Please wait a minute...
Journal of Integrative Agriculture  2024, Vol. 23 Issue (04): 1315-1328    DOI: 10.1016/j.jia.2023.04.008
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |

miR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27

Shengjie Shi1, 2*, Lutong Zhang1, 2*, Liguang Wang1, 2, Huan Yuan1, 2, Haowei Sun1, 2, Mielie Madaniyati1, 2, Chuanjiang Cai2, Weijun Pang1, 2, Lei Gao1, 2#, Guiyan Chu1, 2#

1 College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China

2 Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, Yangling 712100, China

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

卵巢卵泡发育状态直接影响母猪的繁殖性能,颗粒细胞作为卵泡中最大的细胞群,其增殖直接促进卵泡的生长,其凋亡直接导致卵泡的闭锁,因此,探究关键因子对颗粒细胞增殖、凋亡的作用及机制是至关重要的。miR-24-3p在高产大白×长白二元母猪卵巢组织中的表达水平显著高于低产母猪,并且miR-24-3p在各个物种间是高度保守的,KEGGGO分析表明miR-24-3p可能与细胞增殖和凋亡有关,其靶基因也富集在WntmTOR等与颗粒细胞生理功能相关的信号通路上。然而,miR-24-3p颗粒细胞中的作用尚不清楚。在本研究中,我们通过流式细胞术、5-乙基-2′-脱氧尿苷(EdU)染色和细胞计数等试验,发现miR-24-3p促进了颗粒细胞增殖,增加S期细胞比例,上调细胞周期基因CyclinBCyclinDCyclinECDK4)的mRNA和蛋白水平表达。此外,通过流式细胞术、RT-qPCRWestern Blot等方法,表明miR-24-3p能够抑制颗粒细胞凋亡并影响凋亡相关基因(BCL-2BAX)的表达。在机制上,通过miRBase 、RNAhybrid等在线预测网站对miR-24-3p潜在的靶基因进行生物信息学分析,5个靶基因预测网站中取交集,筛选出17个潜在基因。通过RT-qPCRWestern Blot双荧光素酶报告试验并结合基因的生物学功能进行分析,结果显示,周期蛋白依赖性激酶抑制剂1B (P27/CDKN1B) miR-24-3p细胞增殖和凋亡方面的靶基因。此外,恢复试验证明,过表达P27减弱了miR-24-3p颗粒细胞增殖的促进作用,同时也减弱了miR-24-3p对颗粒细胞凋亡的抑制作用。这些结果表明miR-24-3p通过靶向P27抑制其转录水平进而促进颗粒细胞增殖并抑制颗粒细胞凋亡。本研究从产仔数极端表型差异个体中筛选并鉴定影响繁殖性状的关键miRNA,并结合猪原代细胞培养模型、生物信息学分析、分子生物学试验阐明miR-24-3p影响颗粒细胞增殖凋亡的具体作用及机制。



Abstract  

Ovarian follicle development is associated with the physiological functions of granulosa cells (GCs), including proliferation and apoptosis.  The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows.  However, the functions of miR-24-3p on GCs are unclear.  In this study, using flow cytometry, 5-ethynyl-2´-de-oxyuridine (EdU) staining, and cell count, we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes, moreover, miR-24-3p inhibited GC apoptosis.  Mechanistically, on-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B (P27/CDKN1B).  Furthermore, the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression.  These findings suggest that miR-24-3p regulates the physiological functions of GCs.

Keywords:  miR-24-3p        granulosa cells        proliferation       apoptosis   
Received: 12 December 2022   Accepted: 10 March 2023
Fund: This work was supported by the National Natural Science Foundation of China (32272849), the National Key R&D Program of China (2021YFF1000602), and the  earmarked fund for CARS-35-PIG.  

About author:  Shengjie Shi, E-mail: shisj@nwafu.edu.cn; Lutong Zhang, E-mail: zhanglutong1011@foxmail.com; #Correspondence Lei Gao, E-mail: gaolei5414@nwafu.edu.cn; Guiyan Chu, E-mail: guiyanchu@nwafu.edu.cn * These authors contributed equally to this study.

