,, 申雪沂, 牛长敏, 夏静, 孙红亚, 郑英,扬州大学医学院组织学与胚胎学教研室,扬州 225001MicroRNA regulates Sertoli cell proliferation and adhesion
Mengmeng Xia,, Xueyi Shen, Changmin Niu, Jing Xia, Hongya Sun, Ying Zheng,Department of Histology and Embryology, School of Medicine,Yangzhou University, Yangzhou 225001, China通讯作者:
编委: 苗龙
收稿日期:2018-02-27修回日期:2018-06-26网络出版日期:2018-09-20
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Received:2018-02-27Revised:2018-06-26Online:2018-09-20
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夏蒙蒙,硕士研究生,专业方向：生殖医学E-mail:
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夏蒙蒙, 申雪沂, 牛长敏, 夏静, 孙红亚, 郑英. MicroRNA参与调控睾丸支持细胞的增殖与粘附功能[J]. 遗传, 2018, 40(9): 724-732 doi:10.16288/j.yczz.18-050
Mengmeng Xia, Xueyi Shen, Changmin Niu, Jing Xia, Hongya Sun, Ying Zheng.
精子发生是睾丸生精小管内的多细胞参与过程,在睾丸体细胞的精密协调下,各类生精细胞(精原细胞、精母细胞和精子细胞)经过连续地增殖、分裂和分化,最终转变为高度特异的精子。而支持细胞作为生精小管内唯一的体细胞,通过与各类生精细胞直接接触,维持生精细胞形态、发育及迁徙运动,在精子发生中扮演极为重要的角色[1,2]。
支持细胞的增殖与粘附功能是其发挥作用的基础,未成熟的支持细胞通过不断的分裂增殖,保持生精小管内自身数量的平衡,维持限定数量的生精细胞,从而决定雄性生精能力[3,4,5]。支持细胞与相邻细胞间的粘附连接是细胞间进行物质交流与信息传递的桥梁,同时为精子发生提供免疫屏障[5]。支持细胞的增殖与粘附功能的改变,都将扰乱精子发生进程,导致雄性生育力下降甚至不育[6],因此,研究支持细胞增殖与粘附的调控机制具有重要意义。
越来越多的证据表明microRNA (miRNA)可通过靶向支持细胞内相关基因,调控支持细胞的增殖与粘附功能[7,8,9,10,11,12,13,14]。同时,miRNA的表达水平又受到内分泌干扰素、性激素及营养状态等多种因素的 调节[10,11,12,13,14,15,16]。
本文综述了近年来与支持细胞增殖及粘附功能相关的miRNA的研究进展,以期为发现及鉴定更多的与支持细胞相关的miRNA、深入探索支持细胞的调控网络以及诊断支持细胞相关不育症提供理论 基础。
1 支持细胞的增殖与粘附
对睾丸支持细胞的研究始于1865年,Enrico Sertoli首次发现了这类细胞并将其描述为“保姆细胞(nurse cells)”。后来这类细胞被命名为“Sertoli cells”,即支持细胞。支持细胞起始于睾丸生精小管的基膜,不断延伸至生精小管腔面,包绕不同发育阶段的生精细胞及精子,构成生长支架。支持细胞的功能包括：分泌雄激素结合蛋白、类固醇和营养因子等,促进生精细胞发育及精子成熟;参与生精细胞易位,释放成熟精子,同时吞噬发育不良的精子、凋亡生精细胞以及多余胞质[17]。不同物种睾丸支持细胞的数量取决于其增殖能力。啮齿类动物支持细胞增殖发生在胚胎期及新生阶段;在猕猴(Macaca mulatta)睾丸中,支持细胞增殖发生在青春期前;人类睾丸支持细胞增殖发生在新生阶段及青春期前。因此,支持细胞的数量在成年之前就已经决定。支持细胞数量决定了生精细胞数量、睾丸大小及精子产量[18,19],缺少支持细胞可导致生精细胞凋亡、退化,甚至引发雄性不育[6,20]。同时,支持细胞在精子发生中具有粘附功能。单个支持细胞可与相邻细胞粘附,形成粘附连接。相邻支持细胞间粘附可形成紧密连接,紧密连接是血睾屏障的重要组成部分。血睾屏障能合成、分泌、运输生精细胞增殖及分化所需的营养因子,如转铁蛋白。同时,血睾屏障可防止免疫球蛋白、淋巴细胞等免疫因子进入近腔室,避免这些免疫因子识别圆形精子细胞表面的特异性抗原,进而避免自身免疫反应的发生[17,21]。支持细胞与各级生精细胞间也存在粘附连接,生精细胞通过粘附连接锚定在支持细胞隐窝中,接受支持细胞提供的营养及物理支持。支持细胞-生精细胞间粘附结构也具有解聚与重塑功能：一方面促进发育期的生精细胞不断向生精小管管腔迁徙,及时释放成熟的精子;另一方面阻止了未成熟生精细胞的释放[22]。研究表明,支持细胞的生长及相关功能主要受卵泡刺激素的调控[18],但近年来的研究表明,支持细胞的功能还受到miRNA的调控[7,8,9,10,11,12,13,14]。
2 miRNA的生物合成及作用机制
miRNA是一类保守的内源性的小分子RNA,其合成始于细胞核内的初级miRNA (pri-miRNA)。pri-miRNA由RNA聚合酶Ⅱ (polymerase Ⅱ, pol Ⅱ)或RNA聚合酶Ⅲ (polymeraseIII, pol III)转录生成,长度约为几百至几千个核苷酸[23]。pri-miRNA在RNaseⅢ核酸酶-Drosha及RNA结合蛋白-DGCR8构成的微小RNA处理器复合体(microprocessor)中加工、修饰。DGCR8识别并结合N6-甲基腺苷(m6A)甲基化的pri-miRNA[24],招募Drosha酶进行剪切产生前体miRNA(pre-miRNA),长度约60~70个核苷酸;pre-miRNA具有发夹结构,5′端含有磷酸盐基团,3′端含有2个悬垂碱基,核质/细胞质转运蛋白5 (Exportin-5, Exp-5)识别悬垂碱基并与pre-miRNA结合,在Ran-GTP供能下将pre-miRNA转运到细胞质中;胞质中Exp-5释放pre-miRNA,pre-miRNA在另一RNaseIII核酸酶-Dicer切割下去除末端发夹结构,产生miRNA duplex,长度约22个核苷酸[25,26,27]。