miR-200b生物学功能及其对小鼠卵泡发育的影响

doi:10.16431/j.cnki.1671-7236.2025.12.002

摘要/Abstract

摘要： 【目的】挖掘miR-200b生物学功能并探究其对小鼠卵泡发育的影响。【方法】利用NCBI数据库获取人、小鼠、大鼠、斑马鱼、猪和绵羊miR-200b成熟序列，通过在线软件WebLogo进行序列分析，利用TargetScan和miRDB软件预测小鼠miR-200b靶基因，并进一步对靶基因进行GO功能和KEGG通路富集分析；通过腹腔注射miRNA antagomir构建小鼠miR-200b颉颃模型，采集小鼠血液及组织器官样本，利用实时荧光定量PCR检测卵巢中miR-200b表达水平；计算脏器系数，通过HE染色观察卵巢组织形态变化，并对卵泡进行计数；利用ELISA检测血清雌二醇(E₂)水平。【结果】不同物种miR-200b种子序列完全相同、成熟序列末端1～2个碱基存在差异；TargetScan和miRDB软件预测的共有靶基因共607个，其主要显著富集在转录调控、DNA和蛋白结合等GO条目，以及PI3K-Akt、MAPK、长寿调节、雌激素等KEGG信号通路。与Control和antagomir NC组相比，miR-200b颉颃组小鼠卵巢中miR-200b表达水平极显著下调(P＜0.01)，卵巢指数和腔前卵泡数极显著降低(P＜0.01)，有腔卵泡数极显著增多(P＜0.01)，同时伴随卵泡体积增大、颗粒细胞增多；血清雌二醇水平极显著升高(P＜0.01)。【结论】miR-200b对小鼠卵泡颗粒细胞生长及卵泡发育起负向调控作用，研究结果为深入剖析miR-200b对卵泡发育的调控功能提供了依据。

关键词: 小鼠; miR-200b; 生物学功能; 卵泡发育

Abstract: 【Objective】 This study aimed to explore the biological functions of miR-200b and investigate the effect on follicle development in mice. 【Method】 In this study,the mature sequences of miR-200b in human,mice,rat,zebrafish,pig and sheep were obtained from the NCBI database,the sequence analysis was carried out by the online software WebLogo,the miR-200b target genes prediction was carried out by TargetScan and miRDB,and the GO function and KEGG pathway enrichment analysis of these target genes were performed.The miR-200b antagonistic model was constructed by intraperitoneal injection of miRNA antagomir,and blood and samples from various tissues and organs of the mice were collected.The expression level of miR-200b in ovaries was detected by Real-time quantitative PCR. The organ coefficient was counted,the morphological changes of ovarian tissues were observed by HE staining,the follicles were counted,and the serum E₂ level was detected by ELISA. 【Result】 The seed sequences of miR-200b were completely identical across different species,and there were differences in the last 1-2 bases of the mature sequence.A total of 607 target genes were predicted by TargetScan and miRDB software,which were mainly enriched in GO entries such as transcriptional regulation,DNA and protein binding,as well as KEGG signaling pathways such as PI3K-Akt,MAPK,longevity regulation,and estrogen.Compared with control and antagomir NC groups,the expression of miR-200b in ovary of mice in miR-200b antagonist group was extremely significantly downregulated (P＜0.01),the ovarian index and the number of pre-antral follicles were extremely significantly decreased (P＜0.01),and the number of antral follicles was extremely significantly increased (P＜0.01).There was also an increase in follicle volume and granulosa cell number.The serum E₂ level was extremely significantly increased (P＜0.01). 【Conclusion】 miR-200b negatively regulated the growth of granulosa cells and follicle development in mice,which provided new evidence for an in-depth analysis of the regulatory function of miR-200b on follicle development.

Key words: mice; miR-200b; biological function; follicle development

中图分类号:

S814

宋鹏琰, 锡建中, 陈晓勇, 方翟, 邢凤, 周荣艳. miR-200b生物学功能及其对小鼠卵泡发育的影响[J]. 中国畜牧兽医, 2025, 52(12): 5539-5548.

SONG Pengyan, XI Jianzhong, CHEN Xiaoyong, FANG Di, XING Feng, ZHOU Rongyan. Biological Functions of miR-200b and Its Effects on Follicle Development in Mice[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5539-5548.