Cite this article: 

Shengjie Shi, Lutong Zhang, Liguang Wang, Huan Yuan, Haowei Sun, Mielie Madaniyati, Chuanjiang Cai, Weijun Pang, Lei Gao, Guiyan Chu. 2024.

miR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27 . Journal of Integrative Agriculture, 23(04): 1315-1328.

Bertoli C, Skotheim J M, de Bruin R A. 2013. Control of cell cycle transcription during G1 and S phases. Nature Reviews Molecular Cell Biology, 14, 518–528.

Bi Y, Yang S, Wang H, Chang G, Chen G. 2021. Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation. Journal of Integrative Agriculture, 20, 2749–2757.

Cui X, Huang X, Huang M, Zhou S, Le G, Yu W, Duan M, Jiang B, Zeng J, Zhou J, Huang X, Liang P, Zhang P. 2022. MiR-24-3p obstructs the proliferation and migration of human skin fibroblasts after thermal injury by targeting PPAR-beta and positively regulated by NF-kappaB. Experimental Dermatology, 31, 841–853.

Ding H, Liu M, Zhou C, You X, Su T, Yang Y, Xu D. 2020. Integrated analysis of miRNA and mRNA expression profiles in testes of Duroc and Meishan boars. Bmc Genomics, 21, 686.

Fujieda S, Inuzuka M, Tanaka N, Sunaga H, Fan G K, Ito T, Sugimoto C, Tsuzuki H, Saito H. 1999. Expression of p27 is associated with Bax expression and spontaneous apoptosis in oral and oropharyngeal carcinoma. International Journal of Cancer, 84, 315–320.

Guo Y, Sun J, Lai D. 2017. Role of microRNAs in premature ovarian insufficiency. Reproductive Biology and Endocrinology, 15, 38.

Hou T, Ou J, Zhao X, Huang X, Huang Y, Zhang Y. 2014. MicroRNA-196a promotes cervical cancer proliferation through the regulation of FOXO1 and p27Kip1. British Journal of Cancer, 110, 1260–1268.

Hu X, Xing Y, Ren L, Wang Y, Li Q, Fu X, Yang Q, Xu L, Willems L, Li J, Zhang L. 2019. Bta-miR-24-3p controls the myogenic differentiation and proliferation of fetal, bovine, skeletal muscle-derived progenitor cells by targeting ACVR1B. Animals (Basel), 9, 859.

Hu Y, Xu J, Shi S J, Zhou X, Wang L, Huang L, Gao L, Pang W, Yang G, Chu G. 2022. Fibroblast growth factor 21 (FGF21) promotes porcine granulosa cell estradiol production and proliferation via PI3K/AKT/mTOR signaling. Theriogenology, 194, 1–12.

Huang L, Yin Z J, Feng Y F, Zhang X D, Wu T, Ding Y Y, Ye P F, Fu K, Zhang M Q. 2016. Identification and differential expression of microRNAs in the ovaries of pigs (Sus scrofa) with high and low litter sizes. Animal Genetics, 47, 543–551.

Huang W. 2017. MicroRNAs: Biomarkers, diagnostics, and therapeutics. Methods in Molecular Biology, 1617, 57–67.

King K L, Cidlowski J A. 1995. Cell cycle and apoptosis: Common pathways to life and death. Journal of Cellular Biochemistry, 58, 175–180.

Kossowska-Tomaszczuk K, De Geyter C, De Geyter M, Martin I, Holzgreve W, Scherberich A, Zhang H. 2009. The multipotency of luteinizing granulosa cells collected from mature ovarian follicles. Stem Cells, 27, 210–219.

Li H, Wang X, Mu H, Mei Q, Liu Y, Min Z, Zhang L, Su P, Xiang W. 2022. Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell  function via YAP1-mediated mitochondrial function and apoptosis. International Journal of Biological Sciences, 18, 1008–1021.

Li Q, Du X, Wang L, Shi K, Li Q. 2021. TGF-beta1 controls porcine granulosa cell states: A miRNA-mRNA network view. Theriogenology, 160, 50–60.

Li X, Chen H, Zhang Z, Xu D, Duan J, Li X, Yang L, Hua R, Cheng J, Li Q. 2021. Isorhamnetin promotes estrogen biosynthesis and proliferation in porcine granulosa cells via the PI3K/Akt signaling pathway. Journal of Agricultural and Food Chemistry, 69, 6535–6542.

