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外泌体microRNA在猪成熟和闭锁卵泡中的表达差异及功能分析

本站小编 Free考研考试/2021-12-26
陈慧芳,, 黄绮亮, 胡智超, 潘晓婷, 吴志胜, 白银山,*佛山科学技术学院生命科学与工程学院,广东佛山 528231

Expression Differences and Functional Analysis of Exosomes microRNA in Porcine Mature and Atretic Follicles

CHEN HuiFang,, HUANG QiLiang, HU ZhiChao, PAN XiaoTing, WU ZhiSheng, BAI YinShan,*School of Life Science and Engineering, Foshan University, Foshan 528231, Guangdong

通讯作者: 白银山,E-mail： xuefei200403@163.com

责任编辑: 林鉴非
收稿日期:2020-09-27接受日期:2021-04-25
基金资助:广东省畜禽疫病防治研究重点实验室基金项目(YDWS1902)
佛山科学技术学院高层次人才科研启动项目(gg040969)


Received:2020-09-27Accepted:2021-04-25
作者简介 About authors
联系方式：陈慧芳,E-mail： chenhuifang07@163.com。








摘要
【目的】通过分析成熟卵泡液外泌体（mature follicular fiuid Exosomes, mffEXs）和闭锁卵泡液外泌体（atretic follicular fiuid Exosomes, affEXs）miRNA的表达差异,探索卵泡液外泌体（EXs）miRNA在卵泡发育和闭锁过程中的调控作用。【方法】本研究通过抽提4—6 mm猪成熟发育和闭锁卵泡的卵泡液分离外泌体,进行粒径分析及Western Blot检测对EXs进行鉴定,接下来对特征性EXs携带的miRNA测序和功能富集分析,筛选关键信号通路和差异基因。最后,将mffEXs和affEXs作为添加剂进行颗粒细胞培养,利用Q-PCR检测技术分析关键基因的表达,验证两类卵泡液内EXs miRNA在卵泡发育中的调控功能。【结果】成功分离了mffEXs和affEXs,对比mffEXs测序结果,affEXs中有90个miRNA上调表达,220个miRNA下调表达,表明了卵泡液中的miRNA表达水平与调控卵泡发育有关;KEGG富集分析结果显示两类卵泡的差异信号通路主要集中在Ras、cAMP、P53和MAPK等信号通路,涉及调控卵母细胞发育、减数分裂以及颗粒细胞细胞周期等生物学功能。在闭锁卵泡中,上调表达的ssc-let-7a和ssc-miR-133a-3p分别潜在靶向调控细胞周期蛋白依赖性激酶（CDK1）和胰岛素生长因子（IGF1）,抑制了G1和G2/M期的运转和类固醇激素代谢,促使颗粒细胞周期运转受阻和颗粒细胞凋亡,引起卵泡闭锁的发生;下调的ssc-miR-21-5p潜在靶向肿瘤抑癌基因（P53）,抑制细胞周期运转,促使颗粒细胞凋亡。在体外培养的颗粒细胞中分别添加mffEXs和affEXs,Q-PCR结果显示CDK1在mffEXs中显著上调表达,而P53显著下调表达,表明了测序分析结果的可靠性。这些结果均显示了affEXs中miRNA表达水平的变化促使颗粒细胞凋亡和细胞周期阻滞,引起卵泡闭锁。【结论】猪affEXs携带miRNA增加了对CDK1、IGF1和P53的表达调控,抑制颗粒细胞细胞周期运转和类固醇激素代谢等信号通路,引起颗粒细胞凋亡,导致卵泡闭锁。
关键词： 猪;成熟卵泡液;闭锁卵泡液;EXs;miRNA

Abstract
【Objective】 To explore the regulatory role of follicular fluid Exosomes (EXs) miRNA in follicular development and atresia, the difference of miRNA expression between mature follicular fluid Exosomes (mffEXs) and atretic follicular fluid Exosomes (affEXs) were analyzed. 【Method】In this study, the follicular fluid of 4-6 mm porcine mature development and atresia follicles was extracted. Then EXs were identified by particle size analysis and Western Blot detection, respectively. the sequencing analysis of the characteristic EXs carried miRNA and functional enrichment analysis were carried out, and then the key signal pathways and differential genes were screened. Finally, mffEXs and affEXs were used as additives for granular cell culture, and Q-PCR detection technology was used to analyze the expression of key genes to verify and analyze the regulatory functions of EXs miRNA in the two types of follicular fluid in follicular development. 【Result】This study successfully separated mffEXs and affEXs. The sequencing results showed that compared with mffEXs, 90 miRNAs in affEXs were up-regulated and 220 miRNAs were down-regulated, indicating that the level of miRNA expression in follicular fluid could directly regulate follicular development. KEGG enrichment analysis showed that the differential signaling pathways of the two types of follicles were mainly concentrated in the signal pathways, such as Ras, cAMP, P53 and MAPK, which involved in the regulation of biological functions, such as oocyte development, meiosis, and granulosa cell cycle. In atretic follicles, the up-regulated expression of ssc-let-7a and ssc-miR-133a-3p potentially targeted and regulated cyclin-dependent kinase (CDK1) and insulin growth factor (IGF1), which inhibited G1 and G2/M Phase operation, and steroid hormone metabolism promoted the obstruction of granular cell cycle and the apoptosis of granular cells, causing follicular atresia; down-regulated ssc-miR-21-5p potentially targeted tumor suppressor gene (P53) and inhibited cell cycle operation to promote the apoptosis of granular cells. mffEXs and affEXs were added to granular cells cultured in vitro, and Q-PCR results showed that CDK1 was significantly up-regulated in mffEXs, while P53 was significantly down-regulated, indicating the reliability of the sequencing analysis results. These results all showed that changes in miRNA expression levels in affEXs promoted granular cell apoptosis and cell cycle arrest, causing follicular atresia. 【Conclusion】 Porcine affEXs carry miRNAs increased the regulation of CDK1, IGF1 and P53 gene expression, and inhibited the cell cycle of granulosa cells and steroid hormone metabolism and other signal pathways, causing granulosa cell apoptosis and follicular atresia.
Keywords：porcine;mature follicular fluid;atretic follicular fluid;EXs;miRNA


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本文引用格式
陈慧芳, 黄绮亮, 胡智超, 潘晓婷, 吴志胜, 白银山. 外泌体microRNA在猪成熟和闭锁卵泡中的表达差异及功能分析. 中国农业科学, 2021, 54(21): 4664-4676 doi:10.3864/j.issn.0578-1752.2021.21.015
CHEN HuiFang, HUANG QiLiang, HU ZhiChao, PAN XiaoTing, WU ZhiSheng, BAI YinShan. Expression Differences and Functional Analysis of Exosomes microRNA in Porcine Mature and Atretic Follicles. Scientia Agricultura Sinica, 2021, 54(21): 4664-4676 doi:10.3864/j.issn.0578-1752.2021.21.015


