGTGCCCTTCAT 3′	109	59.5
RPL-19	F: 5′ ATCGCCAATGCCAACTC 3′	154	60.0
RPL-19	R: 5′ CCTTTCGCTTACCTATACC 3′	154	60.0