Li Y, Wu X, Miao S, Cao Q. 2022. MiR-383-5p promotes apoptosis of ovarian granulosa cells by targeting CIRP through the PI3K/AKT signaling pathway. Archives of Gynecology and Obstetrics, 306, 501–512.

Lian C, Sun B, Niu S, Yang R, Liu B, Lu C, Meng J, Qiu Z, Zhang L, Zhao Z. 2012. A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing. FEBS Journal, 279, 964–975.

Lynch S M, Mckenna M M, Walsh C P, Mckenna D J. 2016. MiR-24 regulates CDKN1B/p27 expression in prostate cancer. Prostate, 76, 637–648.

Matsuda F, Inoue N, Manabe N, Ohkura S. 2012. Follicular growth and atresia in mammalian ovaries: Regulation by survival and death of granulosa cells. Journal of Reproduction and Development, 58, 44–50.

Matsuda-Minehata F, Inoue N, Goto Y, Manabe N. 2006. The regulation of ovarian granulosa cell death by pro- and anti-apoptotic molecules. Journal of Reproduction and Development, 52, 695–705.

Naruse I, Hoshino H, Dobashi K, Minato K, Saito R, Mori M. 2000. Over-expression of p27kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines. International Journal of Cancer, 88, 377–383.

Pena-Blanco A, Garcia-Saez A J. 2018. Bax, Bak and beyond - Mitochondrial performance in apoptosis. FEBS Journal, 285, 416–431.

Qiu M, Liu J, Han C, Wu B, Yang Z, Su F, Quan F, Zhang Y. 2014. The influence of ovarian stromal/theca cells during in vitro culture on steroidogenesis, proliferation and apoptosis of granulosa cells derived from the goat ovary. Reproduction in Domestic Animals, 49, 170–176.

Reis E D, Roque M, Cordon-Cardo C, Drobnjak M, Fuster V, Badimon J J. 2000. Apoptosis, proliferation, and p27 expression during vessel wall healing: Time course study in a mouse model of transluminal femoral artery injury. Journal of Vascular Surgery, 32, 1022–1029.

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. 2014. 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, 9, e106795.

Salilew-Wondim D, Gebremedhn S, Hoelker M, Tholen E, Hailay T, Tesfaye D. 2020. The role of microRNAs in mammalian fertility: From gametogenesis to embryo implantation. International Journal of Molecular Sciences, 21, 585.

Shi S, Hu Y, Song X, Huang L, Zhang L, Zhou X, Gao L, Pang W, Yang G, Chu G. 2022. Totipotency of miR-184 in porcine granulosa cells. Molecular and Cellular Endocrinology, 558, 111765.

Shi S, Zhou X, Li J, Zhang L, Hu Y, Li Y, Yang G, Chu G. 2020. MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine  granulosa cells. Journal of Animal Science and Biotechnology, 11, 94.

Sinha P B, Tesfaye D, Rings F, Hossien M, Hoelker M, Held E, Neuhoff C, Tholen E, Schellander K, Salilew-Wondim D. 2017. MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation. Journal of Ovarian Research, 10, 37.

Sorensen A E, Wissing M L, Englund A L, Dalgaard L T. 2016. MicroRNA species in follicular fluid associating with polycystic ovary syndrome and related intermediary phenotypes. Journal of Clinical Endocrinology & Metabolism, 101, 1579–1589.

Sun B, Ma Y, Wang F, Hu L, Sun Y. 2019. MiR-644-5p carried by bone mesenchymal stem cell-derived exosomes targets regulation of p53 to inhibit ovarian granulosa cell apoptosis. Stem Cell Research & Therapy, 10, 360.

Tan W, Dai F, Yang D, Deng Z, Gu R, Zhao X, Cheng Y. 2022. MiR-93-5p promotes granulosa cell apoptosis and ferroptosis by the NF-kB signaling pathway in polycystic ovary syndrome. Frontiers in Immunology, 13, 967151.

Tao H, Yang J, Xu M, Liu Z, Liu Y, Xiong Q. 2023. MicroRNA-27a-3p targeting Vangl1 and Vangl2 inhibits cell proliferation in mouse granulosa cells. Biochimica et Biophysica Acta: Gene Regulatory Mechanisms, 1866, 194885.

