BIRC5对山羊睾丸细胞周期、凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.04.015

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (4): 1511-1524.doi: 10.11843/j.issn.0366-6964.2023.04.015

BIRC5对山羊睾丸细胞周期、凋亡的影响

但一昕¹, 杨璐¹, 向华^1*, 张焕容¹, 任玉鹏¹, 杨发龙¹, 何翃闳¹, 朱江江²

1. 西南民族大学畜牧兽医学院, 成都 610041;
2. 青藏高原动物遗传资源保护与利用四川省重点实验室, 成都 610041

收稿日期:2022-09-07 出版日期:2023-04-23 发布日期:2023-04-27
通讯作者: 向华,主要从事畜禽传染病研究,E-mail:xianghua2008411@163.com
作者简介:但一昕(1999-),女,河北唐山人,硕士,主要从事畜禽传染病研究,E-mail:danyx99@163.com;杨璐(1995-),女,四川崇州人,硕士,主要从事畜禽传染病研究,E-mail:1753915524@qq.com。
基金资助:
四川省自然科学基金项目（2022NSFSC0082）；西南民族大学优秀学生培养工程（2022NYXXS025）；动物遗传育种四川省重点实验室开放基金（202202）；四川省科技计划（2020JDJQ0010）；四川省畜禽育种攻关项目（2021YFYZ0003）；浙江省科技计划项目（2022C04017）

Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells

DAN Yixin¹, YANG Lu¹, XIANG Hua^1*, ZHANG Huanrong¹, REN Yupeng¹, YANG Falong¹, HE Honghong¹, ZHU Jiangjiang²

1. College of Animal & Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China;
2. Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Chengdu 610041, China

Received:2022-09-07 Online:2023-04-23 Published:2023-04-27

摘要/Abstract

摘要： 旨在在克隆山羊BIRC5基因的基础上，通过过表达或者敲低BIRC5基因表达揭示BIRC5基因对山羊睾丸细胞周期和凋亡的调控作用，为进一步完善BIRC5基因功能奠定基础。本研究以3只3日龄3 kg左右健康简州大耳羊公羊脾脏组织为模板，利用RT-PCR技术克隆山羊BIRC5基因CDS区序列，并进行生物信息学分析。将克隆回收产物连接真核表达载体，从而构建pcDNA3.1（+）-BIRC5。根据山羊BIRC5基因序列设计并筛选有效siRNA。将pcDNA3.1（+）-BIRC5和siRNA分别转染山羊睾丸细胞后，利用Western blot检测BIRC5蛋白表达，利用流式细胞仪检测细胞周期与凋亡，利用实时荧光定量PCR技术检测对凋亡相关基因Bax、Caspase3、Caspase7、Bcl-2、p53、BCL2L11和PARP1的表达。结果，成功扩增山羊BIRC5基因序列，长度为506 bp，包含5'UTR 28 bp，CDS区429 bp，3'UTR 49 bp，编码142个氨基酸残基，且与牛亲缘关系最近。过表达BIRC5基因后抑制了细胞凋亡，且细胞被阻滞于G2/M+S期；同时可下调Caspase7、p53、BCL2L11、Bcl-2和Bax基因mRNA的表达，但Caspase3和PARP1 mRNA表达无显著变化。而敲低BIRC5基因表达后可显著促进细胞凋亡的发生，且细胞被阻滞于细胞被阻滞于G0/G1期，而Caspase3、Caspase7和PARP1 mRNA表达显著上升，p53和Bcl-2的表达显著下降，Bax和BCL2L11表达无显著变化。BIRC5基因可抑制山羊睾丸细胞的凋亡，并促进山羊睾丸细胞被阻滞于G2/M+S期。研究结果为进一步深入全面研究BIRC5基因的功能提供重要的试验数据。

