绵羊miR-200b对卵泡颗粒细胞周期和凋亡的影响

doi:10.11843/j.issn.0366-6964.2021.012.014

摘要/Abstract

摘要： 旨在揭示miR-200b在绵羊卵泡颗粒细胞的作用。本研究利用在线工具（TargetScan、miRTarBase及miRDB）预测miR-200b的靶基因，并利用KOBAS对其进行GO和KEGG通路富集分析。将分离培养的绵羊卵泡颗粒细胞分为4组，分别转染miR-200b mimic、mimic NC、miR-200b inhibitor和inhibitor NC，每组6个重复。采用CCK8法分别检测转染24、48和72 h颗粒细胞的存活率；采用荧光定量PCR检测miR-200b、CDK4、CDK6、CCND1、CCND2、Bax和Bcl-2基因的表达水平。利用3种在线工具预测到25个miR-200b的共同靶基因，GO和KEGG通路富集分析发现这些基因富集在细胞的增殖分化、细胞周期及生殖发育过程；转染miR-200b mimic、miR-200b inhibitor和NC后，颗粒细胞存活率随时间延长呈“V”型趋势降低，且48 h达最低（P<0.01）；miR-200b inhibitor与inhibitor NC组相比，miR-200b表达量无显著差异（P>0.05）；与mimic NC相比，miR-200b mimic组极显著促进miR-200b表达（P<0.001），显著下调CDK4（P<0.01）、CDK6（P<0.001）和CCND1（P<0.001）、CCND2（P<0.05）和Bcl-2（P<0.01）基因表达水平，Bax表达并无显著变化（P>0.05），且极显著下调Bcl-2与Bax比值（P<0.001）。综上所述，miR-200b抑制绵羊卵泡颗粒细胞细胞周期和增殖并促进其凋亡。

关键词: miR-200b, 细胞周期, 凋亡, 颗粒细胞, 绵羊

Abstract: The aim of study was to reveal the effect of ovine miR-200b on the follicular granulosa cells. The predicted target genes of miR-200b by 3 online tools (TargetScan, miRTarBase and miRDB) were analyzed with KOBAS for GO and KEGG pathway enrichment. The isolated and cultured sheep follicular granulosa cells were divided into 4 groups transfected with miR-200b mimic, mimic NC, miR-200b inhibitor and inhibitor NC,respectively, with 6 replicates in each group. CCK8 was used to detect the proliferation of granulosa cells at 24, 48 and 72 h after transfection. The expression level of miR-200b, CDK4, CDK6, CCND1, CCND2, Bax and Bcl-2 genes were measured by fluorescence quantitative PCR. A total of 25 common target genes of miR-200b were predicted by 3 online tools. These predicted target genes were enriched in cell proliferation and differentiation, cell cycle and reproductive development by GO and KEGG enrichment analysis. The survival rate of granulosa cell with time was a "V" decreased trendency and reached the lowest at 48 h (P<0.01) after transfection. There were no significant difference in miR-200b expression (P>0.05) between miR-200b inhibitor and inhibitor NC groups. Compared with mimic NC group, miR-200b mimic group significantly increased the expression of miR-200b (P<0.001), significantly down-regulated the expression of CDK4 (P<0.01), CDK6 (P<0.001), CCND1 (P<0.001), CCND2 (P<0.05) and Bcl-2 (P<0.01), while the expression of Bax did not significantly change (P>0.05), and extremely significantly decreased the ratio of Bcl-2 to Bax (P<0.001). In summary, miR-200b can inhibit the cell cycle and proliferation, and promote the apoptosis of follicular granulosa cells in sheep.

Key words: miR-200b, cell cycle, apoptosis, granulosa cell, sheep

中图分类号:

S826.3

宋鹏琰, 锡建中, 张振红, 付强, 岳巧娴, 张配颖, 周荣艳. 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 Pengyan, XI Jianzhong, ZHANG Zhenhong, FU Qiang, YUE Qiaoxian, ZHANG Peiying, ZHOU Rongyan. Effects of Ovine miR-200b on Cell Cycle and Apoptosis of Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3471-3479.