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0 引言

【研究意义】卵泡闭锁是指一些卵泡发育到一定阶段后停止发育并发生退化的一种普遍存在的现象。在很多雌性动物卵泡发育过程中,只有极少数卵泡能够发育成熟并且排卵,绝大多数卵泡都会发生闭锁[1,2],这可能与卵泡液所含营养物质的差异[3]、激素[4]、氧化应激[5]和各种细胞因子[6]等相关,它们能够通过不同途径将刺激信号级联放大,引起通路中相关作用基因或转录因子活性增加或减少进而调控细胞凋亡[7,8]。以往研究显示激素和营养物质均能参与调控卵泡闭锁,而关于外泌体（exosomes, EXs）的调控作用,目前还知之甚少[9]。EXs是一类由细胞分泌的直径大小约30—150 nm的细胞外囊泡,因携带多种生物活性物质,可进行细胞间信息传递[10]。已有报道在各种体液中均含有大量EXs[11,12],且EXs可选择性地包裹细胞内的多种活性调节物质（miRNA、LncRNA和CircRNA等）,其中miRNA含量最丰富,广泛进行细胞间物质转运和信号转导,调节功能基因表达[13,14]。近年来,相关卵泡液EXs miRNA的研究越来越多,但关于EXs miRNA对卵泡闭锁的调控机制仍未见报道。【前人研究进展】适宜的卵泡液微环境是促使卵泡发育的主要场所,研究显示卵泡液EXs中的miRNA广泛参与调控颗粒细胞类固醇激素代谢的相关通路[15],涉及调节颗粒细胞增殖和雌二醇的合成与分泌[16]。卵泡液EXs miRNA的相关研究已在多种女性生殖系统疾病中作为检测和治疗的关键靶标[17,18,19,20]。如高水平表达的EXs-miR-199可以抑制芳香化酶CYP19A1的合成与代谢,促使雄激素升高,引起了多囊卵巢综合症的发生发展[21]。卵泡闭锁也可能直接受到卵泡液EXs miRNA的调控,但目前miRNA在卵泡闭锁过程中的调控机制还并不清楚。【本研究切入点】卵泡液携带的EXs miRNA在调控卵泡闭锁方面的研究有待解析。本研究对猪EXs miRNA进行测序分析,通过GO和KEGG功能富集分析,探究EXs miRNA对卵泡闭锁的调控机制。【拟解决的关键问题】通过对比成熟及闭锁卵泡液中的EXs miRNA的表达情况,拟探究猪卵泡液EXs携带的miRNA在调控成熟卵泡发育和闭锁卵泡发生的潜在分子机制。

1 材料与方法

试验于2018年10月至2019年12月于广东省佛山科学技术学院生命科学与工程学院和华南农业大学动物科学学院进行。

1.1 卵泡液的收集

从广州市嘉禾屠宰场采集猪卵巢,装入38.5℃含有青霉素和链霉素生理盐水中的保温瓶内,2 h内运回实验室。用37℃添加青链霉素的生理盐水清洗3遍,随后使用10 mL注射器吸取直径为4—6 mm卵泡中的液体,4℃,12 000 r/min离心3 min去除细胞碎片;然后使用0.22 μm过滤器过滤,进行EXs提取。

1.2 外泌体的分离与鉴定

使用ExoQuick Exosome Isolation Kit（System Bioscience, SBI）分离mffEXs和affEXs,详细步骤均参照试剂盒说明书。分离完成后,一部分用于检测鉴定,另一部分置于-80℃冰箱中保存备用。

1.3 外泌体粒径分析（NTA）

取一部分mffEXs和affEXs,用PBS重悬,随后将液体添加到Nanosight（NS300）纳米颗粒跟踪分析仪（NTA）中,利用NTA对分离出的EXs的大小和形态进行分析。

1.4 Western Blot技术检测

将RIPA裂解液混合加入mffEXs和affEXs中,进行裂解和变性。根据Western-Blot步骤检测外泌体表面标志蛋白CD9、CD63和CD81（Rabbit anti-CD9, 1:2000 Dilution, ab92726 Abcam; Rabbit anti-CD63, 1:2000 Dilution, ab231975, Abcam和Rabbit anti-CD81, 1:2000 Dilution, ab109201, Abcam）的表达,4℃过夜孵育,1×TBST洗脱3次后,添加二抗（sc-2030, Santa Cruz, 1:2000 Dilution）,室温孵育2 h,1×TBST洗脱3次,添加显色剂显色后使用凝胶成像系统拍照[22,23]。

1.5 外泌体总RNA提取、cDNA文库构建和RNA测序

采用Trizol（Life teachnologies）传统法提取mffEXs和affEXs总RNA,通过NanoDrop（Nanodrop 2000,Thermo,USA）以及Agilent 2100（Agilent Technologies, Palo Alto, CA, USA）对提取的总RNA的浓度和完整性进行检测,并构建小RNA文库,使用Illumina HiSeqTM 2000（Illumina, San Diego, CA, USA）进行测序,该部分由广州基迪奥生物科技有限公司完成。

1.6 miRNA的差异分析与靶基因预测

评估miRNA测序质量,计算RNA的长度分布,过滤掉原始数据中低质量序列（质量值<20的碱基数超过1个或含N的序列）,获得高质量的Reads。通过计算TPM（Tags per million）的表达量[24],筛选出mffEXs和affEXs中差异表达的miRNA。使用RNAhybrid（v2.1.2）、Miranda（v3.3a）和TargetScan（Version：7.0）进行miRNA靶基因预测和功能分析,并将获得的靶基因用Cytoscape软件绘制成可视化互作网络图。

1.7 GO和KEGG通路富集分析

筛选出卵泡液EXs中差异表达miRNA对应的靶基因,进行GO（ http://www.geneontology.org/）和KEGG（kyoto encyclopedia of genesand genomes; http://www.kegg.jp/kegg）显著性富集分析[25],分别描述GO的分子功能（molecular function）、细胞组分（cellular component）和生物进程（biological process）,并选择与卵母细胞发育相关的主要调节基因和信号通路进行分析。

1.8 颗粒细胞试验

将配制20 μg·mL-1的mffEXs和affEXs作为添加剂加到颗粒细胞培养基中进行培养,48 h后提取颗粒细胞的RNA,反转录合成cDNA,通过Q-PCR分析CDK1和P53的表达情况,具体反应条件为95℃预变性4 min;95℃变性30 s;59℃退火30 s;72℃延伸15 s,共33个循环。其中CDK1引物（上游AGAGTCGCTGGGGATCTACC;下游：TCATGGC TACCACTTGACCTG,扩增产物134 bp;XM_ 005671013）、P53引物（上游AAGGGAATTTACGGGC CGAG;下游CACGCACCTCAAAGCTGTTC,扩增产物228 bp;NM_213824）和GAPDH内参引物（上游TCGGAGTGAACGGATTTGGC;下游TGACAAG CTTCCCGTTCTCC,扩增产物189 bp;NM_001206359）。