miRNA duplex为双链结构,由引导链(guide strand)和过客链(passenger strand)组成。在miRNA研究中,引导链和过客链也被称为miRNA:miRNA*或miRNA-5p:miRNA-3p[25,26]。miRNA duplex是miRNA的前体形式,必须经过解旋及引导链选择性的保留这两个步骤,才能形成引导基因沉默的成熟miRNA。miRNA duplex的解旋机制较为复杂,且至今仍在不断探索中。目前,将miRNA duplex的解旋机制阐释为两种：切割依赖性/非依赖性解旋和ATP依赖性解旋酶解旋[26,27,28,29,30]。切割依赖性/非依赖性解旋(slicer-dependent/independent unwinding)机制为：miRNA duplex在ATP作用下装载到AGO蛋白上,形成前体RISC(pre-RISC),pre-RISC不具有转录后调控功能[26,27,28]。当引导链和过客链高度互补时,AGO2蛋白在Mg2+参与下切割过客链中特定核苷酸间的磷酸二酯键[26],且Mg2+浓度越高,AGO2切割活力越强[27],从而使过客链断裂,降低miRNA duplex的热力学稳定性,miRNA duplex双链分离,这种解旋方式称为切割依赖性解旋[26];当引导链和过客链之间存在错配,尤其是5′端种子区及3′端的中间序列错配或存在G-U碱基摆动配对时,miRNA duplex双链变形并解旋,这种解旋方式称为切割非依赖性解旋[26,28]。由于大多数miRNA duplx存在错配现象,因此miRNA duplex的解旋多选择切割非依赖性解旋模式[26,28]。ATP依赖性解旋酶解旋是最早提出的miRNA duplex解旋方式[30],即miRNA duplex在RNA解旋酶作用双链分离,引导链与AGO蛋白结合直接形成RISC[30,31]。最近的研究表明,某些RNA解旋酶甚至能解旋pre- RISC中的miRNA duplex,分离引导链和过客链[32]。
miRNA duplex解旋后,过客链降解,引导链保留,pre-RISC转变为成熟的RISC[26]。成熟RISC在引导链的指引下结合靶mRNA,根据引导链和靶mRNA的序列互补程度,选择不同的基因沉默方式。当引导链和靶mRNA的碱基序列完全互补时,靶mRNA被切割并降解;当引导链和靶mRNA的碱基序列不完全互补时,靶mRNA翻译抑制[33]。
3 miRNA参与调控支持细胞的增殖与粘附功能
研究表明,超过60%的人类基因是miRNA的靶基因[34],而许多miRNA倾向于或优先表达于睾丸组织中,提示miRNA在精子生成中发挥着重要的转录后调控作用。随着睾丸支持细胞的分离与纯化、高通量测序及microRNA微阵列技术的发展,研究人员发现了一些在睾丸支持细胞中特异性表达或高表达的miRNA,如：Papaioannou等[6]对分离纯化的小鼠支持细胞进行microRNA微阵列技术分析,发现miR-299、miR-376a、miR-381、miR-409-5p、miR-674*在支持细胞中特异表达,miR-431、miR-341、miR-487b在支持细胞中高表达;Halima等[35]应用microRNA微阵列技术检测出在唯支持细胞征患者睾丸组织中miR-449a、miR-125b、miR-204、miR-22等高表达。近年来,研究人员应用qRT-RCR技术对与支持细胞功能相关的miRNA进行了相对定量分析,结合生物信息学软件分析、荧光素酶报告基因及Western blot等实验,结果发现：部分miRNA可通过调控其靶mRNA的表达水平,进一步影响与支持细胞增殖和粘附功能相关基因的表达,从而参与支持细胞的功能调节(表1)。
Table 1
表1
表1 与支持细胞增殖与粘附功能相关的miRNA
Table 1
| 名称 | 物种 | 靶基因(信号通路) | 功能 | 参考文献 |
|---|---|---|---|---|
| miR-762 | 猪(Sus scrofa) | RNF4 | 支持细胞增殖 | [7] |
| miR-638 | 猪(Sus scrofa) | SPAG1 (PI3K/AKT) | 支持细胞增殖 | [8] |
| miR-133b | 人(Homo sapiens) | GLI3 | 支持细胞增殖 | [9] |
| miR-301b-3p/3584-5p | 大鼠(Rattus norvegicus) | RASD1 (ERK1/2) | 支持细胞增殖 | [10] |
| miR-1285 | 猪(Sus scrofa) | (AMPK) | 支持细胞增殖 | [11] |
| miR-471 | 小鼠(Mus musculus) | FOXD1 | 支持细胞代谢 | [12] |
| DSC1 | 血睾屏障形成 | [12,13] | ||
| miR-23b | 大鼠(Rattus norvegicus) | PTEN | 支持细胞-精子粘附 | [14] |
| EPS15 |
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3.1 与支持细胞增殖相关的miRNA
Luo等[36]通过microRNA微阵列技术比较了大白猪性成熟前与性成熟后睾丸组织中miRNA的表达水平,发现了129种差异表达的miRNA,其中在性成熟阶段有51种miRNA表达上调,78种miRNA表达下调。值得一提的是,miR-762和miR-638在大白猪性成熟睾丸组织中表达显著上调。miR-762通过靶向精子发生相关基因RNF4(ring finger protein 4, RNF4)调控猪未成熟支持细胞的增殖过程。qRT-PCR检测显示RNF4高表达于猪未成熟的睾丸组织,其表达趋势与miR-762正好相反,进一步验证了miR-762与RNF4之间的负调控作用[37]。
RNF4是细胞核内的转录因子,能够结合到雄激素受体(androgen receptor, AR)的DNA结合区域并辅助激活雄激素依赖的转录调节[7]。免疫组织荧光显示,RNF4与AR在猪支持细胞的细胞核共定位,提示两者可能相互作用。在猪支持细胞中过表达RNF4,可促进AR的转录并上调其表达水平,影响AR效应基因的表达。这些结果表明在猪未成熟支持细胞中RNF4也是AR依赖型转录调节共激活因子[7]。RNF4还是一类泛素连接酶,通过泛素化作用在DNA损伤修复及DNA甲基化抑制方面发挥重要的作用[38,39]。miR-762与RNF4的3°UTR区域结合,抑制RNF4的转录及翻译,继而下调AR表达。同时上调细胞核增殖抗原(proliferating cell nuclear antigen, PCNA)的表达,促进支持细胞S期进程,减少支持细胞凋亡。此外,miR-762可降低γ-H2AX(DNA双链损伤标记物)的表达水平,促进支持细胞的DNA损伤修复[7]。
