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

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 Anqi, CHEN Bin

【摘 要】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. 【期刊名称】《农业科学学报（英文版）》 【年(卷),期】2019(018)008 【总页数】12

【关键词】Keywords: miR-10b, DAZAP1, gene expression, proliferation, porcine immature Sertoli cell

Received 28 May, 2018 Accepted 26 November, 2018

WENG Bo, Tel: +86-731-84635385, E-mail: wengbo831@126.com； Correspondence

CHEN

Bin,

Tel:

+86-731-84618176,E-mail:

chenbin7586@126.com

＊ These authors contributed equally to this study.

? 2019 CAAS. Published by Elsevier Ltd. This is an open access article under

the

CC

BY-NC-ND

license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. lntroduction

Spermatogenesis is a complex and strictly regulated process that involves complex interactions between several cell types in the testicular tissue, such as Sertoli cells, Leydig cells and germ cells (Lucas et al. 2014； Tremblay 2015). Of these cells, Sertoli cells have been identified as “nursing cells” in the processes of spermatogenesis. Sertoli cells form the blood-testis barrier and thus create a safe environment that protects spermatozoa (Mruk et al. 2015； Gerber et al. 2016).Sertoli cells also secrete androgen-binding protein (Ma et al.2015) and support developing germ cells with nutrients and regulatory factors. Previous studies demonstrate that each Sertoli cell supports limited number of germ cells, which is supported by the fact that the number of Sertoli cells is related to the daily sperm production per testis (Johnson et al. 2008). Sertoli cells have intense proliferation in fetal period and the prepubertal period testicular tissues but almost lose the ability in the pubertal period (Sharpe et al.2003). Therefore, the proliferation of Sertoli cells in the fetal period or prepubertal period testicular tissue is important for spermatogenesis.

MicroRNAs (miRNAs), a class of non-coding RNA(approximately 22 nt), are abundant and they play important roles in regulating cell growth, development, and apoptosis by targeting the 3′-untranslated region (3′-

UTR) and thus repressing the protein expression of protein-coding gene(Bartel 2009； Eisenberg et al. 2015； Holt et al. 2016； Luo et al. 2016). Several hundreds of known and novel miRNAs have been identified from the developmental processes of testicular tissues in pigs (Sus scrofa), and shown differential expression profiles (Lian et al. 2012； Ran et al. 2015； Li et al.2016). Our previous studies and others have indicated that miR-10b is highly expressed during the development of testicular tissues in several species, including swine (Ran et al. 2015), human (Abu-Halima et al. 2014), mouse (Nixon et al. 2015), and goat (Wu et al. 2014). Furthermore, a total of 560 protein-coding genes, including DAZ-associated protein 1 (DAZAP1) gene, are identified as the target genes of miR-10b by the integrated analysis of miRNA and mRNA expression profiles in developing porcine testicular tissues(Ran et al. 2015). The DAZAP1 gene was widely expressed in mammalian testicular tissues with high mRNA and protein expression levels (Dai et al. 2001； Pan et al. 2005). In addition, previous studies demonstrated that DAZAP1 gene plays crucial roles in spermatogenesis as well as the normal growth and development of testicular tissue in mice (Hsu et al. 2008) and human (Smith et al. 2011).

These above-mentioned clues suggested that the regulatory roles of miR-10b might be related to porcine testicular tissue development or spermatogenesis process,while the mechanisms of the action of miR-

10b were unclear.Therefore, we hypothesized that miR-10b might promote Sertoli cells proliferation through targeting DAZAP1 gene and repressing DAZAP1 protein expression. The objective of the present study was to investigate the effects of miR-10b and DAZAP1 gene on proliferation, apoptosis, and also the regulatory role of miR-10b on the DAZAP1 gene expression in porcine immature Sertoli cells to test the above hypothesis.

2. Materials and methods

2.1. Experimental design and treatments

In the present study, we used a completely randomized design to conduct six experiments. In experiment 1, a total of seven treatments were set in different days of age,including 1, 30, 60, 90, 120, 150, and 180 days of age. In addition, a total of nine treatments were set in different tissues, including lungs, liver, heart, kidney, spleen, muscle,adipose, intestine, and testicular tissues. Each treatment contained three replicates with one pig in per replicate. In experiment 2, there were a total of four cell co-transfection treatments including miR-10b mimic+3′-UTR-WT, miR-10b mimic+3′-UTR-MT, mimic NC+3′-UTR-WT, and mimic NC+3′-UTR-MT (WT means wild and MT means mutant).In experiment 3, there were two cell transfection treatments including miR-10b mimic and mimic NC. In experiment 4,there were two cell transfection treatments including miR-10b inhibitor and inhibitor