miR-26a-5p通过靶向PTEN基因调控山羊卵巢颗粒细胞的增殖

doi:10.16431/j.cnki.1671-7236.2025.08.020

Abstract

Abstract: 【Objective】 Granulosa cells (GCs) proliferation is regulated by many factors,such as miRNAs,miR-26a-5p was significantly enriched in the growing follicles of goats,which might have a regulated role during the folliclar development.This study was aimed to reveal the functions of miR-26a-5p in goat GCs,in order to enrich the molecular regulatory network in follicular development. 【Method】 Real-time quantitative PCR was used to analyze the expression of miR-26a-5p in goat follicles with different sizes.The ovarian GCs were cultured and identified in vitro.The proliferation of GCs was detected using EdU and MTT assays after with overexpressed and inhibited miR-26a-5p expression.The interaction between miR-26a-5p and the target gene PTEN was verified using a dual luciferase reporter system and Western blotting,respectively.Western blotting was used to analyze the expression of proteins related to PI3K/Akt signaling pathway in GCs after overexpression or interference with miR-26a-5p expression. 【Result】 The expression of miR-26a-5p was detected in goat follicles of varying sizes,including in oocytes.miR-26a-5p was significantly enriched in GCs of small-to-medium follicles (2-3 mm in diameter) compared with GCs from follicles of other diameters (＜2,4-5,and ＞5 mm) (P＜0.05).In in vitro cultured GCs,the proportion of proliferating cells was significantly higher in miR-26a-5p overexpression group than that in control group (transfected with NC mimics) (P＜0.05).Conversely,transfection with miR-26a-5p inhibitor significantly reduced the proliferation rate compared with NC inhibitor group (P＜0.05).Dual-luciferase reporter assays revealed that compared with mutant group,miR-26a-5p overexpression significantly reduced luciferase activity by targeting the seed sequence in the 3'-UTR of PTEN gene (P＜0.05).Western blotting results showed that compared with NC mimics group, miR-26a-5p overexpression significantly downregulated the expression of PTEN protein in GCs (P＜0.05).Furthermore,elevated phosphorylated the expression of p-AKT protein and an increased p-AKT/AKT ratio were still detectable 96 h after miR-26a-5p overexpression (P＜0.05). 【Conclusion】 miR-26a-5p directly targeted and suppressed the expression of PTEN gene in ovarian GCs,thereby activated the AKT protein and PI3K/Akt signaling pathway,which promoted GCs proliferation.

Key words: miR-26a-5p; PTEN gene; granulosa cell proliferation; PI3K/Akt signaling pathway

CLC Number:

S814.8

WANG Yaoyue, SHI Qianqian, LUO Qi, GAO Linna, WU Hao, ZHANG Jiangli, DING Qiang. miR-26a-5p Targets PTEN Gene to Regulate the Proliferation of Goat Ovarian Granulosa Cells[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3715-3725.

