Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (21): 4664-4676.doi: 10.3864/j.issn.0578-1752.2021.21.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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()   

  1. School of Life Science and Engineering, Foshan University, Foshan 528231, Guangdong
  • Received:2020-09-27 Accepted:2021-04-25 Online:2021-11-01 Published:2021-11-09
  • Contact: YinShan BAI E-mail:chenhuifang07@163.com;xuefei200403@163.com

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

Fig. 1

Isolation 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"

Table 1

The main differential expression miRNA in follicular fluid EXs"

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

Fig. 2

Go enrichment analysis of differentially expressed genes in follicular fluid EXs miRNA"

Fig. 3

Enrichment analysis of KEGG function"

Table 2

Information 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)

Fig. 4

Target gene interaction network of differentially expressed miRNA"

Fig. 5

Regulatory 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"

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