遗传 ›› 2018, Vol. 40 ›› Issue (7): 572-584.doi: 10.16288/j.yczz.18-028

• 研究报告 • 上一篇    下一篇

miR-362靶向ZNF644基因调控猪未成熟支持细胞的增殖和凋亡

冉茂良1,2,董莲花1,2,翁波1,2,曹蓉1,2,彭馥芝1,2,高虎1,2,罗荟1,2,陈斌1,2()   

  1. 1. 湖南农业大学动物科学技术学院,长沙 410128
    2. 畜禽遗传改良湖南省重点实验室,长沙 410128
  • 收稿日期:2018-01-29 修回日期:2018-03-21 出版日期:2018-07-20 发布日期:2018-06-25
  • 作者简介:冉茂良,博士,讲师,研究方向：猪的分子遗传学。E-mail:ranmaoliang0903@126.com|董莲花,硕士研究生,专业方向：动物遗传育种。E-mail:846419902@qq.com
  • 基金资助:
    国家现代农业产业技术体系建设专项资金项目(CARS-36);湖南省自然科学基金项目(2018JJ3219);湖南农业大学省级优秀博士论文培育基金项目(YB2015001)

miR-362 regulates the proliferation and apoptosis of porcine immature Sertoli cells through targeting the ZNF644 gene

Maoliang Ran1,2,Lianhua Dong1,2,Bo Weng1,2,Rong Cao1,2,Fuzhi Peng1,2,Hu Gao1,2,Hui Luo1,2,Bin Chen1,2()   

  1. 1. College of Animal Science and Technology, Hunan Agricultural University, Changsha410128, China
    2. Hunan Provincial Key Laboratory for Genetic Improvement of Domestic Animal, Changsha410128, China
  • Received:2018-01-29 Revised:2018-03-21 Online:2018-07-20 Published:2018-06-25
  • Supported by:
    Supported by the China Agriculture Research System(CARS-36);Hunan Provincial Natural Science Foundation of China(2018JJ3219);the Excellent Doctoral Dissertation Cultivating Fund of Hunan Agricultural University(YB2015001)

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摘要：

睾丸组织中未成熟支持细胞的增殖能力决定成熟支持细胞的数量,进而制约成年雄性动物的精子生成能力。研究表明microRNA (miRNA)参与调控猪未成熟支持细胞的增殖和凋亡,但大部分鉴定出的miRNA功能仍不明确。本文基于前期RNA-seq数据筛选结果,研究了miR-362对猪未成熟支持细胞增殖和凋亡的调控作用。首先利用生物信息学方法预测miR-362的靶基因,通过qRT-PCR技术检测miR-362和ZNF644基因在不同发育阶段的猪睾丸组织中的表达水平以及在猪未成熟支持细胞中过表达或抑制表达miR-362后ZNF644基因的表达水平,采用双荧光素酶报告基因系统验证miR-362与ZNF644基因之间的靶向关系。结果显示,miR-362与ZNF644基因3′UTR具有一个潜在的结合位点,miR-362和ZNF644基因在猪睾丸组织中的mRNA表达水平显著负相关(r=-0.723, P<0.01),miR-362和psiCHECK2-ZNF644-WT 3′UTR共转染组的双荧光活性显著降低,且miR-362显著调节ZNF644基因的表达水平,表明miR-362靶向ZNF644基因并抑制其表达水平。为进一步检测过表达miR-362或抑制表达ZNF644基因对猪未成熟支持细胞增殖和凋亡的影响,通过流式细胞术检测细胞周期,CCK8和EdU试剂盒检测细胞增殖情况,Annexin V-FITC/PI方法和qRT-PCR技术检测细胞凋亡情况及凋亡相关基因的表达水平。结果表明,过表达miR-362后,猪未成熟支持细胞周期被阻滞在G1期,抑制表达ZNF644基因后,猪未成熟支持细胞被阻滞在G2期,细胞增殖能力显著减弱,细胞凋亡率显著提高,细胞凋亡相关基因呈促进凋亡的差异表达。本研究结果证实miR-362靶向ZNF644基因抑制猪未成熟支持细胞的增殖而促进其凋亡,为深入研究miR-362在猪精子生成过程中的生物学功能提供了理论基础。

关键词: miR-362, ZNF644, 猪, 未成熟支持细胞, 增殖, 凋亡

Abstract:

In testicular tissue, immature Sertoli cell proliferation ability determines the size of mature Sertoli cell populations, which further regulates the spermatogenesis in the adult male animals. Studies have demonstrated that microRNAs (miRNAs) participate in the regulation of the immature Sertoli cell proliferation and apoptosis, but the functions of most identified miRNAs remain unclear. In this study, based on previous RNA-seq results, we analyzed the regulatory role (s) of miR-362 in proliferation and apoptosis of porcine immature Sertoli cells. The ZFN644 gene was predicted to be a target gene of miR-362 using bioinformatics methods. The expression levels of miR-362 and ZNF644 gene were measured using qRT-PCR assay in developing porcine testicular tissues and in immature Sertoli cells transfected with either miR-362 mimic or miR-362 inhibitor. The dual luciferase reporter assay was used to determine the regulatory relationship between miR-362 and ZNF644. The results showed that a putative target site of miR-362 was located in the 3′UTR of ZNF644. The expression of miR-362 was significantly and negatively correlated with ZNF644 expression in the developing porcine testicular tissues. Co-transfection of miR-362 and psiCHECK2-ZNF644-WT 3′UTR luciferase vector significantly suppressed luciferase activity. The ZNF644 gene expression level was significantly regulated by miR-362, demonstrating that miR-362 targets ZNF644 gene and inhibits its expression in porcine immature Sertoli cells. Flow cytometry, CCK8, and EdU assays were used to measure the effects of over-expression of miR-362 or knockdown of ZNF644 on porcine immature Sertoli cell proliferation; Annexin V-FITC/PI staining assays and qRT-PCR technology were used to test the apoptosis and the expression levels of cell survival-related genes, respectively. Over-expression of miR-362 and knockdown of ZNF644 arrested the porcine immature Sertoli cells in G1 and G2 phases of the cell cycle, respectively, and inhibited proliferation, enhanced apoptosis in the porcine immature Sertoli cells, and significantly regulated the expression levels of cell survival-related genes. Taken together, these data indicate that miR-362 inhibits proliferation and promotes apoptosis in porcine immature Sertoli cells by targeting the ZNF644 gene, thereby providing the scientific basis for further study on the function(s) of miR-362 in the porcine spermatogenesis.

Key words: miR-362, ZNF644, porcine, immature Sertoli cell, proliferation, apoptosis