敲低组蛋白甲基转移酶ASH1L基因对牛卵丘细胞表达谱的影响

doi:10.11843/j.issn.0366-6964.2022.01.015

摘要/Abstract

摘要： 旨在研究干扰类缺失的、小的、同源异形1（absent，small，or homeotic 1-like，ASH1L）甲基转移酶基因表达对牛卵丘细胞表达谱的影响。本研究采用经过筛选的siRNA干扰牛卵丘细胞中ASH1L基因的表达，分别收集干扰组和对照组细胞（野生型）进行RNA测序及基因差异表达、基因功能、信号通路、可变剪切和单核苷酸多态性（single nucleotide polymorphisms，SNP）等生物信息学分析；采用实时定量PCR方法验证测序结果；每个试验3个重复。结果显示，干扰组与对照组细胞共筛选出2 046个差异表达基因，其中上调基因1 078个，下调基因968个；差异表达基因主要富集到2 802个GO条目，如细胞分裂、细胞连接和组蛋白甲基转移酶活性；涉及到白细胞跨内皮迁移、PI3K-Akt、细胞黏附分子等53条KEGG通路。全部表达基因的基因集富集分析结果显示，ASH1L基因参与发育诱导、H3K4特异性组蛋白甲基转移酶活性、组蛋白去甲基化等生物学过程。另外，测序结果鉴出12种可变剪切方式，共117 994个可变剪切事件；同时检测到522 205个SNPs位点，其中碱基转换的发生频率显著高于颠换。综上所述，ASH1L基因干扰的卵丘细胞和野生型卵丘细胞的表达谱存在差异，推测该基因可能通过细胞黏附分子和白细胞跨内皮迁移等通路调节细胞生长，也可通过可变剪切和SNP影响基因表达，本试验为深入研究ASH1L对卵丘细胞增殖和胚胎发育的调控功能提供理论基础。

关键词: ASH1L, 转录组测序, 卵丘细胞, 牛

Abstract: The study aimed to explore the effects of absent, small, or homeotic 1-like (ASH1L) gene knockdown on mRNA profiling in bovine cumulus cell. In this study, the mRNA profiling in cumulus cells with or without interference of ASH1L gene was measured using RNA sequencing. Differentially expressed genes (DEGs) were identified, and bioinformatics analyses (differential expression of genes,function,signaling pathway,alternative splicing (AS) and SNP) were carried out. Transcriptional levels were confirmed using quantitative RT-PCR,3 replicates per trial. Results showed that a total of 2 046 DEGs were screened in cells with or without interfere-nce of ASH1L gene, of which 1 078 were up-regulated and 968 were down-regulated. Those DEGs mainly enriched into 2 802 GO terms including cell division, cell junctions and histone methyltransferase activity. Fifty-three pathways were involved by those DEGs such as leukocyte transendothelial migration, PI3K-Akt and cell adhesion molecule. The results of gene set enrichment analysis showed that ASH1L gene was closely involved in developmental induction, H3K4 specific histone methyltransferase activity, histone demethylation, etc. Moreover, 117 994 AS events with 12 patterns were identified; In the meantime,522 205 SNPs were detected and the frequency of base transitions was significantly higher than that of transversions. In summary, the mRNA profiling between bovine cumulus cells with or without interference of ASH1L gene were different. It was speculated that ASH1L gene may be involved in leukocyte transendothelial migration and cell adhesion molecule to regulate cell proliferation. Also, ASH1L can affect gene expression by AS and SNP, which may provide a theoretical basis for further study on the function of ASH1L in cumulus cell proliferation and embryo development.

Key words: ASH1L, transcriptome sequencing, cumulus cells, cattle

中图分类号:

田雅晴, 崔立欣, 郝海生, 邹惠影, 庞云渭, 赵学明, 朱化彬, 杜卫华. 敲低组蛋白甲基转移酶ASH1L基因对牛卵丘细胞表达谱的影响[J]. 畜牧兽医学报, 2022, 53(1): 151-160.

TIAN Yaqing, CUI Lixin, HAO Haisheng, ZOU Huiying, PANG Yunwei, ZHAO Xueming, ZHU Huabin, DU Weihua. Effects of ASH1L Knockdown on mRNA Profiling in Bovine Cumulus Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 151-160.