参考文献 43

[1]	仝晓丽.睾酮在体外通过促进颗粒细胞凋亡引起卵泡闭锁的机制研究[D].长春:吉林大学, 2020.TONG X L.Study on the mechanism of follicular atresia induced by testosterone in vitro by promoting granulosa cell apoptosis[D].Changchun:Jilin University, 2020. (in Chinese)
[2]	郭亚军, 柳苗苗, 付德海, 等.藏绵羊卵巢组织学及卵泡超微形态的观察[J].畜牧兽医学报, 2021, 52(2):389-398.GUO Y J, LIU M M, FU D H, et al.Observation of ovary histology and ultrastructure of follicles in Tibetan sheep[J].Acta Veterinaria et Zootechnica Sinica, 2021, 52(2):389-398.(in Chinese)
[3]	WANG C L, FAN Y C, TSENG C H, et al.Salmonella Enteritidis infection slows steroidogenesis and impedes cell growth in hen granulosa cells[J].Avian Dis, 2014, 58(4):511-517.
[4]	WU Y Q, ZHANG Z H, LIAO X H, et al.High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development[J].Biochem Biophys Res Commun, 2015, 466(3):599-605.
[5]	HSU C Y, HSIEH T H, TSAI C F, et al.Synthetic steroid hormones regulated cell proliferation through microRNA-34a-5p in human ovarian endometrioma[J].Biol Reprod, 2016, 94(3):60.
[6]	LIU J Y, TU F, YAO W, et al.Conserved miR-26b enhances ovarian granulosa cell apoptosis through HAS2-HA-CD44-Caspase-3 pathway by targeting HAS2[J].Sci Rep, 2016, 6:21197.
[7]	YOSHIDA K, YOKOI A, KAGAWA T, et al.Unique miRNA profiling of squamous cell carcinoma arising from ovarian mature teratoma:comprehensive miRNA sequence analysis of its molecular background[J].Carcinogenesis, 2019, 40(12):1435-1444.
[8]	ZHANG X D, ZHANG L, SHANG J N, et al.Combined microRNAome and transcriptome analysis of follicular phase and luteal phase in porcine ovaries[J].Reprod Domest Anim, 2019, 54(7):1018-1025.
[9]	GU B, LIU H, HAN Y, et al.Integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old Small Tail Han Sheep ovaries reveals that oar-miR-432 downregulates RPS6KA1 expression[J].Gene, 2019, 710:76-90.
[10]	SONG P Y, YUE Q X, FU Q, et al.Integrated analysis of miRNA-mRNA interaction in ovaries of Turpan Black Sheep during follicular and luteal phases[J].Reprod Domest Anim, 2021, 56(1):46-57.
[11]	LIU Y, WU X Q, XIE J, et al.Identification of transcriptome differences in goat ovaries at the follicular phase and the luteal phase using an RNA-Seq method[J].Theriogenology, 2020, 158:239-249.
[12]	PASQUARIELLO R, MANZONI E F M, FIANDANESE N, et al.Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence[J].Theriogenology, 2020, 145:77-85.
[13]	YANG S H, WANG S, LUO A Y, et al.Expression patterns and regulatory functions of microRNAs during the initiation of primordial follicle development in the neonatal mouse ovary[J].Biol Reprod, 2013, 89(5):126.
[14]	LI T T, LIU X Q, GONG X F, et al.microRNA-92b-3p regulates primordial follicle assembly by targeting TSCI in neonatal mouse ovaris[J].Cell Cycle, 2019, 18(8):824-833.
[15]	SONTAKKE S D, MOHAMMED B T, MCNEILLY A S, et al.Characterization of microRNAs differentially expressed during bovine follicle development[J].Reproduction, 2014, 148(3):271-283.
[16]	PANDE H O, TESFAYE D, HOELKER M, et al.MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway[J].J Ovarian Res, 2018, 11(1):34.
[17]	ZHOU R Y, MIAO Y P, LI Y M, et al.MicroRNA-150 promote apoptosis of ovine ovarian granulosa cells by targeting STAR gene[J].Theriogenology, 2019, 127:66-71.
[18]	SHI S J, ZHOU X G, LI J J, et al.MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells[J].J Anim Sci Biotechnol, 2020, 11:94.
[19]	ZHU L, JING J, QIN S Q, et al.miR-130a-3p regulates steroid hormone synthesis in goat ovarian granulosa cells by targeting the PMEPA1 gene[J].Theriogenology, 2021, 165:92-98.
[20]	SINHA P B, TESFAYE D, RINGS F, et al.MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation[J].J Ovarian Res, 2017, 10(1):37.
[21]	EISENBERG I, NAHMIAS N, PERSKY M N, et al.Elevated circulating micro-ribonucleic acid (miRNA)-200b and miRNA-429 levels in anovulatory women[J].Fertil Steril, 2017, 107(1):269-275.
