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

Effects of ASH1L Knockdown on mRNA Profiling in Bovine Cumulus Cells

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2046个差异表达基因,其中上调基因1078个,下调基因968个;差异表达基因主要富集到2802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117994个可变剪切事件;同时检测到522205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。

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 2046 DEGs were screened in cells with or without interfere-nce of ASH1L gene,of which 1078 were up-regulated and 968 were down-regulated.Those DEGs mainly enriched into 2802 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,117994 AS events with 12 patterns were identified;In the meantime,522205 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.