miR-638可靶向生精相关基因SPAG1 (sperm- associated antigen 1, SPAG1)。SPAG1最早在不孕症妇女的血清中被发现,可引起人类精子头对头凝集。免疫组织荧光分析显示,SPAG1与支持细胞有丝分裂中期、末期微管蛋白α-TUBULIN共定位[8],提示SPAG1可能通过参与纺锤体组装调节支持细胞生长。SPAG1抑制后,c-MYC、细胞周期蛋白CCND1、CCNE1、细胞周期蛋白激酶CDK4的表达降低,支持细胞增殖及周期进程阻滞。同时,磷酸化PI3K、AKT的蛋白水平降低,PI3K/AKT信号通路抑制,支持细胞凋亡增加[8]。
Yao等[9]应用microRNA微阵列分析方法分析了唯支持细胞征和梗阻性无精症患者支持细胞miRNA的表达情况,结果筛选出174个差异表达的miRNA。其中,miR-133b在唯支持细胞征患者支持细胞中的表达显著上调,并靶向GLI3基因(GLI family zinc finger 3, GLI3)。GLI3是睾丸组织中重要的转录因子,通过参与HH (hedgrhog, Hh)信号通路,调控细胞增殖与分化[40,41]。GLI3的表达起始于新生支持细胞,在未成熟支持细胞中持续表达,提示其可能在未成熟支持细胞的增殖过程中发挥一定的作用。DHH (Desert Hedgehog, DHH)是HH信号通路中最重要的因子,其缺失将导致雄性不育。DHH高表达于支持细胞,该基因敲除小鼠生精细胞完全缺失,表现为唯支持细胞综合征。因此,GLI3可能通过下调DHH的表达而导致唯支持细胞征[41]。在miR-133b过表达的支持细胞中,GLI3表达下调,SOX9、PCNA、细胞周期蛋白CCNB1及CCND1表达升高,从而促进支持细胞增殖及DNA合成[9]。
Yin等[10]使用邻苯二甲酸单丁酯(mono-butyl phthalate, MBP)处理出生9天后大鼠,结果发现支持细胞增殖明显,数量增多。microRNA微阵列分析显示支持细胞内miR-301b-3p,miR-3584-5p表达升高,且两者靶向同一基因—RASD1 (dexamethasone-induced ras-related protein 1, RASD1)。RASD1是一种特异性高表达于睾丸组织的基因,属RAS小G蛋白家族,参与G蛋白偶联受体-丝裂原活化蛋白激酶/细胞外信号调节激酶的信号转导(G protein-coupled receptors signaling to MAPK/ERK),其相关蛋白DEXRAS1/AGS-1通过抑制Giα 亚基ADP核糖基化,促使Gβγ解偶联,进而抑制RAS/RAF/MEK/ERK级联活化[42]。在支持细胞中,miR-301b-3p和miR- 3584-5p通过靶向RASD1,下调其表达水平,使磷酸化MEK蛋白水平升高,激活ERK1/2信号通路,促进大鼠未成熟支持细胞的增殖[10]。
Zhang等[11]研究发现,miR-1285通过AMPK信号通路参与17β-雌二醇介导的猪未成熟支持细胞的增殖抑制过程。在一定浓度的17β-雌二醇作用下,miR-1285表达水平下降,支持细胞增殖抑制,表现为磷酸化AMPK表达水平升高,导致ATP含量下降,细胞代谢阻滞,生长延缓。此外,mTOR表达抑制后其下游底物p70S6K激酶活化水平降低,导致细胞增殖受阻。miR-1285表达抑制后,P53、P27基因表达水平上升,抑制S期激酶相关蛋白SKP2表达,阻碍S期的DNA合成。
3.2 与支持细胞粘附相关的miRNA
miRNA除了对支持细胞增殖具有调控作用,也参与支持细胞粘附连接。支持细胞特异性Dicer敲除小鼠表现为支持细胞凋亡增加、成熟支持细胞功能受损、支持细胞间连接缺陷、体细胞退化,导致未成熟精子释放、生精细胞及睾丸退化,最终引起小鼠不育[43]。Panneerdoss等[12]通过microRNA微阵列技术比较了雄激素抑制和雄激素替代模型小鼠支持细胞中miRNA的表达水平,结果发现miR-471在雄激素抑制模型小鼠支持细胞中的表达显著上调。进一步研究发现miR-471同时靶向FOXD1 (forkhead/winged- helix transcription factor, FOXD1)和DSC1 (desmocollin 1, DSC1)基因,抑制FOXD1、DSC1基因的表达。FOXD1特异性表达于睾丸,主要表达于支持细胞中,在支持细胞代谢中起重要作用[12,44]。FOXD1还参与性腺激素释放,间接调控精子发生。此外,FOXD1参与SHH (Sonic Hedgehog, SHH)信号通路,调控胚胎发育及生殖系统稳态[45,46]。DSC1同时表达于支持细胞及生精细胞,调节上皮细胞粘附、脱落,其基因敲除小鼠表现为细胞粘附缺陷[47]。在miR- 471-5P转基因小鼠模型中,血睾屏障因子DSC2、闭锁蛋白OCLN、连接蛋白CLDN3表达水平下调,睾丸生精小管中生精细胞脱落,血睾屏障受损,提示miR-471参与调控细胞间粘附及血睾屏障的形成[13]。
Nicholls等[14]应用microRNA微阵列技术分析了雄激素抑制模型大鼠支持细胞中miRNA的表达水平,结果发现miR-23b在雄激素抑制大鼠支持细胞中的表达显著上升,模型大鼠表现为生精小管内精子释放失败。进一步研究发现,miR-23b靶向 PTEN基因(phosphatase and tensin homolog deleted on chromosome Ten, PTEN)及内吞作用因子EPS15 (Epidermal growth factor receptor substrate, EPS15)。PTEN通过抑制磷酸酶基因调控粘着斑激酶FAK的表达,抑制细胞的粘附。大鼠睾丸免疫组化染色显示EPS15定位在管泡复合体(tubulobulbar complex, TBC)[14],TBC是以网格蛋白为骨架的结构,参与支持细胞与生精细胞间粘附连接。研究表明,TBC可能通过内化支持细胞与精子间连接蛋白,促进成熟精子的释放[48,49]。EPS15是网格蛋白介导的内吞途径必要因子之一,参与网格蛋白包被小泡组成及内化作用[50],EPS15表达下调后,精子释放阻滞,提示miR-23b可通过抑制EPS15的表达,干扰TBC内化作用,紊乱精子释放。此外,EPS15基因家族成员EHD1也与精子发生相关,其基因敲除小鼠表现为生精小管内成熟精子滞留,雄性不育[51]。因此,miR-23b可能作为雄激素及支持细胞间的中介,促进支持细胞的粘附功能。
