miR-145-4调控蛋鸭卵泡发育机制的研究

doi:10.16431/j.cnki.1671-7236.2022.03.002

Abstract

Abstract: 【Objective】 The purpose of this study was to reveal the role and regulatory mechanism of miR-145-4 in follicular development for egg duck.【Method】 The egg duck follicular granulosa cells were isolated and cultured.After miR-145-4 mimic and inhibitor were transfected, the expression of cell proliferation and apoptosis related genes were detected by Real-time quantitative PCR.RNAhybrid and Targetscan programs were used to predict the target gene and predict the potential binding sites of miR-145-4 to the 3'-UTR of phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA) gene.The wild type and mutant type dual-luciferase reporter vector of PIK3CA gene 3'-UTR were constructed and co-transfeacted with miR-145-4 mimic and mimic-NC into duck granulose cells.The binding relationship between miR-145-4 and target gene PIK3CA was verified by double luciferase reporting system.And the effect of miR-145-4 on the expression of PIK3CA in granulosa cells of egg ducks was detected by Real-time quantitative PCR.【Result】 The results of Real-time quantitative PCR showed that after over expression of miR-145-4, compared with control group, the expression of CyclinB2 gene was extremely significantly decreased (P<0.01), and the expression of BCL2 gene was extremely significantly increased (P<0.01).After inhibiting miR-145-4, compared with control group, the expression of CyclinB2 gene was extremely significantly increased (P<0.01), and the expression of BCL2 was extremely significantly decreased (P<0.01).The predict results showed that miR-145-4 could be binding to PIK3CA gene 3'-UTR.PCR amplification and sequencing results indicated that the wild type and mutant type vectors of PIK3CA gene 3'-UTR were successfully constructed.The results of double luciferase assay showed that the double luciferase activity of wild-type group was significantly lower than that of mutant and empty vector co-transfection groups (P<0.05), indicating that miR-145-4 could bind to PIK3CA gene 3'-UTR.The results of Real-time quantitative PCR showed that the expression of PIK3CA gene decreased significantly after overexpression of miR-145-4 in egg duck follicular granulosa cells (P<0.05), while the expression of PIK3CA gene increased extremely significantly after inhibition of miR-145-4 (P<0.01).【Conclusion】 miR-145-4 promoted the apoptosis of follicle granulosa cells by positively regulating the target gene PIK3CA, and then regulated the follicular development of egg ducks.

Key words: egg duck; miR-145-4; PIK3CA; granulose cells apoptosis

CLC Number:

S834

WU Yan, PI Jinsong, ZHANG Hao, LIANG Zhenhua, DU Jinping, PAN Ailuan, SHEN Jie, PU Yuejin. Study on the Mechanism of miR-145-4 Regulating Follicle Development in Egg Ducks[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 809-816.

