外泌体microRNA在猪成熟和闭锁卵泡中的表达差异及功能分析

doi:10.3864/j.issn.0578-1752.2021.21.015

摘要/Abstract

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.

Key words: porcine, mature follicular fluid, atretic follicular fluid, EXs, miRNA

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[J]. 中国农业科学, 2021, 54(21): 4664-4676.

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[J]. Scientia Agricultura Sinica, 2021, 54(21): 4664-4676.

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图/表 7

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表2

主要信号通路中miRNA调控靶基因信息表"

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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miRNA名称 miRNA name	mffEXs-TPM mffEXs-Tags per million	affEXs-TPM affEXs-Tags per million	差异倍数 Fold change
ssc-miR-1	191.1357	3683.4041	4.268370509
ssc-miR-133a-3p	125.3327	1137.3101	3.181790898
ssc-miR-874	588.8902	4261.314	2.855227792
ssc-let-7a	359.4472	2278.7335	2.664380439
ssc-let-7f-5p	378.9345	1986.6937	2.39034906
ssc-miR-23b	121.4619	631.3819	2.378009061
ssc-miR-486	810.8585	4166.7095	2.361386438
ssc-miR-6529	548.7143	2743.5294	2.321905957
ssc-miR-26a	2160.5543	9279.4636	2.102639923
ssc-miR-99b	13585.3175	38933.8489	1.51897669
ssc-miR-202-3p	2947.6543	7512.8282	1.349788776
ssc-miR-1271-5p	22519.3101	52612.4199	1.224240778
ssc-miR-21-5p	555532.4026	444467.5	- 0.321793131
ssc-miR-143	1443.9288	621.0989	-1.2171
ssc-miR-10b	163814.113	68106.9853	-1.266184975
ssc-miR-140-3p	5264.5074	2046.3356	-1.363255777
ssc-miR-10a-5p	7077.7606	2453.5461	-1.528424586
ssc-miR-2320-5p	146.8221	49.3588	-1.572689917
ssc-miR-125b	13217.7283	3973.3874	-1.734032879
ssc-miR-16	371.46	109.0008	-1.768868142
ssc-miR-146a-5p	6504.7539	1748.1259	-1.895685384
ssc-miR-145-3p	975.5663	257.0773	-1.924037697
ssc-miR-744	367.4557	94.6045	-1.957589617
ssc-miR-184	584.4855	121.3405	-2.268106073
ssc-miR-30d	3433.5021	707.4768	-2.278926102
ssc-miR-132	301.9197	47.3022	-2.674185705
ssc-miR-125a	18571.3967	1441.6897	-3.687249733