4 调控支持细胞中miRNA表达的因素
支持细胞是生精小管内唯一与生精细胞接触的体细胞,在维持精子发生稳态中发挥重要的作用。同时支持细胞还是许多内外因素作用的靶细胞[18],当内外环境发生改变时,支持细胞内miRNA的表达水平也将发生变化。4.1 激素类
在精子发生过程中,激素直接靶向体细胞[18],因此支持细胞内的miRNA表达主要受激素调控。研究发现,性激素可以调节支持细胞内的miRNA的表达水平,如雌激素、雄激素、卵泡刺激素等[12,13,14,15]。研究表明,雌激素调节支持细胞内与生长相关的miRNA,如小鼠支持细胞系中,一定浓度的雌激素可以使miR-17的表达水平下降,激活MAPK信号,促进支持细胞的增殖[15]。在17β-雌二醇作用下,猪未成熟支持细胞内miR-1285的表达降低,磷酸化AMPK的水平升高,支持细胞生长抑制[11]。雄激素在支持细胞紧密连接及其介导的精子释放过程中有重要作用,研究发现使用睾酮抑制剂及替代剂处理小鼠支持细胞后有218种miRNA表达上调,其中包 括miR-471,提示雄激素可抑制靶向粘附因子的miRNA[12]。卵泡刺激素和雄激素联合作用于原代大鼠支持细胞后有163种miRNA的表达水平发生变化,这些miRNA中有多数与MAPK信号通路、黏着斑基因、细胞骨架蛋白的调节相关。单独使用卵泡刺激素或雄激素抑制,也会影响支持细胞内相关miRNA的表达水平,表明卵泡刺激素和雄激素可协同或独立调节大鼠支持细胞内特定的miRNA的表达[14]。
4.2 内分泌干扰素
内分泌干扰素(endocrine disrupting chemical, EDC)可以通过阻断或模拟体内激素生成,使生殖系统功能紊乱。如邻苯二甲酸单丁酯(Mono-butyl phthalate, MBP)是一种邻苯二甲酸酯,通常作为塑化剂生产各类聚合材料,具有严重的生殖毒性。高浓度的MBP可导致小鼠睾发生氧化应激反应及丙二醛含量增加,同时精子发生重要基因SOX9、DAZL的转录水平明显下降,最终引起精子计数减少、畸形精子症及生精小管退化[52]。在大鼠支持细胞中,高浓度的MBP处理24~48 h,可产生毒性作用,表现为支持细胞数量减少、活力降低;相反,低浓度的MBP可以明显提高大鼠支持细胞活力、降低凋亡率,并通过上调miR-301b-3p及miR-3584-5p的表达水平激活ERK信号通路,促进支持细胞的增殖[10]。4.3 其他因素
除激素、化合物外,热应激、营养状况对支持细胞miRNA的表达也有影响。热应激是雄性生育的危害因素之一,Xu等[53]发现热处理TM4支持细胞后,miR-132、miR-431和miR-543的表达下调,炎性细胞因子中IL-6、IL-1α、IL-1β 的mRNA表达水平上调,干扰了精子发生或睾丸发育。此外,营养状态也能影响支持细胞内miRNA的表达水平。营养不良的绵羊组与对照组相比,miR-99a表达上调,紧密连接蛋白ZO-1基因的表达下调,引起血睾屏障功能受损;miR-98表达上调,使生精细胞凋亡增加;miR-34c、miR-10b的表达水平也发生了变化,影响精子质量[16]。这些体内外因素通过调控支持细胞内miRNA的表达水平,间接影响支持细胞的增殖或粘附功能,最终影响精子发生进程及睾丸发育。
5 结 语
miRNA自发现以来已逐渐成为生物学领域的研究热点之一,有关miRNA的生物合成过程、表达特性以及调控机制仍在不断探索中。已有大量研究阐释了miRNA合成的基本过程,涉及多种核酸酶及其辅助因子的剪切修饰、细胞核质转运及miRNA duplex的解旋等事件。但在miRNA合成方面仍有相关问题亟待解决,如miRNA duplex的解旋中切割依赖性/非依赖性解旋机制和ATP依赖性解旋酶解旋机制谁为主导?在哪些情况下仅存在一种机制或两种并存?ATP依赖性的解旋酶有哪些?这些疑问仍需进一步探索。研究表明,miRNA广泛存在于动、植物体内,在不同种属组织或细胞中特异性表达[54,55]。一些细胞内的miRNA通过对靶基因进行转录调控,在细胞增 殖分化、能量代谢、信号转导等方面发挥重要的作用[56,57,58,59]。近年来,越来越多与睾丸支持细胞相关的miRNA通过生物信息学技术、高通量测序、microRNA微阵列等方法得以发现,其中一些miRNA与支持细胞内的信号通路、靶基因相互作用,形成一个复杂的、多层次的支持细胞增殖与粘附功能的调控网络,间接参与精子发生与雄性生育。如miR-133b通过靶向GLI3,下调其表达,促进人支持细胞增殖及生精细胞缺失,引发唯支持细胞征[9];miR-471可下调DSC1、DSC2的表达,造成睾丸血睾屏障缺损、支持细胞-生精细胞间粘附缺陷[12,13]。因此,研究支持细胞相关的miRNA具有深远意义,不仅能丰富支持细胞生长调控网络,也可为支持细胞功能缺陷引起的不育症的诊断提供新的方法。
目前,支持细胞相关的miRNA的研究处于起步阶段,有关支持细胞特异性表达的miRNA仍少见报道。虽然某些miRNA的调控功能已经明确,但是大部分miRNA调节支持细胞的机制及最终是否与精子发生直接相关仍未阐明。已知的支持细胞相关miRNA的作用机制多由非人类哺乳动物模型或细胞水平得出,这些miRNA是否具有物种间保守性,能否在人体内发挥相同作用尚不明确。
综上所述,在后续的研究中,筛选支持细胞特异性表达的miRNA、对既往的研究结论进一步体内实验或转化验证、应用合适的miRNA分子诊断支 持细胞相关不育症将成为支持细胞miRNA的研究重点。
(责任编委: 苗龙)
