miR-191靶向BDNF基因通过激活PI3K/AKT信号通路促进猪未成熟支持细胞增殖

doi:10.16288/j.yczz.21-154

遗传 ›› 2021, Vol. 43 ›› Issue (7): 680-693.doi: 10.16288/j.yczz.21-154

miR-191靶向BDNF基因通过激活PI3K/AKT信号通路促进猪未成熟支持细胞增殖

唐湘薇(), 楚丹, 颜赛娜, 尹艳飞, 卞桥, 翁波, 陈斌, 冉茂良

湖南农业大学动物科学技术学院,畜禽遗传改良湖南省重点实验室,长沙 410128

收稿日期:2021-04-25 修回日期:2021-06-02 出版日期:2021-07-20 发布日期:2021-06-11
作者简介:唐湘薇,在读硕士研究生,专业方向：猪遗传育种。E-mail: 993808546@qq.com
基金资助:
湖南省自然科学基金项目编号(2020JJ4348);湖南省重点研发计划项目项目编号(2020NK2024);湖南省生猪产业技术体系岗位专家项目资助

MiR-191 promotes the porcine immature Sertoli cell proliferation by targeting the BDNF gene through activating the PI3K/AKT signaling pathway

Xiangwei Tang(), Dan Chu, Saina Yan, Yanfei Yin, Qiao Bian, Bo Weng, Bin Chen, Maoliang Ran

Hunan Provincial Key Laboratory for Genetic Improvement of Domestic Animal, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China

Received:2021-04-25 Revised:2021-06-02 Online:2021-07-20 Published:2021-06-11
Supported by:
Supported by the Natural Science Foundation of Hunan Province No(2020JJ4348);the Key Research and Development Plan Projects of Hunan ProvinceNo(2020NK2024);the Project from Porcine Industry and Technology System of Hunan Province

摘要/Abstract

摘要：

睾丸支持细胞数量是影响精子生成能力的主要因素之一,microRNA (miRNA)参与调控猪未成熟支持细胞的发育过程,然而,大多数被鉴定出的miRNA对支持细胞的作用及其机制尚不明确。基于本课题组前期高内涵筛选结果,本文进一步通过流式细胞术、蛋白免疫印迹和双荧光素酶报告基因等方法,研究了miR-191调控猪未成熟支持细胞增殖和凋亡的作用机理。结果表明：过表达miR-191显著促进细胞周期由G₁期进入S期和G₂期,细胞增殖能力显著增强,细胞凋亡率显著降低;而抑制表达miR-191则与之相反。双荧光素酶报告基因系统验证miR-191直接靶向BDNF基因3′-UTR。抑制表达BDNF基因促进细胞周期进入S期,并促进细胞增殖而抑制细胞凋亡,与过表达miR-191的作用一致。共转染试验结果显示,BDNF基因可以拮抗miR-191对细胞增殖和凋亡的调控作用。此外,过表达miR-191和抑制表达BDNF基因均可显著促进PI3K/AKT信号通路中关键蛋白PI3K和AKT的磷酸化水平,且BDNF基因同样拮抗miR-191对PI3K和AKT蛋白的调控作用。本研究结果证实miR-191靶向BDNF基因,通过激活PI3K/AKT信号通路促进猪未成熟支持细胞增殖且抑制其凋亡,为进一步解析miR-191调控猪精子生成的生物学功能提供了理论基础。

关键词: miR-191, BDNF基因, PI3K/AKT信号通路, 增殖, 猪睾丸支持细胞

Abstract:

The number of Sertoli cells in the testis is a major regulator on the sperm production capacity. MicroRNAs (miRNAs) participate in regulating the proliferation and apoptosis of porcine immature Sertoli cells. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. In the present study, based on our previous results from an EdU-based high-content screening assay, we further studied the mechanism of action of miR-191 on the proliferation and apoptosis of porcine immature Sertoli cells through flow cytometry, Western blotting, and dual-luciferase activity analyses. The results demonstrated that overexpression of miR-191 promoted cell cycle progression from G₁ phase to the S and G₂ phases, enhanced cell proliferation, and inhibited apoptosis in the porcine immature Sertoli cells, whereasmiR-191 inhibition resulted in the opposite effects. The results from a luciferase reporter assay showed that miR-191 directly targeted the 3′-UTR of theBDNF gene. BDNF knockdown also promoted cell cycle progression to the S phase, cell proliferation and inhibited cell apoptosis, which were consistent with the effects of the miR-191overexpression. A co-transfection experiment showed that BDNF knockdown abolished the effects of miR-191 inhibition. Furthermore, both miR-191 overexpression and BDNFinhibition elevated the phosphorylation of PI3K and AKT, the key components of the PI3K/AKT signaling pathway, whereas BDNFinhibition offset the effects of the miR-191 knockdown. Overall, these data indicated that miR-191 promotes cell proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting theBDNF gene through activating the PI3K/AKT signaling pathway. This study provides a novel scientific basis for further investigation on the biological functions of miR-191 on porcine spermatogenesis.

Key words: miR-191, BDNF gene, PI3K/AKT signaling pathway, proliferation, porcine Sertoli cells

唐湘薇, 楚丹, 颜赛娜, 尹艳飞, 卞桥, 翁波, 陈斌, 冉茂良. miR-191靶向BDNF基因通过激活PI3K/AKT信号通路促进猪未成熟支持细胞增殖[J]. 遗传, 2021, 43(7): 680-693.

Xiangwei Tang, Dan Chu, Saina Yan, Yanfei Yin, Qiao Bian, Bo Weng, Bin Chen, Maoliang Ran. MiR-191 promotes the porcine immature Sertoli cell proliferation by targeting the BDNF gene through activating the PI3K/AKT signaling pathway[J]. Hereditas(Beijing), 2021, 43(7): 680-693.

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基因	引物序列(5′→ 3′)
CCND1	F: TACACCGACAACTCCATCCG
CCND1	R: GCCGCCAGGTTCCACTT
CCNE1	F: CCTGCTGAAGATGCCCATAAC
CCNE1	R: TGCTCTGCTTCTTACTGCTCG
CDK-4	F: GTGGCCCTCAAGAGCGTAAG
CDK-4	R: CAGACATCCATCAGCCGGAC
c-MYC	F: AACCCTTGGCTCTCCACGAG
c-MYC	R: ATTCCGACCTTTTGGCAGGG
FGF2	F: ACCAGGTCACTGAGATCCATCCAC
FGF2	R: TTCGGCAACAGCACACCAATCC
BMP4	F: ACCAATCATGCCATCGTTCAGACC
BMP4	R: TGATTCAGCGGCAACCACATCC
GDNF	F: GAGACCGCTGTGTATCGCATTCC
GDNF	R: GCCTTCTTCCTCTTCCTCCTCCTC
PCNA	F: ATTTGGCCATGGGCGTGAAC
PCNA	R: CTAGTGCCAAGGTGTCTGCAT
EGF	F: GCGAGCGATGTCAGCACAGAG
EGF	R: AGGAGCAGCAGCAGGACCAG
TBP	F: GCGATTTGCTGCTGTAATCA
TBP	R: CCCCACCATGTTCTGAATCT

miR-191靶向BDNF基因通过激活PI3K/AKT信号通路促进猪未成熟支持细胞增殖

MiR-191 promotes the porcine immature Sertoli cell proliferation by targeting the BDNF gene through activating the PI3K/AKT signaling pathway

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