参考文献 36

[1]	SOTO-HERAS S,PARAMIO M T.Impact of oxidative stress on oocyte competence for in vitro embryo production programs[J].Res Vet Sci,2020,132:342-350.
[2]	MACAULAY A D,GILBERT I,SCANTLAND S,et al.Cumulus cell transcripts transit to the bovine oocyte in preparation for maturation[J].Biol Reprod,2016,94(1):16.
[3]	ROY S,GANDRA D,SEGER C,et al.Oocyte-derived factors (GDF9 and BMP15) and FSH regulate AMH expression via modulation of H3K27AC in Granulosa cells[J].Endocrinology,2018,159(9):3433-3445.
[4]	ORTIZ-ESCRIBANO N,SMITS K,PIEPERS S,et al.Role of cumulus cells during vitrification and fertilization of mature bovine oocytes:effects on survival,fertilization,and blastocyst development[J].Theriogenology,2016,86(2):635-641.
[5]	SHEARN A,HERSPERGER E,HERSPERGER G.Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations[J].Roux’s Arch Dev Biol,1987,196(4):231-242.
[6]	LEE Y,YOON E,CHO S,et al.Structural basis of MRG15-mediated activation of the ASH1L histone methyltransferase by releasing an autoinhibitory loop[J].Structure,2019,27(5):846-852.e3.
[7]	DORAFSHAN E,KAHN T G,GLOTOV A,et al.Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation[J].EMBO Rep,2019,20(4):e46762.
[8]	BRINKMEIER M L,GEISTER K A,JONES M,et al.The histone methyltransferase gene absent,small,or homeotic discs-1 like is required for normal Hox gene expression and fertility in Mice[J].Biol Reprod,2015,93(5):121.
[9]	CUI L X,TIAN Y Q,HAO H S,et al.Knockdown of ASH1L methyltransferase induced apoptosis inhibiting proliferation and H3K36 methylation in bovine cumulus cells[J].Theriogenology,2021,161:65-73.
[10]	CHOWDHURY H A,BHATTACHARYYA D K,KALITA J K.Differential expression analysis of RNA-seq reads:overview, taxonomy,and tools[J].IEEE/ACM Trans Comput Biol Bioinform,2020,17(2):566-586.
[11]	BIASE F H,KIMBLE K M.Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells[J].BMC Genomics,2018,19(1):351.
[12]	JIA B Y,XIANG D C,QUAN G B,et al.Transcriptome analysis of porcine immature oocytes and surrounding cumulus cells after vitrification and in vitro maturation[J].Theriogenology,2019,134:90-97.
[13]	崔立欣.组蛋白甲基转移酶ASH1L在牛卵丘细胞及胚胎中的表达和功能[D].北京:中国农业科学院,2020.CUI L X.Expression and function of ASH1L methyltransferase in bovine cumulus cells and embryos[D].Beijing:Chinese Academy of Agricultural Sciences,2020.(in Chinese)
[14]	RICHANI D,DUNNING K R,THOMPSON J G,et al.Metabolic co-dependence of the oocyte and cumulus cells:essential role in determining oocyte developmental competence[J].Hum Reprod Update,2021,27(1):27-47.
[15]	FANG Y,DENG S L,ZHANG J L,et al.Melatonin-mediated development of ovine cumulus cells,perhaps by regulation of DNA methylation[J].Molecules,2018,23(2):494.
[16]	SUBRAMANIAN A,TAMAYO P,MOOTHA V K,et al.Gene set enrichment analysis:a knowledge-based approach for interpreting genome-wide expression profiles[J].Proc Natl Acad Sci U S A,2005,102(43):15545-15550.
[17]	崔立欣,田雅晴,郝海生,等.ASH1L甲基转移酶在牛卵丘细胞中的表达与功能研究[J].畜牧兽医学报,2020,51(8):1866-1877.CUI L X,TIAN Y Q,HAO H S,et al.Expression and function of ASH1L methyltransferase in bovine cumulus cells[J].Acta Veterinaria et Zootechnica Sinica,2020,51(8):1866-1877.(in Chinese)
[18]	TANAKA Y,KATAGIRI Z I,KAWAHASHI K,et al.Trithorax-group protein ASH1 methylates histone H3 lysine 36[J].Gene,2007,397(1-2):161-168.