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Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from undifferentiated precursors known as gonocytes and are strictly controlled in a special 'niche' microenvironment in the seminiferous tubules. Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors. Spermatocyte meiosis is another key step of spermatogenesis, which is regulated by Sertoli cells on the luminal side of the blood-testis barrier through paracrine signaling. In this review, we mainly focus on the role of Sertoli cells in the regulation of SSC self-renewal and spermatocyte meiosis, with particular emphasis on paracrine and endocrine-mediated signaling pathways. Sertoli cell growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), as well as Sertoli cell transcription factors, such as ETS variant 5 (ERM; also known as ETV5), nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR), have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis. Other transcription factors and signaling pathways (GDNF-RET-GFRA1 signaling, FGF2-MAP2K1 signaling, CXCL12-CXCR4 signaling, CCL9-CCR1 signaling, FSH-nociceptin/OPRL1, retinoic acid/FSH-NRG/ERBB4, and AR/RB-ARID4A/ARID4B) are also addressed.
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In the rat testis, studies have shown that cell polarity, in particular spermatid polarity, to support spermatogenesis is conferred by the coordinated efforts of the Par-, Crumbs-, and Scribble-based polarity complexes in the seminiferous epithelium. Furthermore, planar cell polarity (PCP) is conferred by PCP proteins such as Van Gogh-like 2 (Vangl2) in the testis. On the other hand, cell junctions at the Sertoli cell–spermatid (steps 8–19) interface are exclusively supported by adhesion protein complexes (for example, α6β1-integrin-laminin-α3,β3,γ3 and nectin-3-afadin) at the actin-rich apical ectoplasmic specialization (ES) since the apical ES is the only anchoring device in step 8–19 spermatids. For cell junctions at the Sertoli cell–cell interface, they are supported by adhesion complexes at the actin-based basal ES (for example, N-cadherin-β-catenin and nectin-2-afadin), tight junction (occludin-ZO-1 and claudin 11-ZO-1), and gap junction (connexin 43-plakophilin-2) and also intermediate filament-based desmosome (for example, desmoglein-2-desmocollin-2). In short, the testis-specific actin-rich anchoring device known as ES is crucial to support spermatid and Sertoli cell adhesion. Accumulating evidence has shown that the Par-, Crumbs-, and Scribble-based polarity complexes and the PCP Vangl2 are working in concert with actin- or microtubule-based cytoskeletons (or both) and these polarity (or PCP) protein complexes exert their effects through changes in the organization of the cytoskeletal elements across the seminiferous epithelium of adult rat testes. As such, there is an intimate relationship between cell polarity, cell adhesion, and cytoskeletal function in the testis. Herein, we critically evaluate these recent findings based on studies on different animal models. We also suggest some crucial future studies to be performed.