References

[1] MATSUDA F, INOUE N, MANABE N, et al.Follicular growth and atresia in mammalian ovaries:Regulation by survival and death of granulosa cells[J].Journal of Reproduction and Development, 2012, 58(1):44-50.
[2] BAUFELD A, VANSELOW J.Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells[J].Cell & Tissue Research, 2013, 354:869-880.
[3] JOHNSON A L, BRIDGHAM J T, WITTY J P, et al. Expression of BCL-2 and NR-13 in hen ovarian follicles during development[J].Biology of Reproduction, 1997, 57(5):1096-1103.
[4] VRIJENS K B V, NAWROT T S. microRNAs as potential signatures of environmental exposure or effect:A systematic review[J].Environmental Health Perspectives, 2015, 123(5):399-411.
[5] HUTVAGNER G, ZAMORE P D.A microRNA in a multi-ple-turnover RNAi enzyme complex[J].Science, 2002, 297(5589):2056-2060.
[6] IMBAR T, EISENBERG I.Regulatory role of microRNAs in ovarian function[J].Fertility & Sterility, 2014, 101(6):1524-1530.
[7] LIN F, LI R, PAN Z X, et al.miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary[J].PLoS One, 2012, 7(6):e38640.
[8] 李润.水牛颗粒细胞凋亡相关基因和miRNA表达及雌二醇对其表达调控的研究[D].南宁:广西大学, 2014. LI R.Study on the expression of apoptosis related genes and miRNA in buffalo granulosa cells and the regulation of estradiol on their expression[D].Nanning:Guangxi University, 2014.(in Chinese)
[9] 侯英萍.FSH处理对鸡卵泡颗粒细胞凋亡的影响及差异表达miRNAs的研究[D].南京:南京农业大学, 2014. HOU Y P.Effect of FSH on chicken granulosa cells apoptosis and identification of differently expressed miRNAs[D].Nanjing:Nanjing Agricultute University, 2014.(in Chinese)
[10] 黄正洋.表皮生长因子参与鹅卵泡颗粒细胞增殖调控机理的研究[D]. 扬州:扬州大学, 2016. HUANG Z Y.Effect of epidermal growth factor on follicular granulosa cell development in laying geese[D].Yangzhou:Yangzhou University, 2016.(in Chinese)
[11] DONADEU F X, MOHAMMED B T, IOANNIDIS J.A miRNA target network putatively involved in follicular atresia[J].Domestic Animal Endocrinology, 2017, 58:76-83.
[12] WU Y, XIAO H, PI J, et al.LncRNA lnc_13814 promotes the cells apoptosis in granulosa cells of duck by acting as apla-miR-145-4 sponge[J].Cell Cycle, 2021, 20(9):927-942.
[13] GILBERT A B, EVANS A J, PERRY M M, et al. A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus)[J].Journal of Reproduction and Fertility, 1977, 50(1):179-181.
[14] LIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2^-ΔΔCt method[J].Methods, 2001, 25(4):402-408.
[15] MATSUDA F, INOUE N, MANABE N, et al.Follicular growth and atresia in mammalian ovaries:regulation by survival and death of granulosa cells[J].Journal of Reproduction & Development, 2012, 58(1):44-50.
[16] WU N, GAUR U, ZHU Q, et al.Expressed microRNA associated with high rate of egg production in chicken ovarian follicles[J].Animal Genetics, 2017, 48(2):205-216.
[17] 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 & Cell Biology, 2017, 36(11):922-929.
[18] YANG B, GUO H F, ZHANG Y L, et al.microRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9[J].PLoS One, 2011, 6(7):e21679.
[19] 张月宁, 康媛, 姬超.miR-145通过调控MMP-2、MMP-9的表达对卵巢癌细胞调亡、增殖、迁移的影响[J].检验医学与临床, 2021, 18(6):755-758. ZHANG Y N, KANG Y, JI C.miR-145 regulates apoptosis, proliferation and migration of ovarian cancer cells through regulation of MMP-2 and MMP-9 expression[J].Laboratory Medicine and Clinic, 2021, 18(6):755-758.(in Chinese)
[20] 张连筱, 郑筱娇, 徐科君, 等.miR-145靶向作用激活素受体ⅠB抑制小鼠卵巢颗粒细胞增殖[J].现代实用医学, 2017, 29(9):1129-1132. ZHANG L X, ZHENG X J, XU K J, et al.miR-145 targeting activator receptor IB inhibits ovarian granulocyte proliferation in mice[J].Modern Practical Medicine, 2017, 29(9):1129-1132.(in Chinese)
[21] HUANG J C, BABAK T, CORSON T W, et al. Using expression profiling data to identify human microRNA targets[J].Nature Methods, 2007, 4(12):1045-1049.
[22] BARTEL D P. microRNAs:Target recognition and regulatory functions[J]. Cell, 2009, 136(2):215-233.
[23] 李蕾, 沈铿.PIK3CA基因在浆液性卵巢癌中的拷贝数扩增分析及其机制探讨[J].现代妇产科进展, 2017, 26(7):481-484. LI L, SHEN K.DNA copy number analysis of PIK3CA in human serous ovarian cancer using TCGA database[J].Progress in Obstetrics and Gynecology, 2017, 26(7):481-484.(in Chinese)
[24] 潘蕾蕾, 江卫兵, 方芳, 等.miR-152-3p靶向PIK3CA基因对乳腺癌细胞生物学特性的调控及其机制[J].新乡医学院学报.2019, 36(12):1130-1136. PAN L L, JIANG W B, FANG F, et al.Regulation and mechanism of miR-152-3p targeting PIK3CA gene on biological characteristics of breast cancer cells[J].Journal of Xinxiang Medical University, 2019, 36(12):1130-1136.(in Chinese)