参考文献

[1] LEE E B,CHAKRAVARTHI V P,WOLFE M W,et al.ERbeta regulation of gonadotropin responses during folliculogenesis[J].International Journal of Molecular Sciences,2021,22(19):10348.
[2] 李琴.microRNA对哺乳动物卵泡发育的影响[J].畜牧兽医学报,2018,49(12):2558-2566.
LI Q.Roles of microRNA on mammalian follicle development[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2558-2566.(in Chinese)
[3] 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.
[4] KIM V N.MicroRNA biogenesis:Coordinated cropping and dicing[J].Nature Reviews Molecular Cell Biology,2005,6(5):376-385.
[5] BARTEL D P.MicroRNAs:Genomics,biogenesis,mechanism,and function[J]. Cell,2004,116(2):281-297.
[6] XU B,ZHANG Y W,ZHENG S X,et al.Expression profile of microRNAs and their targeted pathways in human ovaries detected by next-generation small RNA sequencing[J].DNA and Cell Biology,2016,35(5):226-234.
[7] 于建杰,王相燕,石家祺,等.小鼠原始卵泡发育关键时期卵巢中miRNAs的生物学特征及功能分析[J].黑龙江畜牧兽医,2023,23:7-12.
YU J J,WANG X Y,SHI J Q,et al.Biological characteristics and functional analysis of miRNAs in mouse ovary during the critical period of primordial follicle development[J].Heilongjiang Animal Science and Veterinary,2023,23:7-12.(in Chinese)
[8] HU C,ZHAO X,CUI C,et al.miRNA-29-3p targets PTEN to regulate follicular development through the PI3K/Akt/mTOR signaling pathway[J].Theriogenology,2024,214:173-181.
[9] TU J J,YANG Y Z,ALBERT C H,et al.Conserved miR-10 family represses proliferation and induces apoptosis in ovarian granulosa cells[J].Scientific Reports,2017,7:41304.
[10] YUAN H,LU J,XIAO S Y,et al.miRNA expression analysis of the sheep follicle during the prerecruitment,dominant,and mature stages of development under FSH stimulation[J].Theriogenology,2022,181:161-169.
[11] HASUWA H,UEDA J,IKAWA M,et al.miR-200b and miR-429 function in mouse ovulation and are essential for female fertility[J].Science,2013,341(6141):71-73.
[12] YANG H,LIU X,HU G,et al.Identification and analysis of microRNAs-mRNAs pairs associated with nutritional status in seasonal sheep[J].Biochemical and Biophysical Research Communications,2018,499(2):321-327.
[13] 解领丽,李嫒,黄万龙,等.湖羊卵巢不同发育阶段的miRNA鉴定与分析[J].畜牧兽医学报,2019,50(7):1396-1404.
XIE L L,LI Y,HUANG W L,et al.Identification and analysis of miRNAs at different developmental stages in Hu sheep ovaries[J].Acta Veterinaria et Zootechnica Sinica,2019,50(7):1396-1404.(in Chinese)
[14] 于要升,林杉,王开胜,等.绵羊oar-miR-200b对GNAQ基因的表达调控[J].石河子大学学报(自然科学版),2016,34(4):424-430.
YU Y S,LIN S,WANG K S,et al.Regulation of gene expression of GNAQ by oar-miR-200b in sheep[J].Journal of Shihezi University (Natural Science),2016,34(4):424-430.(in Chinese)
[15] PATINO-GARCIA D,CRUZ-FERNANDES L,BUNAY J,et al.Daily exposure to phthalates and alkylphenols alters miR biogenesis and expression in mice ovaries[J].Journal of Molecular Endocrinology,2020,65(4):175-186.
[16] 宋鹏琰,王思伟,岳巧娴,等.绵羊miR-200启动子鉴定及其对卵泡颗粒细胞线粒体功能的影响[J].畜牧兽医学报,2023,54(12):5066-5076.
SONG P Y,WANG S W,YUE Q X,et al.Identification of oar-miR-200b promoter and effects on mitochondrial function in follicular granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2023,54(12):5066-5076.(in Chinese)
[17] SONG P Y,CHEN X Y,ZHANG P Y,et al.miR-200b/MYBL2/CDK1 suppresses proliferation and induces senescence through cell cycle arrest in ovine granulosa cells[J].Theriogenology,2023,207:19-30.
[18] 宋鹏琰,锡建中,张振红,等.绵羊miR-200b对卵泡颗粒细胞周期和凋亡的影响[J].畜牧兽医学报,2021,52(12):3471-3479.
SONG P Y,XI J Z,ZHANG Z H,et al.Effects of ovine miR-200b on apoptosis and cell cycle of follicular granulosa cell[J].Acta Veterinaria et Zootechnica Sinica,2021,52(12):3471-3479.(in Chinese)