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Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.
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A growing number of reports have revealed that microRNAs (miRNAs) play critical roles in spermatogenesis. Our previous study showed that miR-762 is differentially expressed in immature and mature testes of Large White boars. Our present data shows that miR-762 directly binds the 3′ untranslated region (3′UTR) of ring finger protein 4 (RNF4) and down-regulates RNF4 expression. A single nucleotide polymorphism (SNP) in theRNF43′UTR that is significantly associated with porcine sperm quality traits leads to a change in the miR-762 binding ability. Moreover, miR-762 promotes the proliferation of and inhibits apoptosis in porcine immature Sertoli cells, partly by accelerating DNA damage repair and by reducing androgen receptor (AR) expression. Taken together, these findings suggest that miR-762 may play a role in pig spermatogenesis by regulating immature Sertoli cell growth.
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URLPMID:26755652 [本文引用: 5]
Sertoli cells play critical roles in regulating spermatogenesis and they can be reprogrammed to the cells of other lineages, highlighting that they have significant applications in reproductive and regenerative medicine. The fate determinations of Sertoli cells are regulated precisely by epigenetic factors. However, the expression, roles, and targets of microRNA (miRNA) in human Sertoli cells remain unknown. Here we have for the first time revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between Sertoli-cell-only syndrome (SCOS) patients and obstructive azoospermia (OA) patients with normal spermatogenesis using miRNA microarrays and real time PCR, suggesting that these miRNAs may be associated with the pathogenesis of SCOS. MiR-133b is upregulated in Sertoli cells of SCOS patients compared to OA patients. Proliferation assays with miRNA mimics and inhibitors showed that miR-133b enhanced the proliferation of human Sertoli cells. Moreover, we demonstrated that GLI3 was a direct target of miR-133b and the expression of Cyclin B1 and Cyclin D1 was enhanced by miR-133b mimics but decreased by its inhibitors. Gene silencing of GLI3 using RNA inference stimulated the growth of human Sertoli cells. Collectively, miR-133b promoted the proliferation of human Sertoli cells by targeting GLI3. This study thus sheds novel insights into epigenetic regulation of human Sertoli cells and the etiology of azoospermia and offers new targets for treating male infertility
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URLPMID:3401116 [本文引用: 8]
Abstract Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs--small, non-coding RNAs--are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.
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URLPMID:21325043 [本文引用: 7]
Abstract Spermatogenesis is absolutely dependent on FSH and androgens; suppression of these hormones inhibits germ cell development and thus sperm production. The final release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to hormone suppression. To define the molecular mechanisms that mediate FSH and androgen effects in the Sertoli cell, we investigated the expression and regulation of micro-RNAs (miRNAs), small noncoding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs up-regulated more than 2-fold after hormone suppression in vivo and in vitro in primary Sertoli cell cultures. The regulation of four of these miRNAs (miR-23b, -30c, -30d, and -690) was confirmed by quantitative RT-PCR. Bioinformatic analysis of potential targets of hormonally regulated miRNAs identified genes important for focal adhesion and regulation of the actin cytoskeleton, processes known to be intimately associated with adhesion of spermatids to Sertoli cells. Two of the identified genes, Pten, an intracellular phosphatase, and Eps15, a mediator of endocytosis, were down-regulated by the withdrawal of hormones in vivo and possess miR-23b target sites in their 3'-untranslated regions. Overexpression of miR-23b in vitro resulted in decreased translation of PTEN and EPS15 protein as assessed by Western blot and luciferase analysis. We conclude that FSH and androgens act on Sertoli cells in stage VIII to control the expression of miRNAs that operate in a coordinated manner to regulate cell adhesion pathways and male fertility and that miRNA transcription is a new paradigm in the hormone dependence of spermiation.
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URL [本文引用: 2]
URLPMID:26859601 [本文引用: 2]
The Sertoli cell is important for endocrine and paracrine control of spermatogenesis. Functions attributed to Sertoli cells are: (1) supportive and trophic functions for the cells of the seminiferous epithelium, (2) transport of mature spermatids towards the lumen of seminiferous tubules, (3) secretion of androgen binding protein, (4) production of substances with endocrine or paracrine action for spermatogenesis control and (5) interaction with intertubular endocrine Leydig cells. Inhibin B and anti-M llerian hormone (AMH) are glycoproteins belonging to the transforming growth factor (TGF-) superfamily; they are produced almost exclusively by the Sertoli cells and have been proposed as direct markers of their function and indirect markers of spermatogenesis. Serum inhibin B and AMH concentrations seem to constitute additional diagnostic parameters in male subfertility as they reflect Sertoli cell function. Stimulated concentrations of serum inhibin B and AMH do not add clinically relevant information in subfertile men compared to basal concentrations of these hormones. Serum inhibin B and AMH concentrations correlate with testicular histology/cytology but are not superior to FSH as predictors of the presence of sperm in testicular sperm extraction (TESE)/fine needle aspiration (FNA) biopsy in men with azoospermia.