2 结果

2.1 成熟及闭锁卵泡液EXs的分离鉴定与miRNA测序分析

将分离的成熟卵泡和闭锁卵泡（图1-A,B）EXs进行粒径分析和蛋白检测,结果显示粒径峰值约100 nm（图1-C,D）;Western-Blot结果显示,CD9、CD63和CD81（图1-E）这些标志性蛋白均在分离的mffEXs和affEXs中表达。测序结果显示,mffEXs和affEXs携带的miRNA序列长度主要分布在20—22 nt范围内（图1-F）;miRNA表达谱比较分析显示,90个miRNA在affEXs中显著上调表达,220个miRNA显著下调表达（图1-G）。

图1

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图1外泌体分离检测与miRNA测序

A：闭锁卵泡的形态观察结果;B：成熟卵泡的形态观察结果;C：mffEXs粒径分析结果;D：affEXs粒径分析结果;E：Westem blot检测结果;F：miRNA的序列长度分布;G：mffEXs和affEXs中miRNA的表达差异结果
Fig. 1Isolation and detection of exosomes and sequencing of miRNA

A: Morphological observation of atretic follicles; B: Morphological observation of mature follicles; C: Particle size analysis results of mffexs; D: Particle size analysis results of affexs; E: Results of Westem blot analysis; F: Sequence length distribution of miRNA; G: Differential expression of miRNA in mffEXs and affEXs


2.2 mffEXs和affEXs中主要差异的miRNA

根据mffEXs和affEXs携带的miRNA测序结果,以差异倍数|log2（F/C）|≥1.2,且P<0.05为阈值进行筛选,发现这些miRNA在mffEXs和affEXs中存在显著差异,且可能直接参与调控卵泡的成熟发育及闭锁（表1）。

Table 1
表1
表1卵泡液EXs中主要差异表达的miRNA
Table 1The main differential expression miRNA in follicular fluid EXs
miRNA名称 miRNA namemffEXs-TPM mffEXs-Tags per millionaffEXs-TPM affEXs-Tags per million差异倍数 Fold change
ssc-miR-1191.13573683.40414.268370509
ssc-miR-133a-3p125.33271137.31013.181790898
ssc-miR-874588.89024261.3142.855227792
ssc-let-7a359.44722278.73352.664380439
ssc-let-7f-5p378.93451986.69372.39034906
ssc-miR-23b121.4619631.38192.378009061
ssc-miR-486810.85854166.70952.361386438
ssc-miR-6529548.71432743.52942.321905957
ssc-miR-26a2160.55439279.46362.102639923
ssc-miR-99b13585.317538933.84891.51897669
ssc-miR-202-3p2947.65437512.82821.349788776
ssc-miR-1271-5p22519.310152612.41991.224240778
ssc-miR-21-5p555532.4026444467.5- 0.321793131
ssc-miR-1431443.9288621.0989-1.2171
ssc-miR-10b163814.11368106.9853-1.266184975
ssc-miR-140-3p5264.50742046.3356-1.363255777
ssc-miR-10a-5p7077.76062453.5461-1.528424586
ssc-miR-2320-5p146.822149.3588-1.572689917
ssc-miR-125b13217.72833973.3874-1.734032879
ssc-miR-16371.46109.0008-1.768868142
ssc-miR-146a-5p6504.75391748.1259-1.895685384
ssc-miR-145-3p975.5663257.0773-1.924037697
ssc-miR-744367.455794.6045-1.957589617
ssc-miR-184584.4855121.3405-2.268106073
ssc-miR-30d3433.5021707.4768-2.278926102
ssc-miR-132301.919747.3022-2.674185705
ssc-miR-125a18571.39671441.6897-3.687249733

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2.3 GO功能注释分析

根据差异表达miRNA的靶基因进行GO功能注释分析,结果发现它们主要富集于11个生物过程条目（细胞周期进程、蛋白丝氨酸/苏氨酸激酶活性、MAPK级联和有丝分裂细胞周期的G2/M转变等）、11个细胞组分条目（纺锤、周期蛋白依赖的蛋白激酶全酶复合物、蛋白激酶复合物和泛素连接酶复合物等）和13个分子功能条目（磷酸酶结合、钙调素结合、蛋白磷酸酶结合和腺苷酸环化酶活性等）。进一步研究发现,这些差异基因被划分为多种生物功能类别,包括细胞增殖、代谢和凋亡等。差异表达基因的分布及生物学过程类别如图2所示。

图2

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图2卵泡液EXs中差异miRNA靶基因的GO富集分析

Fig. 2Go enrichment analysis of differentially expressed genes in follicular fluid EXs miRNA



2.4 KEGG富集分析

KEGG通路分析显示差异表达的miRNA涉及调控169个靶基因,主要富集于Ras信号通路（ssc04014）、cAMP信号通路（ssc04024）、P53信号通路（ssc04115）、MAPK信号通路（ssc04010）、卵母细胞减数分裂（ssc04114）、泛素介导的蛋白水解作用（ssc04120）和细胞周期（ssc04110）等通路（图3）,可能参与调控卵泡的成熟与闭锁。

图3

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图3KEGG功能富集分析

Fig. 3Enrichment analysis of KEGG function



2.5 富集关键信号通路中miRNA潜在的靶基因

差异表达的miRNA靶标富集的主要信号通路显示了靶基因的调控作用（表2）。通过富集通路筛选出一些参与颗粒细胞凋亡和卵母细胞发育的关键基因,结合信号传导通路深入研究猪卵泡闭锁调控机制,发现介导卵泡闭锁的途径显著富集于7条信号通路,且这些通路可能直接参与调控卵泡闭锁。

