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Journal of Integrative Agriculture  2017, Vol. 16 Issue (09): 2019-2028    DOI: 10.1016/S2095-3119(16)61582-4
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
MicroRNA-34c regulates porcine granulosa cell function by targeting forkhead box O3a
XU Yuan1, ZHANG Ai-ling2, ZHANG Zhe1, YUAN Xiao-long, CHEN Zan-mou1, ZHANG Hao1, LI Jia-qi1
1 Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, P.R.China
2 College of Biological and Food Engineering, Guangdong University of Education, Guangzhou 510642, P.R.China
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Abstract  Granulosa cells (GCs) are somatic cells of ovary, the behaviors of GCs are important for ovarian function.  MicroRNAs (miRNAs) are a class of endogenous 18–24 nucleotide (nt) non-coding RNAs, some of which have been shown to be important regulators of GCs function.  miR-34c involved in the regulation of various biological processes and was identified to be a pro-apoptotic and anti-proliferative factor in many cell types.  However, the roles of miR-34c in GCs function remain unknown.  In this study, we used Annexin V-FITC and EdU assays to demonstrate that miR-34c exerted pro-apoptotic and anti-proliferative effects in porcine GCs.  Dual-luciferase reporter assays, quantitative real-time PCR (qRT-PCR) and Western blotting identified Forkhead box O3a (FoxO3a) as a direct target gene of miR-34c.  The overexpression of FoxO3a rescued the phenotypic change caused by miR-34c in porcine GCs.  In conclusion, miR-34c regulate the function of porcine GCs by targeting FoxO3a.
Keywords:  porcine        microRNA-34c        FoxO3a        granulosa cell function  
Received: 25 October 2016   Accepted:

This work is supported by the National Natural Science Foundation of China (31201771) and the earmarked fund for the China Agriculture Research System (CARS-36).

Corresponding Authors:  Correspondence LI Jia-qi, Tel: +86-20-85283519, Fax: +86-20-85280740, E-mail:    
About author:  XU Yuan, E-mail:;

Cite this article: 

XU Yuan, ZHANG Ai-ling, ZHANG Zhe, YUAN Xiao-long, CHEN Zan-mou, ZHANG Hao, LI Jia-qi. 2017. MicroRNA-34c regulates porcine granulosa cell function by targeting forkhead box O3a. Journal of Integrative Agriculture, 16(09): 2019-2028.

Ambros V. 2004. The functions of animal microRNAs. Nature, 431, 350–355.

Amodio N, Di Martino M T, Foresta U, Leone E, Lionetti M, Leotta M, Gulla A M, Pitari M R, Conforti F, Rossi M, Agosti V, Fulciniti M, Misso G, Morabito F, Ferrarini M, Neri A, Caraglia M, Munshi N C, Anderson K C, Tagliaferri P, Tassone P. 2012. miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1. Cell Death and Disease, 3, e436.

Bartel D P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 116, 281–297.

Bennett J, Wu Y G, Gossen J, Zhou P, Stocco C. 2012. Loss of GATA-6 and GATA-4 in granulosa cells blocks folliculogenesis, ovulation, and follicle stimulating hormone receptor expression leading to female infertility. Endocrinology, 153, 2474–2485.

Binelli M, Murphy B D. 2010. Coordinated regulation of follicle development by germ and somatic cells. Reproduction, Fertility and Development, 22, 1–12.

Bouhallier F, Allioli N, Lavial F, Chalmel F, Perrard M H, Durand P, Samarut J, Pain B, Rouault J P. 2010. Role of miR-34c microRNA in the late steps of spermatogenesis. RNA, 16, 720–731.

Cai K M, Bao X L, Kong X H, Jiang W, Mao M R, Chu J S, Huang Y J, Zhao X J. 2010. Hsa-miR-34c suppresses growth and invasion of human laryngeal carcinoma cells via targeting c-Met. International Journal of Molecular Medicine, 25, 565–571.

Cannell I G, Bushell M. 2010. Regulation of Myc by miR-34c: A mechanism to prevent genomic instability? Cell Cycle, 9, 2726–2730.

Carletti M Z, Fiedler S D, Christenson L K. 2010. MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells. Biology of Reproduction, 83, 286–295.

Choe N, Kwon J S, Kim Y S, Eom G H, Ahn Y K, Baik Y H, Park H Y, Kook H. 2015. The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor. Cell Signal, 27, 1056–1065.

Corney D C, Flesken-Nikitin A, Godwin A K, Wang W, Nikitin A Y. 2007. MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Research, 67, 8433–8438.

Gao P, Zhong Y Y, Zhang A L. 2014. Separation, culture and identification of sow ovarian granulose cells. Guangdong Agricultural Science, 41, 131–135. (in Chinese)

Hagman Z, Larne O, Edsjo A, Bjartell A, Ehrnstrom R A, Ulmert D, Lilja H, Ceder Y. 2010. miR-34c is downregulated in prostate cancer and exerts tumor suppressive functions. International Journal of Cancer, 127, 2768–2776.

Hillier S G. 2001. Gonadotropic control of ovarian follicular growth and development. Molecular and Cell Endocrinology, 179, 39–46.

Huntzinger E, Izaurralde E. 2011. Gene silencing by microRNAs: Contributions of translational repression and mRNA decay. Nature Reviews Genetics, 12, 99–110.