References

[1] XIAO S,LIU N,YANG X,et al.Polygalacin D suppresses esophageal squamous cell carcinoma growth and metastasis through regulating miR-142-5p/Nrf2 axis[J].Free Radical Biology and Medicine,2021,164:58-75.
[2] BANTOUNAS I,LOPES F M,ROONEY K M,et al.The miR-199a/214 cluster controls nephrogenesis and vascularization in a human embryonic stem cell model[J].Stem Cell Reports,2021,16(1):134-148.
[3] WANG C,DING C,HUA Z,et al.Cangfudaotan decoction alleviates insulin resistance and improves follicular development in rats with polycystic ovary syndrome via IGF-1-PI3K/Akt-Bax/Bcl-2 pathway[J].Mediators of Inflammation,2020,2020:8865647.
[4] CALIN G A,CROCE C M.microRNA signatures in human cancers[J].Nature Reviews Cancer,2006,6(11):857-866.
[5] CHEN Y,YANG W,SHI X,et al.The factors and pathways regulating the activation of mammalian primordial follicles in vivo[J].Frontiers in Cell and Developmental Biology,2020,8:575706.
[6] LV W,AN R,LI X,et al.Multi-omics approaches uncovered critical mRNA-miRNA-lncRNA networks regulating multiple birth traits in goat ovaries[J].International Journal of Molecular Sciences,2024,25(22)：12466.
[7] WANG Y,WANG J,LI Q,et al.Transcriptomic and metabolomic data of goat ovarian and uterine tissues during sexual maturation[J].Scientific Data,2024,11(1):777.
[8] SUN X,KLINGER F G,LIU J,et al.miR-378-3p maintains the size of mouse primordial follicle pool by regulating cell autophagy and apoptosis[J].Cell Death & Disease,2020,11(9):737.
[9] LEI L,JIN S,GONZALEZ G,et al.The regulatory role of Dicer in folliculogenesis in mice[J].Molecular and Cellular Endocrinology,2010,315(1-2):63-73.
[10] DU X,ZHANG L,LI X,et al.TGF-beta signaling controls FSHR signaling-reduced ovarian granulosa cell apoptosis through the SMAD4/miR-143 axis[J].Cell Death & Disease,2016,7(11):e2476.
[11] FENG G,LIU J,LU Z,et al.miR-450-5p and miR-202-5p synergistically regulate follicle development in Black goat[J].International Journal of Molecular Sciences,2022,24(1)：401.
[12] DING Q,JIN M,KALDS P,et al.Comparison of microRNA profiles in extracellular vesicles from small and large goat follicular fluid[J].Animals,2021,11(11)：3190.
[13] DING Q,JIN M,WANG Y,et al.Transactivation of miR-202-5p by steroidogenic factor 1 (SF1) induces apoptosis in goat granulosa cells by targeting TGFbetaR2[J].Cells,2020,9(2)：445.
[14] EMORI C,SUGIURA K.Role of oocyte-derived paracrine factors in follicular development[J].Animal Science Journal,2014,85(6):627-633.
[15] 郭妍岩,张羽欣,陆瑞,等.哺乳动物卵泡发育阶段颗粒细胞增殖与分化的研究进展[J].畜牧兽医学报,2025,56(4):1484-1493. GUO Y Y,ZHANG Y X,LU R,et al.Research progress on the proliferation and differentiation of granulosa cells at various follicular development stages in mammal[J].Acta Veterinaria et Zootechnica Sinica,2025,56(4):1484-1493.(in Chinese)
[16] KONG Y,ZHANG Y,MAO F,et al.Inhibition of EZH2 enhances the antitumor efficacy of metformin in prostate cancer[J].Molecular Cancer Therapeutics,2020,19(12):2490-2501.
[17] WANG Z,LIU T,XUE W,et al.ARNTL2 promotes pancreatic ductal adenocarcinoma progression through TGF/BETA pathway and is regulated by miR-26a-5p[J].Cell Death & Disease,2020,11(8):692.
[18] LI Y,GUO D,ZHAO Y,et al.Long non-coding RNA SNHG5 promotes human hepatocellular carcinoma progression by regulating miR-26a-5p/GSK3beta signal pathway[J].Cell Death & Disease,2018,9(9):888.
[19] KANG L,YANG C,WU H,et al.miR-26a-5p regulates TNRC6A expression and facilitates theca cell proliferation in chicken ovarian follicles[J].DNA and Cell Biology,2017,36(11):922-929.
[20] YU Q,PENG C,YE Z,et al.An estradiol-17beta/miRNA-26a/cyp19a1a regulatory feedback loop in the protogynous hermaphroditic fish,Epinephelus coioides[J].Molecular and Cellular Endocrinology,2020,504:110689.
[21] LI C,LI Y,LU Y,et al.miR-26 family and its target genes in tumorigenesis and development[J].Critical Reviews in Oncology Hematology,2021,157:103124.
[22] MA J,FAN Y,ZHANG J,et al.Testosterone-dependent miR-26a-5p and let-7 g-5p act as signaling mediators to regulate sperm apoptosis via targeting PTEN and PMAIP1[J].International Journal of Molecular Sciences,2018,19(4)：1233.
[23] ZHU Q,ZHANG X,ZAI H,et al.circSLC8A1 sponges miR-671 to regulate breast cancer tumorigenesis via PTEN/PI3k/Akt pathway[J].Genomics,2021,113(1 Pt1):398-410.
[24] WENG L,BROWN J,ENG C.PTEN induces apoptosis and cell cycle arrest through phosphoinositol-3-kinase/Akt-dependent and-independent pathways[J].Human Molecular Genetics,2001,10(3):237-242.
[25] BADDELA V S,MICHAELIS M,TAO X,et al.Comparative analysis of PI3K-Akt and MEK-ERK1/2 signaling-driven molecular changes in granulosa cells[J].Reproduction,2025,169(2):e240317.
[26] 李娇娇,焦亚飞,石德顺.PI3K/Akt信号通路对卵泡发育和卵母细胞成熟作用的研究进展[J].中国畜牧杂志,2021,57(8):33-37. LI J J,QIAO Y F,SHI D S.Research progress on the role of PI3K/Akt signaling pathway in follicular development and oocyte maturation[J].Chinese Journal of Animal Science,2021,57(8):33-37.(in Chinese)
[27] LI L,SHI X,SHI Y,et al.The signaling pathways involved in ovarian follicle development[J].Frontiers in Physiology,2021,12:730196.
[28] ZHU Y,HOELL P,AHLEMEYER B,et al.PTEN:A crucial mediator of mitochondria-dependent apoptosis[J].Apoptosis,2006,11(2):197-207.
[29] MISSIROLI S,MORGANTI C,GIORGI C,et al.Methods to study PTEN in mitochondria and endoplasmic reticulum[J].Methods in Molecular Biology,2016,1388:187-212.
[30] BONONI A,BONORA M,MARCHI S,et al.Identification of PTEN at the ER and MAMs and its regulation of Ca²⁺ signaling and apoptosis in a protein phosphatase-dependent manner[J].Cell Death and Differentiation,2013,20(12):1631-1643.
[31] AZIMI Y,HAJIBABAEI S,AZIMI G,et al.Inhibitory effect of miR-377 on the proliferative and invasive behaviors of prostate cancer cells through the modulation of MYC mRNA via its interaction with BCL-2/Bax,PTEN,and CDK4[J].Genes Cancer,2024,15:28-40.

miR-26a-5p Targets PTEN Gene to Regulate the Proliferation of Goat Ovarian Granulosa Cells

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