Table 2
表2
表2主要信号通路中miRNA调控靶基因信息表
Table 2Information table of miRNA regulatory target genes in main significant pathways
信号通路 Signaling pathways靶基因 Target gene list
Ras信号通路
Ras signaling pathway (ssc04014)		AKT2 (miR-148b-5p), CALM1 (miR-143-3p), IGF1 (let-7a), INS (miR-424-5p), KRAS (miR-181a), MAP2K1 (miR-1271-5p), MAPK10 (miR-148a-3p), PIK3CB (miR-126-5p), PRKACB (miR-146b), RAF1 (miR-125b)
cAMP信号通路
cAMP signaling pathway (ssc04024)		ADCY7 (miR-125b), AKT2 (miR-148b-5p), BRAF (let-7a), CALM1 (miR-130a), CAMK2A (miR-10a-5p), CAMK2G (miR-371-5p), GNAI2 (miR-124a), MAPK10 (miR-1), PDE3B (miR-126-5p), PIK3CG (miR-30a-3p), PPP1CC (miR-1343), PRKACB (miR-181a), RAF1 (miR-424-5)
P53信号通路
P53 signaling pathway (ssc04115)		CCNE2 (miR-1), IGF1 (miR-133a-3p), CDK1 (let-7a), CCNB (miR-9791-3p), P53 (miR-21), P53I3 (ssc-miR-7-5p)
MAPK信号通路
MAPK signaling pathway (ssc04010)		AKT2 (miR-10a-3p), BRAF (miR-18a), CDC25B (miR-146a-3p), KRAS (miR-181d-5p), MAPK10 (miR-199b-3p), MAPK8 (miR-199a-3p), PPP3CB (miR-202-3p), PRKACB (miR-143-5p), RAF1 (miR-424-5p), RPS6KA3 (miR-125a), P53 (miR-21)
卵母细胞减数分裂
Oocyte meiosis (ssc04114)		ADCY7 (miR-125b), ANAPC1 (miR-326), AR (miR-124a), AURKA (miR-125a), BTRC (miR-1271-5p), CALM1 (miR-181b), CAMK2G (miR-371-5p), CCNE2 (miR-140-3p), CDC26 (miR-218), CDK1 (miR-143-3p), CPEB3 (miR-199b-3p), ESPL1 (miR-141), FBXO11 (miR-129a-3p), IGF1 (miR-133a-3p), ITPR1 (miR-200b), MAP2K1 (miR-143-5p), MAPK12 (miR-125b), MOS (miR-155-3p), PGR (miR-101), PKMYT1 (miR-106a) PLCZ (miR-124a), PPP3CD (miR-199a), PRKACB (miR-143-5p), PTTG1 (miR-1224), RBX (miR-218-3p), REC8 (miR-199b-3p), RPS6KA3 (let-7a), SGOL1 (miR-126-5p), SKP1 (miR-222), SMC1A (miR-128), SMC 3 (miR-9820-5P), STAG3 (miR-27b-3p), YWHAZ (miR-1)
泛素介导的蛋白水解作用
Ubiquitin mediated proteolysis (ssc04120)		IGF1 (miR-133a-3p), BTRC (miR-1), FZR1 (miR-874), CDC27 (miR-1271-5), FBXW11 (miR-671-5p), RBX1 (miR-1224), SKP1 (miR-148a-3p)
细胞周期
Cell cycle (ssc04110）		IGF1 (miR-133a-3p), BUB1 (miR-338), CCNE (miR-140-3p), CDC26 (miR-143-5p), CDK1 (let-7a), FZR1 (miR-138), MAD1L1 (miR-199b-5p), PKMYK1 (miR-106a), PLK1 (miR-18a), SMC1A (miR-132), SMC (miR-9820-5p), YWHAZ (miR-1), P53 (miR-21)

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2.6 构建miRNA-靶基因互作网络

将筛选获得的与卵泡成熟发育和闭锁相关且差异表达显著的miRNA及靶基因进行了共表达网络分析。在互作网络中,我们检测了3个特异性miRNA（ssc-miR-21-5p、ssc-miR-133a-3p和ssc-let-7a）,发现它们分别作用于P53、IGF1和CDK1等基因（图4）。其中,CDK1是一种调节细胞周期运转的重要激酶,它能在特定时间被激活,通过底物磷酸化结合cyclin B驱动细胞周期运转,而ssc-let-7a潜在靶向CDK1,抑制了细胞G2/M期的运转,抑制其增殖,诱导颗粒细胞凋亡;P53是调控细胞凋亡的关键因子,进一步分析发现affEXs中ssc-miR-21-5p下调潜在靶向调控P53表达,调控颗粒细胞凋亡;胰岛素生长因子（IGF1）作为ssc-miR-133a-3p的潜在靶基因,在IGFBP-1作用下参与调控,增强了卵巢颗粒细胞芳香化酶的活性,抑制了类固醇激素代谢,诱导颗粒细胞凋亡,揭示了这些miRNA的靶标可能潜在调控卵泡的成熟发育和闭锁。

图4

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图4差异性表达miRNA的靶基因互作网络

Fig. 4Target gene interaction network of differentially expressed miRNA



2.7 卵泡液EXs添加颗粒细胞的验证

根据以上mffEXs和affEXs的差异表达及功能富集分析结果,我们将mffEXs和affEXs分别添加到颗粒细胞中培养48 h。镜检结果显示,mffEXs添加的颗粒细胞显示出增殖状态,长势较好;affEXs添加颗粒细胞,形态发生拉长,部分出现凋亡现象（图5-A、B）。Q-PCR结果显示CDK1在mffEXs中显著上调表达,而P53在affEXs中显著上调（图5-C、D）,表明affEXs可能潜在促进了颗粒细胞凋亡的发生。

图5

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图5mffEXs和affEXs对猪颗粒细胞调控作用

A：添加mffEXs培养的颗粒细胞结果;B：添加affEXs培养颗粒细胞的结果;C和D：Q-PCR检测CDK1和P53表达结果
Fig. 5Regulatory effects of mffEXs and affEXs on pig granulosa cells

A: Results of granulosa cells cultured with mffEXs; B: Results of affEXs culture of granulosa cells; C&D: Expression results of CDK1 and P53 detected by Q-PCR


3 讨论

卵泡闭锁是一种通过凋亡机制产生的生理性程序性细胞死亡,最终引起卵母细胞发育停滞和颗粒细胞凋亡,其中颗粒细胞凋亡是引起闭锁卵泡发生的主要原因[26]。大量研究报道EXs介导细胞间信号转导,涉及多种生理功能调控。已有研究显示,卵泡液中的EXs携带大量miRNA[27,28],参与卵泡发育调控。但EXs miRNA参与调控卵泡闭锁的调控机制还不清楚。本研究通过抽提卵泡液,发现猪成熟及闭锁卵泡中均含有粒径峰值约100 nm,且表面表达特异性标志蛋白CD9、CD63和CD81的EXs,这与先前的研究结果一致[29]。EXs是细胞向胞外分泌的一种囊泡小体[30],在卵泡液[31]、输卵管[32]和子宫液[33]中均存在。卵泡液中EXs携带大量调控分子可以直接调控卵母细胞和颗粒细胞,构成卵母细胞和颗粒细胞生存的微环境,促使卵泡发育[34]。miRNA是一类EXs中含量最丰富的RNA小分子物质,广泛参与基因表达调控[35,36],促使完成动物机体的各项生命活动[37]。

在卵泡发育过程中,EXs介导的信号传递发挥重要的调控作用,不仅影响卵母细胞发育,而且还会影响卵母细胞受精和胚胎质量等[20,38-40]。本文通过对猪成熟和闭锁卵泡液中的EXs miRNA进行测序,发现许多miRNA呈现显著差异表达,且参与介导关键基因和信号转导调控,显示了在卵泡发育过程中的重要调控作用。两类差异表达的EXs miRNA功能富集分析发现这些miRNA对颗粒细胞凋亡、细胞周期运转和类固醇代谢等方面具有潜在的调控作用。表明了EXs中的miRNA可能直接参与调控卵泡的成熟发育和闭锁[38,41]。

