Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (9): 1868-1876.doi: 10.3864/j.issn.0578-1752.2022.09.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep

LIU YuFang1,2(),CHEN YuLin1,2,ZHOU ZuYang1,2,CHU MingXing1,*()   

  1. 1Institute of Animal Science, Chinese Academy of Agricultural Sciences/Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and Rural Affairs, Beijing 100193
    2College of Life Science and Food Engineering, Hebei University of Engineering, Handan 056001, Hebei
  • Received:2021-03-04 Revised:2021-10-31 Online:2022-05-01 Published:2022-05-19
  • Contact: MingXing CHU E-mail:aigaiy@126.com;mxchu@263.net

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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

Table 1

The sequences, amplified products and Tm（annealing temperature）of qPCR"

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
R: 5′ CCAAAGGGTTGTAACCCTTCTT 3′
XIAP F: 5′ CCAGCATCGTGAGGGTCAAT 3′ 156 59.5
R: 5′ TTCAGAGCAAGGCACACACT 3′
Fas F: 5′ ATGCCAGCCTGGTGAACG 3′ 109 59.5
R: 5′ ACTTCTAAACCGTGCCCTTCAT 3′
RPL-19 F: 5′ ATCGCCAATGCCAACTC 3′ 154 60.0
R: 5′ CCTTTCGCTTACCTATACC 3′

Fig. 1

The interaction sites of miR-221-3p and BCL2L11 was predicted and vector constructed"

Fig. 2

The expression profile of BCL2L11 in different tissues A: Semi-quantitative analysis of BCL2L11; B: The relative expression of BCL2L11. M: Marker; 1-10: Heart, liver, spleen, lung, stomach, hypothalamus, pituitary, ovary, uterus, oviduct"

Fig. 3

Relative expression of BCL2L11 and miR-221-3p in the LP and FP stages in Small Tail Han sheep LP"

Fig. 4

Dual-luciferase reporter assay of targeting relationship between miR-221-3p and BCL2L11"

Fig. 5

Overexpression of miR-221-3p inhibits BCL2L11 expression"

Fig. 6

Overexpression of miR-221-3p inhibits the granulosa cells apoptosis A：Overexpression of miR-221-3p inhibits XIAP and Fas expression; B: EdU assay of granulosa cells apoptosis (**P<0.01)"

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