Study on the Mechanism of miR-145-4 Regulating Follicle Development in Egg Ducks

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZHANG He, LIU Dapeng, TANG Hehe, LIU Tong, YU Daxin, YU Simeng, GAI Kai, HOU Shuisheng, ZHOU Zhengkui. Identification and Analysis of the Lipidome of the Breast Muscle of Pekin Duck and Liancheng White Duck [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 904-912.
[2]	LIU Zhenni, LEI Xiaowen, LI Jianjun, ZENG Qingyuan, TAN Donghai, ZHONG Yunping, ZHANG Qiang, XIE Hualiang. Effects of Fermented Navel Orange Pomace on Intestinal Morphology,Intestinal Mucosal Immune Function and Microbial Diversity of Muscovy Ducks [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 573-585.
[3]	ZHANG Qiang, LIU Zhenni, LIAO Qiang, LEI Xiaowen, KONG Zhiwei, XIE Hualiang, TAN Donghai, LI Jianjun, ZHONG Yunping. Effects of Fermented Andrographis Powder on Production Performance, Serum Biochemical Indexes, Immune Related Indexes, Antioxidant Indexes and Fatty Acid Contents of Muscle in Muscovy Ducks [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4614-4624.
[4]	JIA Wenqian, ZHUANG Zhong, BAI Hao, WANG Zhixiu, CHANG Guobin, CHEN Guohong, JIANG Yong. Research Progress on Threonine in Pekin Duck [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4197-4206.
[5]	YU Simeng, GUO Zhanbao, HU Jian, ZHOU Zhengkui, HOU Shuisheng. Analysis of Genetic Differentiation Between IAS Grassland Ducks and Cherry Valley Ducks [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 801-808.
[6]	SANG Qiangqiang, SUN Yunxiao, LIU Ying, HOU Zhuocheng. Estimation of Heritability and Genetic Parameters of Related Traits in Striped Duck Eggs [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 830-836.
[7]	LUO Shiqi, ZHANG Xiuwen, SU Jianyu, CHEN Longhai, YANG Lin, WANG Wence. Advances on Intestinal Microorganisms in Waterfowl [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 845-856.
[8]	YANG Suan, ZHANG Yiyu, TAN Yuancheng, WANG Yingtong, YOU Minfang, LIAO Chaomei. Genetic Effects Analysis of Polymorphism and Expression of SLC4A9 Gene on Eggshell Quality in Sansui Ducks [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 2082-2090.
[9]	SHU Jingting, JI Gaige, TU Yunjie, ZHANG Ming, JU Xiaojun, SHAN Yanju, LIU Yifan, ZOU Jianmin. Tissue Expression Analysis of Wnt3a Gene and Genetic Effects of SNPs on Feather Follicle Density in Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1672-1680.
[10]	LIU Bo, ZHAO Mingming, WU Qiong, ZHAO Jiaping, TU Jianfeng. Analysis and Evaluation of Nutrient Composition of Muscles of Two Kinds of Mallard [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 4166-4173.
[11]	LIU Zhenni, LEI Xiaowen, KONG Zhiwei, ZHONG Yunping, XIE Hualiang, TAN Donghai, WU Yijian. Effects of Fermented Peanut Vine on Production Performance,Slaughter Performance, Serum Biochemical Indexes and Feces' Routine Ingredients in Muscovy Ducks [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3493-3501.
[12]	HUANG Huilan, LI Wenjun, XIE Zimin, YANG Huihu, CUI Huiyi, HUANG Shujian, ZHANG Xuelian. Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3713-3721.
[13]	JIANG Yong, YANG Tingshuo, TANG Jing, XIE Ming, CHEN Guohong, HOU Shuisheng. Effects of Threonine in Low Protein Diet on Growth Performance and Plasma Biochemical Index of Pekin Duck [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3176-3182.
[14]	ZHANG Lei, ZHANG Haibo, ZHANG Jingqi, ZHANG Yang, CHANG Guobin, CHEN Guohong, ZHOU Lijun, WANG Jian. SNP Screening and Analysis of Ovaries Between Gaoyou Ducks with High and Low Yield Double-yolk Egg Based on RNA-Seq [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3495-3503.
[15]	ZHAO Wenwen, WANG Lei, YUAN Wenhua, TIAN Yong, CHEN Bindan, DIAO Xinping, LU Lizhi. Effects of Astragalus Polysaccharides and Clostridium butyricum on Growth Performance and Serum Biochemical Indexes of Ducklings [J]. , 2018, 45(12): 3447-3454.