卵泡闭锁是从颗粒细胞凋亡开始的[42],P53属于一种促细胞凋亡基因,其功能主要涉及调控G1和G2/M期的运转,促进细胞凋亡[40,43]。研究通过对猪[44]和牛[45,46]进行miRNA表达谱分析,发现miR-21-5p在卵泡发育过程中呈现不同表达[45];在培养小鼠颗粒细胞时发现miR-21-5p属于一种抗凋亡因子,可以直接靶向P53同源基因,促进细胞增殖[47],证明了在颗粒细胞凋亡发生过程中起到重要调控作用[48];本研究显示affEXs中下调表达的ssc-miR-21-5p,潜在靶向P53,促使P53表达增加,引起颗粒细胞凋亡;在体外培养颗粒细胞时添加affEXs可增加P53的表达,引起卵巢颗粒细胞的凋亡,这说明了affEXs中ssc-miR-21-5p下调可以引起卵母细胞凋亡;也有研究显示miR-21高表达受到抑制时,体外胚胎发育停滞[49,50]。

let-7家族（let-7a、let-7b和let-7c等）在猪早期卵泡闭锁和逐步闭锁过程中特异性表达,且卵巢卵泡的数量也会随卵泡闭锁的发生逐渐减少[51]。随着研究的深入,let-7a在动物生长发育、细胞增殖、分化和凋亡等方面发挥着重要调控作用[52]。相关报道显示let-7a通过下调STAT3抑制哮喘气道平滑肌细胞的增殖,促进其凋亡[53];还可以在细胞周期蛋白-D1的调控作用下,抑制肺腺癌细胞增殖[54];在骨肉瘤细胞中let-7a潜在靶向Aurora-B,促使细胞生长和肺转移受到抑制[55,56];let-7a还可以通过抑制Bcl-xl和YAP1表达诱导滋养层细胞凋亡[57];还有研究显示let-7a靶向凋亡基因Fas、FasL和caspase-3发挥调控作用[58,59]。miR-133a-3p在多种细胞中都起到了抑制增殖、侵袭,阻滞细胞周期等作用,促进细胞凋亡[60,61],如在口腔鳞状细胞癌研究中发现miR-133a-3p下调COL1A1表达,抑制癌细胞的增殖、侵袭和转移[62,63];它还可以通过阻断自噬介导的谷氨酰胺分解,抑制胃癌细胞的生长和转移等[64];在视网膜母细胞瘤中通过靶向CREB1,促进细胞凋亡,阻滞细胞周期[65];还有研究显示胰岛素生长因子（IGF）被认为是卵泡发育所必须的生长因子[66],显然IGF缺失会对卵泡闭锁的发生产生一定的影响。我们发现与mffEXs相比,affEXs中上调表达的ssc-let-7a和ssc-miR-133a-3p潜在靶向CDK1和IGF1,催化底物磷酸化,抑制了G2/M期的运转,降低了细胞的增殖能力,并使卵泡细胞与LH作用,诱导雄激素释放,促使卵母细胞降解和颗粒细胞凋亡或其受IGFBP-1调控,增强卵巢颗粒细胞芳香化酶的活性,抑制类固醇激素代谢,解释了EXs携带的miRNA上调表达可以促使颗粒细胞凋亡,进而诱导卵泡闭锁的发生,并且有望成为解析卵泡发育规律的分子机制[67]。

4 结论

近年来EXs已成为动物生殖发育领域的研究热点,许多潜在的功能机制逐渐被人们揭晓,然而卵泡液EXs携带的miRNA对猪卵泡发育调控作用仍然不清楚。本文研究显示卵泡液EXs携带的miRNA（ssc-miR-133-3p、ssc-miR-21-5p和ssc-let-7a）在颗粒细胞凋亡、细胞周期调控和类固醇激素代谢等过程中具有重要的调控作用。这将为猪卵泡发育和闭锁相关的主要调控机制提供重要数据。

（责任编辑 林鉴非）

参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

JONG E D. Weaning practices and culling policy: Critical steps for optimal reproductive performance of female breeding pigs.
2014.
[本文引用: 1]

FORTUNE J E. Ovarian follicular growth and development in mammals
Biology of Reproduction, 1994, 50(2):225-232.
DOI:10.1095/biolreprod50.2.225URL [本文引用: 1]

DISKIN M G, MACKEY D R, ROCHE J F, SREENAN J M. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle
Animal Reproduction Science, 2003, 78(3/4):345-370. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/s0378-4320(03)00099-x.
URL [本文引用: 1]

HSUEH A J, BILLIG H, TSAFRIRI A. Ovarian follicle atresia: a hormonally controlled apoptotic process
Cancer Management and Research, 1994, 15(6):707-724. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1210/edrv-15-6-707.
[本文引用: 1]

TILLY J L, TILLY K I. Inhibitors of oxidative stress mimic the ability of follicle-stimulating hormone to suppress apoptosis in cultured rat ovarian follicles
Microorganisms, 1995, 136(1):242-252. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1210/endo.136.1.7828537.
[本文引用: 1]

PERSANI L, ROSSETTI R, CACCIATORE C, FABRE S. Genetic defects of ovarian TGF-β-like factors and premature ovarian failure
Journal of Endocrinological Investigation, 2011, 34(3):244-251. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/BF03347073.
URL [本文引用: 1]

SOHEL M M, HOELKER M, NOFERESTI S S, SALILEW- WONDIM D, THOLEN E, LOOFT C, RINGS F, UDDIN M J, SPENCER T E, SCHELLANDER K, TESFAYE D. Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: Implications for bovine oocyte developmental competence
PLoS ONE, 2013, 8(11):e78505. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1371/journal.pone.0078505.
URL [本文引用: 1]

LUO F, JIA R, YING S, WANG Z, WANG F. Analysis of genes that influence sheep follicular development by different nutrition levels during the luteal phase using expression profiling
Animal Genetics, 2016, 47(3):354-364. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1111/age.12427.
URL [本文引用: 1]

胡军和, 唐涛, 谭显胜, 曾智, 吴娟. 外泌体调控卵泡中母细胞发育的研究进展
中国农学通报, 2019(27):153-157.
[本文引用: 1]

HU J H, TANG T, TAN X S, ZENG Z, WU J. The advance of exosomes regulating oocyte development in ovarian follicle
Chinese Agricultural Science Bulletin, 2019(27):153-157. (in Chinese)
[本文引用: 1]

THÉRY C, OSTROWSKI M, SEGURA E. Membrane vesicles as conveyors of immune responses
Nature Reviews Immunology, 2009, 9(8):581-593. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1038/nri2567.
URL [本文引用: 1]

