miR-215-5p在雷州黑鸭组织中的表达及其对卵泡颗粒细胞增殖和凋亡的影响

doi:10.11843/j.issn.0366-6964.2025.04.021

摘要/Abstract

摘要：

为探究miR-215-5p在雷州黑鸭组织中的表达规律，验证miR-215-5p对卵泡颗粒细胞增殖凋亡的调控，本研究分别选取体重与健康情况相似的43周龄产蛋期与51周龄休产期雷州黑鸭各3只，利用RT-qPCR检测了其不同组织中miR-215-5p的表达水平。通过构建模拟物和干扰片段载体转染卵泡颗粒细胞，结合CCK-8和流式细胞术等技术检测细胞增殖和凋亡，每组均包含了3个重复。结果显示，miR-215-5p在产蛋期雷州黑鸭的表达普遍高于休产期，其在卵巢、膨大部和胸腿肌肉中相对高表达，在漏斗部、子宫和心脏中相对低表达；CCK-8和EdU试验结果显示，miR-215-5p抑制卵泡颗粒细胞增殖；流式细胞术检测发现，miR-215-5p促进卵泡颗粒细胞凋亡；RT-qPCR检测增殖凋亡标志基因表达结果显示，转染miR-215-5p模拟物后，CCND1和CDK2表达量均极显著降低(P < 0.01)，Caspase-3极显著上升(P<0.01)；而转染miR-215-5p干扰片段后，CCND1显著上升(P < 0.05)，CDK2极显著上升(P < 0.01)，Caspase-3极显著降低(P < 0.01)，TP63显著降低(P < 0.05)。雷州黑鸭miR-215-5p可以抑制卵泡颗粒细胞增殖，促进其凋亡。

关键词: 雷州黑鸭, miR-215-5p, miRNA, 颗粒细胞, 增殖凋亡

Abstract:

The study aimed to investigate the expression pattern of miR-215-5p in tissues of Leizhou black ducks and verify the regulatory effect of miR-215-5p on the proliferation and apoptosis of follicular granulosa cells. Three 43-week-old egg-laying and three 51-week-old non-laying Leizhou black ducks with similar body weight and health conditions were selected. The expression levels of miR-215-5p in different tissues were detected using RT-qPCR. By constructing mimics and interference fragment vectors and transfecting them into follicular granulosa cells, cell proliferation and apoptosis were detected using CCK-8 and flow cytometry techniques, with each group containing 3 replicates. The results showed that the expression of miR-215-5p in egg-laying Leizhou black ducks was generally higher than that in non-laying ducks, with relatively high expression in the ovary, infundibulum, and breast leg muscles, and relatively low expression in the funnel part, uterus, and heart. CCK-8 and EdU assays showed that miR-215-5p inhibited the proliferation of follicular granulosa cells; flow cytometry detected that miR-215-5p promoted the apoptosis of follicular granulosa cells; RT-qPCR detection of proliferation and apoptosis marker genes expression showed that after transfection with miR-215-5p mimics, the expression levels of CCND1 and CDK2 were both significantly decreased (P < 0.01), and Caspase-3 was significantly increased (P < 0.01); while after transfection with miR-215-5p interference fragments, CCND1 was significantly increased (P < 0.05), CDK2 was significantly increased (P < 0.01), Caspase-3 was significantly decreased (P < 0.01), and TP63 was significantly decreased (P < 0.05). miR-215-5p in Leizhou black duck has the ability to restrain the proliferation of follicular granulosa cells and promote their apoptosis.

Key words: Leizhou black duck, miR-215-5p, miRNA, granulosa cells, proliferation and apoptosis

中图分类号:

S834.3

叶润根, 刘渊博, 路丽丽, Collins Amponsah Asiamah, 苏瑛. miR-215-5p在雷州黑鸭组织中的表达及其对卵泡颗粒细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2025, 56(4): 1722-1730.

YE Rungen, LIU Yuanbo, LU Lili, Collins Amponsah Asiamah, SU Ying*. Expression of miR-215-5p in Leizhou Black Duck Tissues and Its Effect on Follicular Granulosa Cells Proliferation and Apoptosis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1722-1730.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence
miR-215-5p	RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTCTG F: GCGCGATGACCTATGAATTGA R: AGTGCAGGGTCCGAGGTATT
U6	RT: AACGCTTCACGAATTTGCGT F: CTCGCTTCGGCAGCACA R: AACGCTTCACGAATTTGCGT
miR-215-5p mimics	F: AUGACCUAUGAAUUGACAGCC R: CUGUCAAUUCAUAGGUCAUUU
mimics NC	F: UUCUCCGAACGUGUCACGUTT R: ACGUGACACGUUCGGAGAATT
miR-215-5p inhibitor	GGCUGUCAAUUCAUAGGUCAU
inhibitor NC	CAGUACUUUUGUGUAGUACAA

引物名称 Primer name	引物序列(5′→3′) Primer sequence	片段长度/bp Fragment length	Tm /℃	NCBI ID
TP63-F	GCCCAACCTCGCTAAGAA	197	55.1	101794454
TP63-R	CAACCACCATCCCTGACG
Caspase3-F	CTGGTATTGAGGCAGACAGTGG	158	58.8	101805296
Caspase3-R	CAGCACCCTACACAGAGACTGAA
CCND1-F	AACCCACCTTCCATGATCGC	159	57.5	101800639
CCND1-R	CTGTTCTTGGCAGGCTCGTA
CDK2-F	GTACAAGGCCCGGAACAAGG	168	55.3	101789539
CDK2-R	TTCTCCGTGTGGATCACGTC
β-actin-F	CGCAAATGCTTCTAAACC	167	48.8	101800437
β-actin-R	AGACTGCTGCTGATACCTT

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