Kress T R, Cannell I G, Brenkman A B, Samans B, Gaestel M, Roepman P, Burgering B M, Bushell M, Rosenwald A, Eilers M. 2011. The MK5/PRAK kinase and Myc form a negative feedback loop that is disrupted during colorectal tumorigenesis. Molecular Cell, 41, 445–457.

Kruger J, Rehmsmeier M. 2006. RNAhybrid: MicroRNA target prediction easy, fast and flexible. Nucleic Acids Research, 34, W451–W454.

Lee Y S, Kim H K, Chung S, Kim K S, Dutta A. 2005. Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. Journal of Biological Chemistry, 280, 16635–16641.

Li Y Q, Ren X Y, He Q M, Xu Y F, Tang X R, Sun Y, Zeng M S, Kang T B, Liu N, Ma J. 2015. MiR-34c suppresses tumor growth and metastasis in nasopharyngeal carcinoma by targeting MET. Cell Death and Disease, 6, e1618.

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. 2012. miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary. PLoS ONE, 7, e38640.

Liu J, Yao W, Yao Y, Du X, Zhou J, Ma B, Liu H, Li Q, Pan Z. 2014. MiR-92a inhibits porcine ovarian granulosa cell apoptosis by targeting Smad7 gene. FEBS Letters, 588, 4497–4503.

Liu L, Rajareddy S, Reddy P, Du C, Jagarlamudi K, Shen Y, Gunnarsson D, Selstam G, Boman K, Liu K. 2007. Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a. Development, 134, 199–209.

Liu X, Feng J, Tang L, Liao L, Xu Q, Zhu S. 2015. The regulation and function of miR-21-FOXO3a-miR-34b/c signaling in breast cancer. International Journal of Molecular Sciences, 16, 3148–3162.

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

Matzuk M M, Burns K H, Viveiros M M, Eppig J J. 2002. Intercellular communication in the mammalian ovary: Oocytes carry the conversation. Science, 296, 2178–2180.

McGee E A, Hsueh A J. 2000. Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews, 21, 200–214.

Miranda K C, Huynh T, Tay Y, Ang Y S, Tam W L, Thomson A M, Lim B, Rigoutsos I. 2006. A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes. Cell, 126, 1203–1217.

Nagaraja A K, Andreu-Vieyra C, Franco H L, Ma L, Chen R, Han D Y, Zhu H, Agno J E, Gunaratne P H, DeMayo F J, Matzuk M M. 2008. Deletion of Dicer in somatic cells of the female reproductive tract causes sterility. Molecular Endocrinology, 22, 2336–2352.

Pelusi C, Ikeda Y, Zubair M, Parker K L. 2008. Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells. Biology of Reproduction, 79, 1074–1083.

Reddy P, Shen L, Ren C, Boman K, Lundin E, Ottander U, Lindgren P, Liu Y X, Sun Q Y, Liu K. 2005. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Developmental Biology, 281, 160–170.

Richards J S, Russell D L, Ochsner S, Hsieh M, Doyle K H, Falender A E, Lo Y K, Sharma S C. 2002. Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Progress in Hormone Research, 57, 195–220.

Rusinov V, Baev V, Minkov I N, Tabler M. 2005. MicroInspector: A web tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids Research, 33, W696–W700.

Shimono Y, Zabala M, Cho R W, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula S P, Chiao E, Dirbas F M, Somlo G, Pera R A, Lao K, Clarke M F. 2009. Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell, 138, 592–603.

Wang C, Yang C, Chen X, Yao B, Yang C, Zhu C, Li L, Wang J, Li X, Shao Y, Liu Y, Ji J, Zhang J, Zen K, Zhang C Y, Zhang C. 2011. Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility. Clinical Chemistry, 57, 1722–1731.

Xu Y, Zhang A L, Xiao G, Zhang Z, Chen Z M, Zhang H, Li J Q. 2016. p53 and NFκB regulate microRNA-34c expression in porcine ovarian granulosa cells. Journal of Integrative Agriculture, 8, 1816–1824.

Yan G, Zhang L, Fang T, Zhang Q, Wu S, Jiang Y, Sun H, Hu Y. 2012. MicroRNA-145 suppresses mouse granulosa cell proliferation by targeting activin receptor IB. FEBS Letters, 586, 3263–3270.

Yang X, Zhou Y, Peng S, Wu L, Lin Y, Wang S, Wang H. 2012. Differentially expressed plasma microRNAs in premature ovarian failure patients and the potential regulatory function of mir-23a in granulosa cell apoptosis. Reproduction, 144, 235–244.

Yao G, Yin M, Lian J, Tian H, Liu L, Li X, Sun F. 2010. MicroRNA-224 is involved in transforming growth factor-beta-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4. Molecular Endocrinology, 24, 540–551.

Yin M, Lu M, Yao G, Tian H, Lian J, Liu L, Liang M, Wang Y, Sun F. 2012. Transactivation of microRNA-383 by steroidogenic factor-1 promotes estradiol release from mouse ovarian granulosa cells by targeting RBMS1. Molecular Endocrinology, 26, 1129–1143.

Yu M, Mu H, Niu Z, Chu Z, Zhu H, Hua J. 2014. miR-34c enhances mouse spermatogonial stem cells differentiation by targeting Nanos2. Journal of Cellular Biochemistry, 115, 232–242.

Zhang Q, Sun H, Jiang Y, Ding L, Wu S, Fang T, Yan G, Hu Y. 2013. MicroRNA-181a suppresses mouse granulosa cell proliferation by targeting activin receptor IIA. PLoS ONE, 8, e59667.
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