VLASSOV A V, MAGDALENO S, SETTERQUIST R, CONRAD R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials
Biochimica et Biophysica Acta, 2012, 1820(7):940-948. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.bbagen.2012.%2003.017.
[本文引用: 1]

KELLER S, RUPP C, STOECK A, RUNZ S, FOGEL M, LUGERT S, HAGER H D, ABDEL B MS, GUTWEIN P, ALTEVOGT P. CD24 is a marker of exosomes secreted into urine and amniotic fluid
Kidney International, 2007, 72(9):1095-1102.
DOI:10.1038/sj.ki.5002486URL [本文引用: 1]

DE LA TORRE GOMEZ C, GOREHAM R V, BECH SERRA J J, NANN T, KUSSMANN M. “exosomics”-A review of biophysics, biology and biochemistry of exosomes with a focus on human breast milk
Frontiers in Genetics, 2018, 9:92. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3389/fgene.2018.00092.
URL [本文引用: 1]

BORIACHEK K, UMER M, ISLAM M N, GOPALAN V, LAM A K, NGUYEN N T, SHIDDIKY M J A. An amplification-free electrochemical detection of exosomal miRNA-21 in serum samples
The Analyst, 2018, 143(7):1662-1669. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1039/c7an01843f.
URL [本文引用: 1]

TAO W, SUN L, SHI H, CHENG Y, JIANG D, FU B, CONTE M A, GAMMERDINGER W J, KOCHER T D, WANG D. Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation
BMC Genomics, 2016, 17(1):328.
DOI:10.1186/s12864-016-2636-zURL [本文引用: 1]

SANG Q, YAO Z, WANG H, FENG R, WANG H, ZHAO X, XING Q, JIN L, HE L, WU L, WANG L. Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo
The Journal of Clinical Endocrinology and Metabolism, 2013, 98(7):3068-3079. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1210/jc.2013-1715.
URL [本文引用: 1]

WILLIS G R, CONNOLLY K, LADELL K, DAVIES T S, GUSCHINA I A, RAMJI D, MINERS K, PRICE D A, CLAYTON A, JAMES P E, REES D A. Young women with polycystic ovary syndrome have raised levels of circulating annexin v-positive platelet microparticles
Human Reproduction, 2014, 29(12):2756-2763.
DOI:10.1093/humrep/deu281URL [本文引用: 1]

SØRENSEN A E, WISSING M L, ENGLUND A L M, DALGAARD L T. MicroRNA species in follicular fluid associating with polycystic ovary syndrome and related intermediary phenotypes
The Journal of Clinical Endocrinology & Metabolism, 2016, 101(4):1579-1589.
DOI:10.1210/jc.2015-3588URL [本文引用: 1]

杨倩, 刘兰心, 黄荷凤. 多囊卵巢综合征患者卵泡液外泌体的提取鉴定及其miRNAs的提取和检测
上海交通大学学报(医学版), 2017, 37(8):1085-1089. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3969/j.issn.1674-8115.2017.08.007.
[本文引用: 1]

YANG Q, LIU L X, HUANG H F. Extraction and identification of exosomes in follicular fluid from patients with polycystic ovary syndrome and isolation and detection of miRNAs in exosomes
Journal of Shanghai Jiao Tong University (Medical Science), 2017, 37(8):1085-1089. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3969/j.issn.1674-8115.2017.08.007. (in Chinese)
[本文引用: 1]

COTICCHIO G, DAL CANTO M, MIGNINI RENZINI M, GUGLIELMO M C, BRAMBILLASCA F, TURCHI D, NOVARA P V, FADINI R. Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization
Human Reproduction Update, 2015, 21(4):427-454. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1093/humupd/dmv011.
URL [本文引用: 2]

刘凯鲁, 胡梦婷, 蔡令波, 李涵, 杨玮杰, 刘嘉茵, 崔毓桂, 千日成. 多囊卵巢综合征患者卵泡液中6种miRNAs表达的检测
国际生殖健康/计划生育杂志, 2018, 37(1):5-10. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3969/j.issn.1674-1889.2018.01.001.
[本文引用: 1]

LIU K L, HU M T, CAI L B, LI H, YANG W J, LIU J Y, CUI Y G, QIAN R C. Expressions of six MiRNAs in follicular fluid of patients with polycystic ovary syndrome. International Journal of Reproductive Health/
Family Planning, 2018, 37(1):5-10. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3969/j.issn.1674-1889.2018.01.001. (in Chinese)
[本文引用: 1]

詹小舒, 罗惠娜, 罗冬章, 陈胜锋, 王丙云, 白银山, 陈志胜, 刘璨颖, 计慧琴. 犬脐带间充质干细胞来源外泌体对血管内皮细胞增殖、迁移和凋亡的调控作用
中国组织工程研究, 2019, 23(29):4637-4643. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3969/j.issn.2095-4344.1808.
[本文引用: 1]

ZHAN X S, LUO H N, LUO D Z, CHEN S F, WANG B Y, BAI Y S, CHEN Z S, LIU C Y, JI H Q. Effects of exosomes derived from canine umbilical cord mesenchymal stem cells on proliferation, migration and apoptosis of vascular endothelial cells
Journal of Clinical Rehabilitative Tissue Engineering Research, 2019, 23(29):4637-4643. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3969/j.issn.2095-4344.1808. (in Chinese)
[本文引用: 1]

ZHANG H, XU S, LIU X. microRNA profiling of plasma exosomes from patients with ovarian cancer using high-throughput sequencing
Oncology Letters, 2019, 17(6):5601-5607. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3892/ol.2019.10220.
[本文引用: 1]

WAGNER G P, KIN K, LYNCH V J. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples
Theory in Biosciences, 2012, 131(4):281-285. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/s12064-012-0162-3.
URL [本文引用: 1]

YOUNG M D, WAKEFIELD M J, SMYTH G K, OSHLACK A. Gene ontology analysis for RNA-seq: Accounting for selection bias
Proceedings Biological Sciences, 2010, 11(2):R14. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1186/gb-%202010-11-2-r14.
[本文引用: 1]

WANG C L, FAN Y C, CHUN-HSIEN TSENG, CHIU C H, TSAI H J, CHOU C H. Salmonella Enteritidis infection slows steroidogenesis and impedes cell growth in hen granulosa cells
Avian Diseases, 2014, 58(4):511-517. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1637/10846-041414-reg.1.
URL [本文引用: 1]

DA SILVEIRA J, ANDRADE G M, PERECIN F, MEIRELES F V, WINGER Q A, BOUMA G J. Isolation and analysis of exosomal microRNAs from ovarian follicular fluid
Methods in Molecular Biology (Clifton, N J), 2018, 1733:53-63. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/978-1-4939-7601-0_4.
[本文引用: 1]