[19]	BANNISTER A J,SCHNEIDER R,KOUZARIDES T.Histone methylation:dynamic or static?[J].Cell,2002,109(7):801-806.
[20]	YUAN W,WU T,FU H,et al.Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation[J]. Science,2012,337(6097):971-975.
[21]	DE CONTO E,MATTE U,CUNHA-FILHO J S.BMP-6 and SMAD4 gene expression is altered in cumulus cells from women with endometriosis-associated infertility[J].Acta Obstet Gynecol Scand,2021,100(5):868-875.
[22]	EL-MAGD M A,GHONIEM A M,HELMY N M,et al.Effect of myostatin inhibitor (myostatin pro-peptide) microinjection on in vitro maturation and subsequent early developmental stages of buffalo embryo[J].Theriogenology,2019,126:230-238.
[23]	YOON J D,HWANG S U,KIM E,et al.GDF8 activates p38 MAPK signaling during porcine oocyte maturation in vitro[J]. Theriogenology,2017,101:123-134.
[24]	CHANG H M,FANG L L,CHENG J C,et al.Growth differentiation factor 8 down-regulates pentraxin 3 in human granulosa cells[J]. Mol Cell Endocrinol,2015,404:82-90.
[25]	SALUSTRI A,GARLANDA C,HIRSCH E,et al.PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization[J].Development,2004,131(7):1577-1586.
[26]	欧阳效晴,杨淑青,张春强,等.生长分化因子9在牛卵丘卵母细胞复合体体外成熟过程中的表达[J].畜牧兽医学报, 2013,44(10):1569-1575.OUYANG X Q,YANG S Q,ZHANG C Q,et al.Expression of growth differentiation factor-9 in bovine COCs during in vitro maturation[J].Acta Veterinaria et Zootechnica Sinica,2013,44(10):1569-1575.(in Chinese)
[27]	KAYAMORI T,KOSAKA N,MIYAMOTO A,et al.The differential pathways of bone morphogenetic protein (BMP)-4 and -7 in the suppression of the bovine granulosa cell apoptosis[J].Mol Cell Biochem,2009,323(1-2):161-168.
[28]	YUAN J S,DENG Y,ZHANG Y Y,et al.BMP4 inhibits goose granulosa cell apoptosis via PI3K/AKT/Caspase-9 signaling pathway[J].Anim Reprod Sci,2019,200:86-95.
[29]	DE VASCONCELOS DA CUNHA E,DE SOUZA G B,DE SOUSA PASSOS J R,et al.Effects of bone morphogenetic protein 4 (BMP4) on in vitro development and survival of bovine preantral follicles enclosed in fragments ovarian tissue[J]. Zygote, 2017,25(3):256-264.
[30]	ZHAO X D,SUN Q,DOU C W,et al.BMP4 inhibits glioblastoma invasion by promoting E-cadherin and claudin expression[J].Front Biosci (Landmark Ed),2019,24(6):1060-1070.
[31]	WANG L L,XUE Y,SHEN Y H,et al.Claudin 6:a novel surface marker for characterizing mouse pluripotent stem cells[J].Cell Res,2012,22(6):1082-1085.
[32]	LI X S,LI H M,LIU C X,et al.CLDN6-mediates SB431542 action through MMPs to regulate the invasion,migration,and EMT of breast cancer cells[J].Int J Clin Exp Pathol,2020,13(7):1590-1600.
[33]	MORIWAKI K,TSUKITA S,FURUSE M.Tight junctions containing claudin 4 and 6 are essential for blastocyst formation in preimplantation mouse embryos[J].Dev Biol,2007,312(2):509-522.
[34]	TURKSEN K,TROY T C.Permeability barrier dysfunction in transgenic mice overexpressing claudin 6[J].Development, 2002,129(7):1775-1784.
[35]	TURKSEN K,TROY T C.Claudin-6:a novel tight junction molecule is developmentally regulated in mouse embryonic epithelium[J].Dev Dyn,2001,222(2):292-300.
[36]	沈朋雷,秦希玲,尹倩,等.水牛卵母细胞GV/MII期对应卵丘细胞miRNA差异表达分析[J].畜牧兽医学报, 2017, 48(5):836-845.SHEN P L,QIN X L,YIN Q,et al.Characterization of the expression of miRNAs in cumulus cells during GV/MII stages of buffalo oocyte[J].Acta Veterinaria et Zootechnica Sinica,2017,48(5):836-845.(in Chinese)