ASH1L甲基转移酶在牛卵丘细胞中的表达与功能研究

Expression and Function of ASH1L Methyltransferase in Bovine Cumulus Cells

旨在探究缺失、小的、同源异形1(absent,small,or homeotic 1-like,ASH1L)甲基转移酶在牛卵丘细胞中的表达与功能。本研究通过免疫荧光染色在健康母牛卵丘细胞中对ASH1L甲基转移酶进行定位,并分析细胞的组蛋白H3第36位赖氨酸(histone H3 lysine36,H3K36)甲基化修饰模式;合成靶向Ash 1L基因的siRNA,对siRNA-1、siRNA-2、siRNA-3及对照组进行荧光定量PCR和蛋白质免疫印迹,筛选有效siRNA;采用荧光定量PCR分析干扰Ash 1L表达对处理组及对照组中凋亡相关基因及多梳抑制复合体(polycomb repressive complex 2,PRC2)组成基因的表达水平的影响。结果显示,ASH1L甲基转移酶位于牛卵丘细胞的细胞核中,呈点状分布。成功筛选到能有效干扰牛Ash 1L基因的siRNA-2,其干扰效率为60%~70%。将siRNA-2转染卵丘细胞后,该干扰组细胞中H3K36的单甲基化、二甲基化及三甲基化3种甲基化水平均显著低于对照组(P<0.05);干扰Ash 1L导致凋亡相关基因Bax、Bcl-2及caspase-3表达水平显著上调,凋亡基因Bax和caspase-3表达量高于抗凋亡相关基因Bcl-2(1.311和1.179 vs 1.146);同时,干扰Ash 1L基因表达也引起PRC2蛋白亚基EZH 2和Suz 12基因的mRNA表达量显著升高(P<0.05)。综上所述,本研究探讨了ASH1L甲基转移酶在牛卵丘细胞中的表达和功能,ASH1L在牛卵丘细胞中呈点状分布,且Ash 1L基因的抑制导致H3K36me1/2/3水平均显著下降及凋亡基因和PRC2蛋白相关亚基EZH 2和Suz 12基因表达的升高,为进一步研究其对家畜胚胎的调控作用提供技术和理论基础。

The study aimed to investigate the expression and function of absent,small,or homeotic 1-like(ASH1L)methyltransferase in healthy bovine cumulus cells.Location of ASH1L methyltransferase and the methylation patterns of histone H3 Lysine 36(H3K36)were detected using immunofluorescence staining in healthy bovine cumulus cells.The quantitative PCR(qPCR)and Western blotting were used to screen the effective siRNA targeting Ash 1L gene in siRNA-1,siRNA-2,siRNA-3 and control group.The expression level of apoptosis-related genes and polycomb repressive complex 2(PRC 2)genes were estimated by qPCR after the Ash 1L gene was interfered with siRNA in treatment and control groups.The results showed that ASH1L methyltransferase was located in the nucleus of bovine cumulus cells and distributed in a dotted pattern.The effective siRNA(siRNA-2)targeting the bovine Ash 1L gene was successfully screened and its interfering efficiency reached 60%-70%.The monomethylation,dimethylation and trimethylation levels of H3K36 in cumulus cells transfected with siRNA-2 were significantly lower than those in the control group(P<0.05).Additionally,interfering Ash 1L gene could significantly up-regulated the expressions of apoptosis-related genes Bax,Bcl-2 and caspase-3.However,the expression levels of apoptosis-related genes Bax and caspase-3 were higher than that of anti-apoptosis-related gene Bcl-2(1.311 and 1.179 vs 1.146).At the same time,the mRNA expressions of PRC2 protein subunit EZH 2 and Suz 12 were significantly increased after interfering Ash 1L(P<0.05).The expression and function of ASH1L methyltransferase in bovine cumulus cells were studied,ASH1L showed dot distribution in bovine cumulus cells,and the inhibition of Ash 1L gene expression resulted in a significant decrease in H3K36me1/2/3 levels and an increase in the expressions of apoptosis-related genes and PRC2 protein related subunits EZH 2 and Suz 12 genes,which will provide technical and theoretical basis for further studying the effects of ASH1L on embryos development in livestock animals.