HU J, TANG T, ZENG Z, WU J, TAN X S, YAN J. The expression of small RNAs in exosomes of follicular fluid altered in human polycystic ovarian syndrome
PeerJ, 2020, 8:e8640.
DOI:10.7717/peerj.8640URL [本文引用: 1]

VICTOR N T, YONATHAN G, GIOVANNI C, MARIA F B. Extracellular vesicles: new players in lymphomas
International Journal of Molecular Sciences, 2018, 20(1). doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3390/%20ijms20010041.
[本文引用: 1]

LÖTVALL J, HILL A F, HOCHBERG F, BUZÁS E I, VIZIO D D, GARDINER C, GHO Y S, KUROCHKIN I V, MATHIVANAN S, QUESENBERRY P. Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles
Journal of Extracellular Vesicles, 2014, 3(1):26913.
DOI:10.3402/jev.v3.26913URL [本文引用: 1]

HUNG W T, NAVAKANITWORAKUL R, KHAN T, ZHANG P, DAVIS J S, MCGINNIS L K, CHRISTENSON L K. Stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation
Biology of Reproduction, 2017, 97(4):644-655. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1093/biolre/iox106.
URL [本文引用: 1]

HUNG W T, HONG X, CHRISTENSON L K, MCGINNIS L K. Extracellular vesicles from bovine follicular fluid support cumulus expansion
Biology of Reproduction, 2015, 93(5):117. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1095/%20biolreprod.115.132977.
[本文引用: 1]

AL-DOSSARY A A, STREHLER E E, MARTIN-DELEON P A. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine Estrus: Association with oviductal exosomes and uptake in sperm
PLoS ONE, 2013, 8(11):e80181.
DOI:10.1371/journal.pone.0080181URL [本文引用: 1]

FIELD S L, DASGUPTA T, CUMMINGS M, ORSI N M. Cytokines in ovarian folliculogenesis, oocyte maturation and luteinisation
Molecular Reproduction and Development, 2014, 81(4):284-314. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1002/mrd.22285.
URL [本文引用: 1]

BENTWICH I, AVNIEL A, KAROV Y, AHARONOV R, GILAD S, BARAD O, BARZILAI A, EINAT P, EINAV U, MEIRI E, SHARON E, SPECTOR Y, BENTWICH Z. Identification of hundreds of conserved and nonconserved human microRNAs
Nature Genetics, 2005, 37(7):766-770. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1038/ng1590.
URL [本文引用: 1]

KROL J, LOEDIGE I, FILIPOWICZ W. The widespread regulation of microRNA biogenesis, function and decay
Nature Reviews Genetics, 2010, 11(9):597-610. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1038/nrg2843.
URL [本文引用: 1]

MASOUMI-DEHGHI S, BABASHAH S, SADEGHIZADEH M. microRNA-141-3p-containing small extracellular vesicles derived from epithelial ovarian cancer cells promote endothelial cell angiogenesis through activating the JAK/STAT3 and NF-κB signaling pathways
Journal of Cell Communication and Signaling, 2020, 14(2):233-244. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/s12079-020-00548-5.
URL [本文引用: 1]

MACHTINGER R, RODOSTHENOUS R S, ADIR M, MANSOUR A, RACOWSKY C, BACCARELLI A A, HAUSER R. Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: An exploratory study
Journal of Assisted Reproduction and Genetics, 2017, 34(4):525-533. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/s10815-017-0876-8.
URL [本文引用: 2]

LIANG M, YAO G, YIN M, LÜ M, TIAN H, LIU L, LIAN J, HUANG X, SUN F. Transcriptional cooperation between p53 and NF-κB p65 regulates microRNA-224 transcription in mouse ovarian granulosa cells
Molecular and Cellular Endocrinology, 2013, 370(1/2):119-129. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.mce.2013.02.014.
URL

DA SILVEIRA J C, VEERAMACHANENI D N, WINGER Q A, CARNEVALE E M, BOUMA G J. Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: A possible new form of cell communication within the ovarian follicle
Biology of Reproduction, 2012, 86(3):71. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1095/biolreprod.111.093252.
[本文引用: 2]

PIETRO C D. Exosome-mediated communication in the ovarian follicle
Journal of Assisted Reproduction and Genetics, 2016, 33(3):303-311.
DOI:10.1007/s10815-016-0657-9URL [本文引用: 1]

KRYSKO D V, DIEZ-FRAILE A, CRIEL G, SVISTUNOV A A, VANDENABEELE P, D’HERDE K. Life and death of female gametes during oogenesis and folliculogenesis
Apoptosis, 2008, 13(9):1065-1087. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/s10495-008-0238-1.
URL [本文引用: 1]

TILLY J L, KOWALSKI K I, JOHNSON A L, HSUEH A J. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression
Thrombosis and Haemostasis, 1991, 129(5):2799-2801. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1210/endo-129-5-2799.
[本文引用: 1]

LIN F, LI R, PAN Z X, ZHOU B, YU D B, WANG X G, MA X S, HAN J, SHEN M, LIU H L. miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary
PLoS ONE, 2012, 7(6):e38640. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1371/journal.pone.0038640.
URL [本文引用: 1]

SALILEW-WONDIM D, AHMAD I, GEBREMEDHN S, SAHADEVAN S, HOSSAIN M D, RINGS F, HOELKER M, THOLEN E, NEUHOFF C, LOOFT C, SCHELLANDER K, TESFAYE D. The expression pattern of microRNAs in granulosa cells of subordinate and dominant follicles during the early luteal phase of the bovine estrous cycle
PLoS ONE, 2014, 9(9):e106795. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1371/journal.%20pone.0106795.
URL [本文引用: 2]

SONTAKKE S D, MOHAMMED B T, MCNEILLY A S, DONADEU F X. Characterization of microRNAs differentially expressed during bovine follicle development
Reproduction (Cambridge, England), 2014, 148(3):271-283. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1530/rep-14-0140.
URL [本文引用: 1]

CARLETTI M Z, FIEDLER S D, CHRISTENSON L K. microRNA 21 blocks apoptosis in mouse periovulatory granulosa cells
Molecules and Cells, 2010, 83(2):286-295. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1095/biolreprod.109.081448.
[本文引用: 1]

周桔, 罗荣保, 汤长发, 瞿树林. Bcl-2蛋白家族和p53基因在细胞凋亡中的调控效应
中国组织工程研究与临床康复, 2007, 11(10):1950-1952. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3321/j.issn:1673-8225.2007.10.048.
[本文引用: 1]

ZHOU J, LUO R B, TANG C F, QU S L. Effect of Bcl-2 protein family and p53 gene on regulating and controlling cell apoptosis
Journal of Clinical Rehabilitative Tissue Engineering Research, 2007, 11(10):1950-1952. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3321/j.issn:1673-8225.2007.10.048. (in Chinese)
[本文引用: 1]