Tiwari M, Prasad S, Tripathi A, Pandey A N, Ali I, Singh A K, Shrivastav T G, Chaube S K. 2015. Apoptosis in mammalian oocytes: A review. Apoptosis, 20, 1019–1025.

Vishnoi A, Rani S. 2017. MiRNA biogenesis and regulation of diseases: An overview. Methods in Molecular Biology, 1509, 1–10.

Wang L, Wang Y, Lin J. 2018. MiR-152-3p promotes the development of chronic myeloid leukemia by inhibiting p27. European Review for Medical and Pharmacological Sciences, 22, 8789–8796.

Yan L, Ma J, Zhu Y, Zan J, Wang Z, Ling L, Li Q, Lv J, Qi S, Cao Y, Liu Y, Cao L, Zhang Y, Qi Z, Nie L. 2018. MiR-24-3p promotes cell migration and proliferation in lung cancer by targeting SOX7. Journal of Cellular Biochemistry, 119, 3989–3998.

Zhang H, Luo Q, Lu X, Yin N, Zhou D, Zhang L, Zhao W, Wang D, Du P, Hou Y, Zhang Y, Yuan W. 2018. Effects of hPMSCs on granulosa cell apoptosis and AMH expression and their role in the restoration of ovary function in premature ovarian failure mice. Stem Cell Research & Therapy, 9, 20.

Zhang H W, Shi Y, Liu J B, Wang H M, Wang P Y, Wu Z J, Li L, Gu L P, Cao P S, Wang G R, Ma Y S, Fu D. 2021. Cancer-associated fibroblast-derived exosomal microRNA-24-3p enhances colon cancer cell resistance to MTX by down-regulating CDX2/HEPH axis. Journal of Cellular and Molecular Medicine, 25, 3699–3713.

Zhang Y, Wang Z, Lan D, Zhao J, Wang L, Shao X, Wang D, Wu K, Sun M, Huang X, Yan M, Liang H, Rong X, Diao H, Guo J. 2022. MicroRNA-24-3p alleviates cardiac fibrosis by suppressing cardiac fibroblasts mitophagy via downregulating PHB2. Pharmacological Research, 177, 106124.

Zhen J, Li J, Li X, Wang X, Xiao Y, Sun Z, Yu Q. 2021. Downregulating lncRNA NEAT1 induces proliferation and represses apoptosis of ovarian granulosa cells in polycystic ovary syndrome via microRNA-381/IGF1 axis. Journal of Biomedical Science, 28, 53.

Zhu L, Jing J, Qin S Q, Lu J N, Zhu C Y, Zheng Q, Liu Y, Fang F G, Li Y S, Zhang Y H, Ling Y H. 2022. miR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells. Journal of Integrative Agriculture, 21, 1137–1145.

[1] Shanshan Su, Hongwu Liu, Junrong Zhang, Puying Qi, Yue Ding, Ling Zhang, Linli Yang, Liwei Liu, Xiang Zhou, Song Yang.

Discovery and structure-activity relationship studies of novel tetrahydro-β-carboline derivatives as apoptosis initiators for treating bacterial infections [J]. >Journal of Integrative Agriculture, 2024, 23(04): 1259-1273.

