miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

doi:10.1016/S2095-3119(19)62564-5

Journal of Integrative Agriculture ›› 2019, Vol. 18 ›› Issue (8): 1924-1935.DOI: 10.1016/S2095-3119(19)62564-5

收稿日期:2018-05-28 出版日期:2019-08-01 发布日期:2019-08-02

miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

WENG Bo*, RAN Mao-liang*, Cao Rong, PENG Fu-zhi, LUO Hui, GAO Hu, TANG Xiang-wei, Yang An-qi, CHEN Bin

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

Received:2018-05-28 Online:2019-08-01 Published:2019-08-02
Contact: Correspondence CHEN Bin, Tel: +86-731-84618176, E-mail: chenbin7586@126.com
About author:WENG Bo, Tel: +86-731-84635385, E-mail: wengbo831@126.com; * These authors contributed equally to this study.
Supported by:
This work was financially supported by the earmarked fund for China Agriculture Research System (CARS-36) and the Hunan Provincial Natural Science Foundation of China (2018JJ2176 and 2018JJ3219).

摘要/Abstract

Abstract:

MicroRNAs (miRNAs) have been widely identified in porcine testicular tissues and implicated as crucial regulators of proliferation, apoptosis, and differentiation in porcine spermatogenesis related cells. However, the function roles of most of the miRNAs that have been identified in Sertoli cells are poorly understood. In the present study, six experiments were conducted to study the regulatory role of miR-10b in porcine immature Sertoli cells. In experiment 1, the results showed that the relative mRNA expression level of miR-10b in porcine testicular tissues decreased quadratically (P<0.001) with increasing age, while the relative mRNA expression level of DAZAP1 gene increased (P<0.001). In addition, the mRNA expression of miR-10b was negatively (P<0.01) correlated with DAZAP1 mRNA expression (r=–0.550). In experiment 2, the results from the bioinformatic analysis and a luciferase reporter assay demonstrated that miR-10b directly targeted the DAZAP1 gene in porcine immature Sertoli cells. DAZAP1 mRNA and protein expressions were both regulated (P<0.05) by miR-10b. In experiments 3 to 5, the over-expression of miR-10b or the siRNA-mediated knockdown of the DAZAP1 gene promoted (P<0.05) porcine immature Sertoli cell proliferation, as determined by the Cell Counting Kit-8 (CCK-8) assay and the 5-Ethynyl-2´-deoxyuridine (EdU) assay. However, an annexin V-FITC/PI staining assay and the expression of cell survival-related genes indicated that over-expression of miR-10b or knockdown of DAZAP1 had no effect (P>0.05) on porcine immature Sertoli cell apoptosis. In experiment 6, the co-transfection treatment results showed that miR-10b promoted (P<0.05) porcine immature Sertoli cell proliferation by targeting DAZAP1 gene. Overall, these experiments demonstrated that miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene.

Key words: miR-10b , DAZAP1 , gene expression , proliferation , porcine immature Sertoli cell

. [J]. Journal of Integrative Agriculture, 2019, 18(8): 1924-1935.

WENG Bo, RAN Mao-liang, Cao Rong, PENG Fu-zhi, LUO Hui, GAO Hu, TANG Xiang-wei, Yang An-qi, CHEN Bin.

miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

[J]. Journal of Integrative Agriculture, 2019, 18(8): 1924-1935.

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miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

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