周桔, 罗荣保, 汤长发, 瞿树林. Bcl-2蛋白家族和p53基因在细胞凋亡中的调控效应
中国组织工程研究与临床康复, 2007(10):1950-1952.
[本文引用: 1]

ZHOU J, LUO R B, TANG C F, QU S L. Effect of Bcl-2 protein family and p53 gene on regulating and controlling cell apoptosis
Journal of Clinical Rehabilitative Tissue Engineering Research, 2007(10):1950-1952.(in Chinese)
[本文引用: 1]

LV C, YU W X, WANG Y, YI D J, ZENG M, XIAO H. MiR-21 in extracellular vesicles contributes to the growth of fertilized eggs and embryo development in mice
Bioscience Reports, 2018, 38(4): BSR20180036.
[本文引用: 1]

KUSAMA K, NAKAMURA K, BAI R, NAGAOKA K, SAKURAI T, IMAKAWA K. Intrauterine exosomes are required for bovine conceptus implantation
Biochemical and Biophysical Research Communications, 2018, 495(1):1370-1375. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.bbrc.2017.11.176.
URL [本文引用: 1]

CAO R, WU W J, ZHOU X L, XIAO P, WANG Y, LIU H L. Expression and preliminary functional profiling of the let-7 family during porcine ovary follicle atresia
Molecules and Cells, 2015, 38(4):304-311. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.14348/molcells.2015.2122.
URL [本文引用: 1]

WANG S, TANG Y, CUI H, ZHAO X, LUO X, PAN W, HUANG X, SHEN N. Let-7/miR-98 regulate Fas and Fas-mediated apoptosis
Genes and Immunity, 2011, 12(2):149-154. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1038/gene.2010.53.
URL [本文引用: 1]

CHEN Y, QIAO L, ZHANG Z, HU G, ZHANG J, LI H. Let-7a inhibits proliferation and promotes apoptosis of human asthmatic airway smooth muscle cells
Experimental and Therapeutic Medicine, 2019, 17(5):3327-3334. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3892/etm.2019.7363.
[本文引用: 1]

ZHAO W, HU J X, HAO R M, ZHANG Q, GUO J Q, LI Y J, XIE N, LIU L Y, WANG P Y, ZHANG C, XIE S Y. Induction of microRNA-let-7a inhibits lung adenocarcinoma cell growth by regulating cyclin D1
Oncology Reports, 2018, 40(4):1843-1854. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.3892/or.2018.6593.
[本文引用: 1]

YU J J, PI W S, CAO Y, PENG A F, CAO Z Y, LIU J M, HUANG S H, LIU Z L, ZHANG W. Let-7a inhibits osteosarcoma cell growth and lung metastasis by targeting Aurora-B
Cancer Management and Research, 2018, 10:6305-6315. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.2147/cmar.s185090.
URL [本文引用: 1]

WANG G, WANG J, KHAN M F. Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen
Toxicology Mechanisms and Methods, 2017, 27(7):511-517. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1080/15376516.2017.1324932.
URL [本文引用: 1]

ZHA W, GUAN S, LIU N, LI Y, TIAN Y, CHEN Y, WANG Y, WU F. Let-7a inhibits Bcl-xl and YAP1 expression to induce apoptosis of trophoblast cells in early-onset severe preeclampsia
The Science of the Total Environment, 2020, 745:139919. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.scitotenv.2020.139919.
URL [本文引用: 1]

YU Y, LIAO L, SHAO B, SU X, SHUAI Y, WANG H, SHANG F, ZHOU Z, YANG D, JIN Y. Knockdown of microRNA let-7a improves the functionality of bone marrow-derived mesenchymal stem cells in immunotherapy
Molecular Therapy, 2017, 25(2):480-493. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.ymthe.2016.11.015.
URL [本文引用: 1]

TSANG W P, KWOK T T. Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3
Apoptosis, 2008, 13(10):1215-1222. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/s10495-008-0256-z.
URL [本文引用: 1]

CHANG L L, WANG H C, TSENG K Y, SU M P, WANG J Y, CHUANG Y T, WANG Y H, CHENG K I. Upregulation of miR-133a-3p in the sciatic nerve contributes to neuropathic pain development
Molecular Neurobiology, 2020, 57(9):3931-3942. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1007/s12035-020-01999-y.
URL [本文引用: 1]

HUANG Y, WANG Y, LIN L, WANG P, JIANG L, LIU J, WANG X. Overexpression of miR-133a-3p inhibits fibrosis and proliferation of keloid fibroblasts by regulating IRF5 to inhibit the TGF-β/Smad2 pathway
Molecular and Cellular Probes, 2020, 52:101563. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.mcp.2020.101563.
URL [本文引用: 1]

HE B, LIN X, TIAN F, YU W, QIAO B. miR-133a-3p inhibits oral squamous cell carcinoma (OSCC) proliferation and invasion by suppressing COL1A1
Journal of Cellular Biochemistry, 2018, 119(1):338-346. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1002/jcb.26182.
URL [本文引用: 1]

YIN Y, DU L, LI X, ZHANG X, GAO Y. miR-133a-3p suppresses cell proliferation, migration, and invasion and promotes apoptosis in esophageal squamous cell carcinoma
Journal of Cellular Physiology, 2019, 234(8):12757-12770. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1002/jcp.27896.
URL [本文引用: 1]

ZHANG X, LI Z, XUAN Z, XU P H, WANG W Z, CHEN Z, WANG S, SUN G L, XU J H, XU Z K. Novel role of miR-133a-3p in repressing gastric cancer growth and metastasis via blocking autophagy-mediated glutaminolysis
Journal of Experimental & Clinical Cancer Research, 2018, 37(1):320.
[本文引用: 1]

LI J, LIU X, WANG W, LI C. miR-133a-3p promotes apoptosis and induces cell cycle arrest by targeting CREB1 in retinoblastoma
Archives of Medical Science, 2020, 16(4):941-956. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.5114/aoms.2019.86901.
URL [本文引用: 1]

HAN Y, WANG S M, WANG Y Z, ZENG S M. IGF-1 inhibits apoptosis of porcine primary granulosa cell by targeting degradation of BimEL
International Journal of Molecular Sciences, 2019, 20(21):5356.
DOI:10.3390/ijms20215356URL [本文引用: 1]

SANTONOCITO M, VENTO M, GUGLIELMINO M R, BATTAGLIA R, WAHLGREN J, RAGUSA M, BARBAGALLO D, BORZÌ P, RIZZARI S, MAUGERI M, SCOLLO P, TATONE C, VALADI H, PURRELLO M, DI PIETRO C. Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: BIoinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation
Fertility and Sterility, 2014, 102(6): 1751-61.e1. doi: https://www.chinaagrisci.com/article/2021/0578-1752/10.1016/j.fertnstert.2014.08.005.
[本文引用: 1]

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