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URLPMID:28779347 [本文引用: 1]
Abstract In recent decades, infertility has been considered a major widespread public health issue of very high concern. Currently, almost 50% of infertility cases are due to male factors, including semen disorders, obstructions, cryptorchidism, varicocele and testicular failures, which can occur due to malfunctions in both somatic and germ cells. In this context, besides other approaches, different miRNAs have been used as biomarkers for the diagnosis of male infertility, with different pathologic conditions such as Sertoli cell-only syndrome, mixed atrophy, and germ cell arrest. However, most studies related to male fertility do not point out the functions and cell targets of the described miRNAs. Initial investigations using experimental assays in murine and porcine models were performed, providing the first evidence of the influence of miRNAs on Sertoli cell function including, for instance, proliferation, maturation and hormone responses of these cells. The aim of this mini-review is therefore to summarize our present knowledge of this relevant subject and to highlight the importance of future investigations concerning the miRNA influence in the control of Sertoli cells, spermatogenesis and male fertility.
URL [本文引用: 1]
URL [本文引用: 1]
URL [本文引用: 1]
睾丸支持细胞(Sertoli cell)是曲细精管内唯一与生精细胞直接接触的体细胞,在生精过程中起免疫屏障、支持、营养和调节作用。相邻支持细胞、支持细胞与生精细胞之间的连接类型包括紧密连接、锚定连接和缝隙连接。这些连接结构与精子发生过程紧密联系,连接结构紊乱或异常,会干扰精子发生过程中的信号通路、生精细胞迁移、精子形态形成和精子极性维持等,引起生精功能障碍,导致男性生育力下降,甚至不育。
URL [本文引用: 1]
睾丸支持细胞(Sertoli cell)是曲细精管内唯一与生精细胞直接接触的体细胞,在生精过程中起免疫屏障、支持、营养和调节作用。相邻支持细胞、支持细胞与生精细胞之间的连接类型包括紧密连接、锚定连接和缝隙连接。这些连接结构与精子发生过程紧密联系,连接结构紊乱或异常,会干扰精子发生过程中的信号通路、生精细胞迁移、精子形态形成和精子极性维持等,引起生精功能障碍,导致男性生育力下降,甚至不育。
URLPMID:5054211 [本文引用: 1]
MicroRNAs (miRNAs) are approximately 22-nucleotide-long non-coding RNAs that are important regulators of gene expression in eukaryotes. miRNAs are first transcribed as long primary transcripts, which then undergo a series of processing steps to produce the single-stranded mature miRNAs. This article reviews our current knowledge of the mechanism and regulation of mammalian miRNA expression and points out areas of research that may enhance our understanding of how the specificity and efficiency of miRNA production is controlledin vivo.
URLPMID:25799998 [本文引用: 1]
Abstract The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N(6)-methyladenosine (m(6)A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m(6)A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.
URLPMID:29615475 [本文引用: 2]
MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that are integral to a wide range of cellular processes mainly through the regulation of translation and mRNA stability of their target genes. The placenta is a transient organ that exists throughout gestation in mammals, facilitating nutrient and gas exchange and waste removal between the mother and the fetus. miRNAs are expressed in the placenta, and many studies have shown that miRNAs play an important role in regulating trophoblast differentiation, migration, invasion, proliferation, apoptosis, vasculogenesis/angiogenesis and cellular metabolism. In this review, we provide a brief overview of canonical and non-canonical pathways of miRNA biogenesis and mechanisms of miRNA actions. We highlight the current knowledge of the role of miRNAs in placental development. Finally, we point out several limitations of the current research and suggest future directions.
URLPMID:22233755 [本文引用: 9]
Small RNAs, such as microRNAs and small interfering RNAs, act through Argonaute (Ago) proteins as a part of RNA-induced silencing complexes (RISCs). To make RISCs, Ago proteins bind and subsequently unwind small RNA duplexes, finally leaving one strand stably incorporated. Here we identified the N domain of human AGO2 as the initiator of duplex unwinding during RISC assembly. We discovered that a functional N domain is strictly required for small RNA duplex unwinding but not for precedent duplex loading or subsequent target cleavage. We postulate that RISC assembly is tripartite, comprising (i) RISC loading, whereby Ago undergoes conformational opening and loads a small RNA duplex, forming pre-RISC; (ii) wedging, whereby the end of the duplex is pried open through active wedging by the N domain, in preparation for unwinding; and (iii) unwinding, whereby the passenger strand is removed through slicer-dependent or slicer-independent unwinding, forming mature RISC.
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URLPMID:4627090 [本文引用: 4]
Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1–4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that ‘slicer-dependent unwinding’ via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that ‘slicer-independent unwinding’ plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that ‘slicer-dependency’ for the unwinding was affected primarily by certain parameters such as temperature and Mg2+. We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system inDrosophila.
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URLPMID:11701122 [本文引用: 3]
We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive 65360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5′ phosphates on siRNAs. A 5′ phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.
URLPMID:27251421 [本文引用: 1]
RNA helicases comprise the largest family of enzymes involved in the metabolism of mRNAs, the processing and fate of which rely on their packaging into messenger ribonucleoprotein particles (mRNPs). In this Review, we describe how the capacity of some RNA helicases to either remodel or lock the composition of mRNP complexes underlies their pleiotropic functions at different steps of the gene expression process. We illustrate the roles of RNA helicases in coordinating gene expression steps and programmes, and propose that RNA helicases function as molecular drivers and guides of the progression of their mRNA substrates from one RNA-processing factory to another, to a productive mRNA pool that leads to protein synthesis or to unproductive mRNA pools that are stored or degraded.
URLPMID:28827400 [本文引用: 1]
Abstract While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of 50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins. 2017 Elbarbary et al.; Published by Cold Spring Harbor Laboratory Press.