[22]	DONADEU F X, SANCHEZ J M, MOHAMMED B T, et al.Relationships between size, steroidogenesis and miRNA expression of the bovine corpus luteum[J].Theriogenology, 2020, 145:226-230.
[23]	贺小云, 刘秋月, 储明星.miRNA调控哺乳动物卵泡发育和卵母细胞成熟的研究进展[J].畜牧兽医学报, 2019, 50(11):2175-2185.HE X Y, LIU Q Y, CHU M X.Advances in miRNA regulating mammalian follicular development and oocyte maturation[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(11):2175-2185.(in Chinese)
[24]	SHI L, LIU S, ZHAO W Q, et al.miR-483-5p and miR-486-5p are down-regulated in cumulus cells of metaphase II oocytes from women with polycystic ovary syndrome[J].Reprod Biomed Online, 2015, 31(4):565-572.
[25]	HUMPHRIES B, YANG C F.The microRNA-200 family:small molecules with novel roles in cancer development, progression and therapy[J].Oncotarget, 2015, 6(9):6472-6498.
[26]	ZOU X, LU T T, ZHAO Z F, et al.Comprehensive analysis of mRNAs and miRNAs in the ovarian follicles of uniparous and multiple goats at estrus phase[J].BMC Genomics, 2020, 21(1):267.
[27]	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)
[28]	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.
[29]	XIONG S T, MA W G, JING J, et al.An miR-200 cluster on chromosome 23 regulates sperm motility in zebrafish[J]. Endocrinology, 2018, 159(5):1982-1991.
[30]	段新崇, 魏彦辉, 李阳, 等.小尾寒羊间情期和发情期microRNAs差异表达分析[J].畜牧兽医学报, 2016, 47(7):1324-1332.DUAN X C, WEI Y H, LI Y, et al.Analyzing the differential expression of microRNAs in estrus and diestrus of Small Tailed Han Sheep[J].Acta Veterinaria et Zootechnica Sinica, 2016, 47(7):1324-1332.(in Chinese)
[31]	HE T T, SUN Y F, ZHANG Y C, et al.MicroRNA-200b and microRNA-200c are up-regulated in PCOS granulosa cell and inhibit KGN cell proliferation via targeting PTEN[J].Reprod Biol Endocrinol, 2019, 17(1):68.
[32]	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)
[33]	WANG H, LI X Y, ZHOU R Y, et al.Genome-wide transcriptome profiling in ovaries of small-tail Han sheep during the follicular and luteal phases of the oestrous cycle[J].Anim Reprod Sci, 2018, 197:212-221.
[34]	FU X, HE Y L, WANG X F, et al.MicroRNA-16 promotes ovarian granulosa cell proliferation and suppresses apoptosis through targeting PDCD4 in polycystic ovarian syndrome[J].Cell Physiol Biochem, 2018, 48(2):670-682.
[35]	DANG Y J, WANG X Y, HAO Y J, et al.MicroRNA-379-5p is associated with biochemical premature ovarian insufficiency through PARP1 and XRCC6[J].Cell Death Dis, 2018, 9(2):106.
[36]	ZHANG J B, XU Y X, LIU H L, et al.MicroRNAs in ovarian follicular atresia and granulosa cell apoptosis[J].Reprod Biol Endocrinol, 2019, 17(1):9.
[37]	LIU J Y, LI X Y, YAO Y, et al.miR-1275 controls granulosa cell apoptosis and estradiol synthesis by impairing LRH-1/CYP19A1 axis[J].Biochim Biophys Acta Gene Regul Mech, 2018, 1861(3):246-257.
[38]	ZHONG P, LIU J, LI H, et al.MicroRNA-204-5p regulates apoptosis by targeting Bcl2 in rat ovarian granulosa cells exposed to cadmium[J].Biol Reprod, 2020, 103(3):608-619.
[39]	STEFANZL G, BERGER D, CERNY-REITERER S, et al.The pan-BCL-2-blocker obatoclax (GX15-070) and the PI3-kinase/mTOR-inhibitor BEZ235 produce cooperative growth-inhibitory effects in ALL cells[J].Oncotarget, 2017, 8(40):67709-67722.
[40]	张纯, 徐晓娟, 姚莉娟, 等.改良多囊卵巢综合征大鼠模型卵泡颗粒细胞中凋亡调控蛋白Bcl-2 Bax表达的研究[J].四川中医, 2016, 34(6):34-37.ZHANG C, XU X J, YAO L J, et al.Research on the expression of apoptosis regulatory proteins Bcl-2, Bax in ovarian follicle granulosa cells of improved PCOS rat model[J].Journal of Sichuan Traditional Chinese Medicine, 2016, 34(6):34-37.(in Chinese)
[41]	TILLY J L, KOWALSKI K I, JOHNSON A L, et al.Involvement of apoptosis in ovarian follicular atresia and postovulatory regression[J].Endocrinology, 1991, 129(5):2799-2801.
[42]	BILLIG H, FURUTA I, HSUEH A J.Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis[J].Endocrinology, 1993, 133(5):2204-2212.
[43]	YU Y S, SUI H S, HAN Z B, et al.Apoptosis in granulosa cells during follicular atresia:relationship with steroids and insulin-like growth factors[J].Cell Res, 2004, 14(4):341-346.