[2] ZHAO Jing, WU Ya-mei, ZHANG Yao, TANG Shu-yue, HAN Shun-shun, CUI Can, TAN Bo, YU Jie, KANG Hou-yang, CHEN Guang-deng, MA Meng-gen, ZHU Qing, YIN Hua-dong. miR-27b-5p regulates chicken liver disease via targeting IRS2 to suppress the PI3K/AKT signal pathway[J]. >Journal of Integrative Agriculture, 2023, 22(11): 3500-3516.
[3] Saif ULLAH, Sheeraz MUSTAFA, Wael ENNAB, Muhammad JAN, Muhammad SHAFIQ, Ngekure M. X. KAVITA, Lü Zeng-peng, MAO Da-gan, SHI Fang-xiong. A protective role of resveratrol against the effects of immobilization stress in corpora lutea of mice in early pregnancy[J]. >Journal of Integrative Agriculture, 2020, 19(7): 1857-1866.
[4] LI Fu-hua, ZHENG Shao-jie, ZHAO Ji-chun, LIAO Xia, WU Su-rui, MING Jian. Phenolic extract of Morchella angusticeps peck inhibited the proliferation of HepG2 cells in vitro by inducing the signal transduction pathway of p38/MAPK[J]. >Journal of Integrative Agriculture, 2020, 19(11): 2829-2838.
[5] WANG Jun, SHI xin-jin, SUN Hai-wei, CHEN Hong-jun. Insights into African swine fever virus immunoevasion strategies[J]. >Journal of Integrative Agriculture, 2020, 19(1): 11-22.
[6] WENG Bo, RAN Mao-liang, Cao Rong, PENG Fu-zhi, LUO Hui, GAO Hu, TANG Xiang-wei, Yang An-qi, CHEN Bin.
miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene  
[J]. >Journal of Integrative Agriculture, 2019, 18(8): 1924-1935.
[7] RAN Mao-liang, WENG Bo, CAO Rong, PENG Fu-zhi, LUO Hui, GAO Hu, CHEN Bin. miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene[J]. >Journal of Integrative Agriculture, 2019, 18(2): 449-459.
[8] ZHANG Feng, GAO Peng, GE Xin-na, ZHOU Lei, GUO Xin, YANG Han-chun. Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4[J]. >Journal of Integrative Agriculture, 2017, 16(11): 2573-2585.
[9] PAN Chuan-ying, YU Shuai, ZHANG Peng-fei, WANG Bo, ZHU Zhen-dong, LIU Ying-ying, ZENG Wen-xian . Effect of sucrose on cryopreservation of pig spermatogonial stem cells[J]. >Journal of Integrative Agriculture, 2017, 16(05): 1120-1129.
[10] HU Ting-xi, ZHU Hua-bin, SUN Wei-jun, HAO Hai-sheng, ZHAO Xue-ming, DU Wei-hua, WANG Zong-li. Sperm pretreatment with glutathione improves IVF embryos development through increasing the viability and antioxidative capacity of sex-sorted and unsorted bull semen[J]. >Journal of Integrative Agriculture, 2016, 15(10): 2326-2335.
[11] YU De-bing, YU Min-li, LIN Fei, JIANG Bao-chun, YANG Li-na, WANG Si-yu, ZHAO Ying , WNAG Zheng-chao. Morphological and Hormonal Identification of Porcine Atretic Follicles and Relationship Analysis of Hormone Receptor Levels During Granulosa Cell Apoptosis In vivo[J]. >Journal of Integrative Agriculture, 2014, 13(5): 1058-1064.
[12] SHI Ya-ran, WANG Zhan-he, CAO Yong-chun, LU Yan, TIAN Jin-ling, ZhANG Chao, JIA Zi-ye, CHEN Wu. Relationships Between Icariin and Anti-Apoptotic miRNA-21 in Mouse Blastocyst Development In vitro[J]. >Journal of Integrative Agriculture, 2013, 12(4): 663-669.
[13] PAN Yao-qian, LIU Xing-you, MENG Li-ping, ZHU Guang-rui, XIA Yin-ke, CHEN Jin-shan , Yoshikawa Takashi. Pathogenesis of Demyelinating Encephalopathy in Dogs with Spontaneous Acute Canine Distemper[J]. >Journal of Integrative Agriculture, 2013, 12(2): 334-343.
[14] SUN Gui-rong, LI Yan, KANG Xiang-tao, TIAN Ya-dong, ZHANG Hu , LI Kui. Effect of Beak Trimming Stress on the Apoptosis and Its Related Protein Expression of Chicken Spleen[J]. >Journal of Integrative Agriculture, 2012, 12(4): 639-645.
[15] YOU Hua, ZHU Jin-feng, SHE Rui-ping, CHANG Ling-ling, SHI Rui-han, DING Ye, CHI Li-juan, LIUBin , YUE Zhuo, TIAN Ji-jing, MAO Jing-jing , SU Li-fang. Induction ofApoptosis in the ImmatureMouse Testes by aMixture ofMelamine and CyanuricAcid[J]. >Journal of Integrative Agriculture, 2012, 12(12): 2058-2066.
No Suggested Reading articles found!