URLPMID:23500488 [本文引用: 1]
78 Network-level perspective of miRNA activity. 78 A summary of the structure of eukaryotic Argonaute. 78 A comparison of miRNA function in vitro versus in vivo. 78 A description of four miRNA families: miR-290–295, let-7, miR-17–92, and miR-34.
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[D].
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Abstract RNF4 is an E3 ubiquitin ligase originally identified as a transcription co-activator. The mechanism by which RNF4 promotes transcription remains unclear. In this study, I found that RNF4 antagonizes transcriptional repression mediated by DNA methylation. RNF4 does not promote DNA demethylation, but mediates the ubiquitination of MeCP2, a methyl-CpG-binding domain (MBD) protein. Removal of MeCP2 from gene promoters activates transcription. This study thus not only uncovers how RNF4 functions as a transcription activator, but also reveals the mechanism by which MeCP2 protein stability is regulated. 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
URLPMID:26766492 [本文引用: 1]
Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.
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URLPMID:16958114 [本文引用: 2]
Hedgehog (Hh) signalling is known to regulate many aspects of normal development as well as being upregulated in various cancers. Signalling is mediated by the Gli family of zinc finger transcription factors. Based on observations that deletion of one of the three Hh genes, Dhh , leads to male infertility, we hypothesized that regulated expression of Hh signalling components would be a feature of adult spermatogenesis. We used in situ hybridization to characterise Gli gene expression in juvenile and adult mouse testes. In the first wave of spermatogenesis, mRNAs encoding all three Glis are detected in spermatogonia and Sertoli cells. In adult mouse testes, these transcripts are observed in spermatogonia and spermatocytes, with reduced signal intensity in round spermatids. The mRNAs encoding key effectors of Hh signalling, Ptc2, Smo , and Fu , are also most apparent in spermatogonia, spermatocytes, and to a lower extent in round spermatids. In contrast, mRNA encoding SuFu , a negative regulator of Hh signalling, was most predominant in round spermatids and the protein is evident in round and elongating spermatids, suggesting that SuFu protein may switch off Hh signalling in haploid germ cells. Overall, the coordinated expression pattern of these genes in adult mouse testis indicates a role for Hh signalling in spermatogenesis.Developmental Dynamics 235:3063-3070, 2006. 2006 Wiley-Liss, Inc.
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Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced by pituitary gonadotrope cells and are required for steroidogenesis, the maturation of ovarian follicles, ovulation, and spermatogenesis. Synthesis of LH and FSH is tightly regulated by a complex network of signaling pathways activated by hormones including gonadotropin-releasing hormone, activin and sex steroids. Members of the forkhead box (FOX) transcription factor family have been shown to act as important regulators of development, homeostasis and reproduction. In this review, we focus on the role of four specific FOX factors (FOXD1, FOXL2, FOXO1 and FOXP3) in gonadotropin hormone production and discuss our current understanding of the molecular function of these factors derived from studies in mouse genetic and cell culture models.
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Abstract Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, 2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones.
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URLPMID:26165742 [本文引用: 1]
Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1, IL-1, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment.
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URLMagsci [本文引用: 1]
MicroRNA(miRNA)是一类长约22 nt的非编码小RNA,广泛存在于各种生物中,调节生物体生长、发育和凋亡等过程。研究表明,miRNA在猪肌肉、脂肪、生殖系统以及免疫系统等的发育过程中发挥着重要的调控作用。此外,高通量的新一代测序技术在猪miRNA的挖掘和差异表达研究中发挥着巨大的作用。文章综述了高通量的新一代测序技术在挖掘猪miRNA中的应用以及一些miRNA在猪脂肪代谢、肌肉发育、卵母细胞成熟和B、T淋巴细胞发育中的调控作用,旨在为猪miRNA的研究提供参考,为利用miRNA调控和改善猪肉品质、生长性能、繁殖性能以及免疫机能提供理论基础和研究思路。
URLMagsci [本文引用: 1]
MicroRNA(miRNA)是一类长约22 nt的非编码小RNA,广泛存在于各种生物中,调节生物体生长、发育和凋亡等过程。研究表明,miRNA在猪肌肉、脂肪、生殖系统以及免疫系统等的发育过程中发挥着重要的调控作用。此外,高通量的新一代测序技术在猪miRNA的挖掘和差异表达研究中发挥着巨大的作用。文章综述了高通量的新一代测序技术在挖掘猪miRNA中的应用以及一些miRNA在猪脂肪代谢、肌肉发育、卵母细胞成熟和B、T淋巴细胞发育中的调控作用,旨在为猪miRNA的研究提供参考,为利用miRNA调控和改善猪肉品质、生长性能、繁殖性能以及免疫机能提供理论基础和研究思路。
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URLPMID:29278767 [本文引用: 1]
Abstract The nutritional value of goat milk largely depends on its fatty acid content and composition. MicroRNAs (miRNAs) are a class of RNA molecules 18-25nt in length that regulate gene expression and play crucial roles in several biological processes, including fatty acid metabolism. In this study, we analyzed the correlation between differentially expressed miRNAs in goat mammary tissue and the fatty acid composition of goat milk by using Pearson correlations. Results revealed that levels of miR-183 were highly and positively correlated with the fatty acid content in the milk. In addition, we demonstrated that overexpression of miR-183 inhibits milk fat metabolism and inhibition of miR-183 promotes milk fat metabolism. Using Western blot, we demonstrate that MST1, one of the major elements of the Hippo signaling pathway, is a target of miR-183. Immunofluorescence assays revealed that miR-183 targets MST1 in the cytoplasm. In summary, data indicate that miR-183 inhibits the metabolism of milk fat by targeting the MST1 gene in the cytoplasm in goat mammary epithelial cells.