[19] 任红艳,刘楠,陶聪,等.小鼠体型控制相关microRNA-200b(miR-200b)的表达谱及靶标分析[J].农业生物技术学报,2013,21(1):47-54.
REN H Y,LIU N,TAO C,et al.The expression profile and target gene analysis of mouse (Mus musculus) body size regulation related microRNA-200b (miR-200b)[J].Chinese Journal of Agricultural Biotechnology,2013,21(1):47-54.(in Chinese)
[20] 金夏阳.绵羊脂肪细胞分化中miRNAs的鉴定及miR-148a和miR-200b功能研究[D].兰州:甘肃农业大学,2021.
JIN X Y.miRNAs profiles and functions of miR-148a and miR-200b in ovine adipogenesis[D].Lanzhou:Gansu Agricultural University,2021.(in Chinese)
[21] 李雪.miR-200b在大鼠角膜新生血管形成中的作用及其机制研究[D].重庆:重庆医科大学,2020.
LI X.Regulation and mechanism of miR-200b in corrneal neovascularization in rats[D].Chongqing:Chongqing Medical University,2020.(in Chinese)
[22] 李健宇.miR-200a对辽宁绒山羊毛色的影响及其靶基因鉴定[D].长春:吉林大学,2021.
LI J Y.miRNA-200a regulated pigmentation by targeting WNT5A and FZD4 in Cashmere goat[D].Changchun:Jilin University,2021.(in Chinese)
[23] 熊舒婷.miR-200家族在斑马鱼生殖发育中的功能研究[D].武汉:华中农业大学,2018.
XIONG S T.Functional analysis of miR-200 family in zebrafish reproductive development[D].Wuhan:Huazhong Agricultural University,2018.(in Chinese)
[24] WANG H,LI X,ZHOU R,et al.Genome-wide transcriptome profiling in ovaries of Small-tail Han sheep during the follicular and luteal phases of the oestrous cycle[J].Animal Reproduction Science,2018,197:212-221.
[25] 解一凡.miR-200c介导MAPK8基因调控哈萨克羊卵泡发育的机制研究[D].石河子:石河子大学,2019.
XIE Y F.Mechanism of miR-200c-mediated MAPK8 gene regulating follicular development in Kazakh sheep[D].Shihezi:Shihezi University,2019.(in Chinese)
[26] 张英,秦江月,贾慧卓,等.人microRNA-200家族靶基因预测及生物信息学分析[J].西部医学,2020,32(1):9-13.
ZHANG Y,QIN J Y,JIA H Z,et al.Target gene prediction and bioinformatics analysis of human microRNA-200 family[J].Medical Journal of West China,2020,32(1):9-13.(in Chinese)
[27] HILL L,BROWNE G,TULCHINSKY E.ZEB/miR-200 feedback loop:At the crossroads of signal transduction in cancer[J].International Journal of Cancer,2013,132(4):745-754.
[28] NISHIJIMA N,SEIKE M,SOENO C,et al.miR-200/ZEB axis regulates sensitivity to nintedanib in non-small cell lung cancer cells[J].International Journal of Oncology,2016,48(3):937-944.
[29] PERDIGAO-HENRIQUES R,PETROCCA F,ALTSCHULER G,et al.miR-200 promotes the mesenchymal to epithelial transition by suppressing multiple members of the Zeb2 and Snail1 transcriptional repressor complexes[J].Oncogene,2016,35(2):158-172.
[30] 刘淑娟,安小鹏,曹斌云.miR-486通过PI3K/AKT/mTOR通路抑制奶山羊卵巢颗粒细胞增殖的研究[J].家畜生态学报,2025,46(1):26-31.
LIU S J,AN X P,CAO B Y.Study on miR-486 inhibiting the proliferation of ovarian granulosa cells in dairy goats by suppressing the PI3K/Akt/mTOR pathway[J].Journal of Domestic Animal Ecology,2025,46(1):26-31.(in Chinese)
[31] WANG L,HE H,ZHAI R,et al.Investigation of the mechanism by which FOXJ2 inhibits proliferation,metastasis and cell cycle progression of ovarian cancer cells through the PI3K/Akt signaling pathway[J]. European Journal of Medical Research,2025,30(1):152.
[32] ZHANG T T,WANG Y,ZHANG X W,et al.miR-200c-3p regulates DUSP1/MAPK pathway in the nonalcoholic fatty liver after laparoscopic sleeve gastrectomy[J].Frontiers in Endocrinology (Lausanne),2022,13:792439.
[33] ZHU M,MIAO S,ZHOU W,et al.MAPK,Akt/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle,proliferation and apoptosis of chicken follicular granulosa cells[J].Ecotoxicology and Environmental Safety,2021,214:112091.
[34] KOMATSU K,WEI W,MURASE T,et al.17beta-estradiol and cathepsins control primordial follicle growth in mouse ovaries[J].Reproduction,2021,162(4):277-287.
[35] KEZELE P,SKINNER M K.Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone:Endocrine model of follicle assembly[J].Endocrinology,2003,144(8):3329-3337.