关键词: 山羊, BIRC5基因, 过表达, 干扰, 细胞周期, 细胞凋亡

Abstract: The aim of the present study was to clone goat BIRC5, and to reveal the role of BIRC5 gene in regulating the cycle and apoptosis of goat testis cells through the overexpression or interference of BIRC5. These data will be beneficial for exploring the role of BIRC5 gene in goat. The BIRC5 gene sequence was cloned by RT-PCR from spleen tissue of 3 three-day-old healthy Jianzhou male goats with 3 kg of body weight, and sequenced for further bioinformatics analysis using online softwares. The clone recovery product was ligated to a eukaryotic expression vector to construct pcDNA3.1(+)-BIRC5. Effective siRNA was designed and screened based on goat BIRC5 gene sequence. After pcDNA3.1(+)-BIRC5 and siRNA were respectively transfected into goat testicular cells, the expression of BIRC5 protein was detected by Western blot, and cell cycle and apoptosis were detected by flow cytometry. Meanwhile, the expression of apoptosis-related genes Bax, Caspase3, Caspase7, Bcl-2, p53, BCL2L11 and PARP1 were detected by RT-qPCR. A length of 506 bp BIRC5 gene sequence was cloned successfully, including 28 bp of 5'UTR, 429 bp of CDS region, and 49 bp of 3'UTR,encoding 142 amino acids. The phylogenetic tree showed that BIRC5 had the closest relative to Bos taurus. The overexpression of BIRC5 gene inhibited the apoptosis of cells, and the cells were arrested in G2/M+S phase; it also down-regulated the expression of Caspase7, p53, BCL2L11, Bcl-2 and Bax gene mRNA, but did not significantly change the mRNA expression of Caspase3 and PARP1. siRNA interference of BIRC5 gene promoted the apoptosis of cells, and the cells were arrested in G0/G1 phase; it also up-regulated the expression of Caspase3, Caspase7 and PARP1 gene mRNA, and down-regulated the expression of p53 and Bcl-2 gene mRNA, but did not significantly change the mRNA expression of Bax and BCL2L11. Goat BIRC5 gene could inhibit the apoptosis of testis cells and promote the arrest of goat testicular cells in G2/M+S phase. These data may lay a foundation for further study of BIRC5 gene function.

Key words: goat, BIRC5 gene, overexpression, interference, cell cycle, cell apoptosis

中图分类号:

S827.3

但一昕, 杨璐, 向华, 张焕容, 任玉鹏, 杨发龙, 何翃闳, 朱江江. BIRC5对山羊睾丸细胞周期、凋亡的影响[J]. 畜牧兽医学报, 2023, 54(4): 1511-1524.

DAN Yixin, YANG Lu, XIANG Hua, ZHANG Huanrong, REN Yupeng, YANG Falong, HE Honghong, ZHU Jiangjiang. Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1511-1524.

参考文献 10

[1]	OH K, WILLETT W C, WU K N, et al.Calcium and vitamin D intakes in relation to risk of distal colorectal adenoma in women[J].Am J Epidemiol, 2007, 165(10):1178-1186.
[2]	刘俊.Survivin在不同年龄牦牛组织器官内表达检测及其多克隆抗体的制备[D].兰州:甘肃农业大学, 2019.LIU J.Expression of survivin in organs of different age yak and preparation of survivin polyclonal antibody[D].Lanzhou:Gansu Agricultural University, 2019.(in Chinese)
[3]	PAHLAVAN Y, KAHROBA H, SAMADI N, et al.Survivin modulatory role in autoimmune and autoinflammatory diseases[J].J Cell Physiol, 2019, 234(11):19440-19450.
[4]	ADAMS R R, CARMENA M, EARNSHAW W C.Chromosomal passengers and the (aurora) ABCs of mitosis[J].Trends Cell Biol, 2001, 11(2):49-54.
[5]	EBRAHIMIYAN H, ASLANI S, REZAEI N, et al.Survivin and autoimmunity; the ins and outs[J].Immunol Lett, 2018, 193:14-24.
[6]	SOHN H J, LEE J Y, LEE H J, et al.Simultaneous in vitro generation of CD8 and CD4 T cells specific to three universal tumor associated antigens of WT1, survivin and TERT and adoptive T cell transfer for the treatment of acute myeloid leukemia[J]. Oncotarget, 2017, 8(27):44059-44072.
[7]	ZHANG L, LIANG H, CAO W, et al.Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells[J].Braz J Med Biol Res, 2014, 47(7):548-553.
[8]	SERPELONI J M, SPECIAN A F L, RIBEIRO D L, et al.Fridericia platyphylla (Cham.) L.G. Lohmann root extract exerts cytotoxic and antiproliferative effects on gastric tumor cells and downregulates BCL-XL, BIRC5, and MET genes[J].Hum Exp Toxicol, 2020, 39(3):338-354.
[9]	NARIMANI M, SHARIFI M, JALILI A.Knockout of BIRC5 gene by CRISPR/Cas9 induces apoptosis and inhibits cell proliferation in leukemic cell lines, HL60 and KG1[J].Blood Lymphat Cancer, 2019, 9:53-61.
[10]	YANG Y, LI D, LI Y L, et al.Low-temperature plasma suppresses proliferation and induces apoptosis in lung cancer cells by regulating the miR-203a/BIRC5 axis[J].Onco Targets Ther, 2020, 13:5145-5153.

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BIRC5对山羊睾丸细胞周期、凋亡的影响

Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells

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