过表达组蛋白甲基转移酶ASH1L对牛卵丘细胞增殖和凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.08.021

摘要/Abstract

摘要： 旨在探究缺失的、小的、同源异形1（absent，small，or homeotic 1-like，ASH1L）过表达对牛卵丘细胞（cumulus cells，CCs）增殖和凋亡的影响。本研究将牛ASH1L SET结构域的cDNA序列克隆到pcDNA3.1上，构建牛ASH1L过表达载体；以导入pcDNA3.1+ASH1L载体的牛CCs为过表达组（ASH1L-OE），野生型牛CCs为对照组（Control），采用免疫荧光染色检测两组细胞中ASH1L表达水平和H3K36me1/2/3甲基化水平；通过流式细胞术分析ASH1L过表达细胞的凋亡和增殖情况；采用荧光定量PCR检测两组细胞中凋亡、增殖相关基因的mRNA表达水平。以牛CCs cDNA为模板，PCR扩增获得长2 487 bp的牛ASH1L SET结构域DNA片段，与pcDNA3.1载体连接；经Nhe I/Not I双酶切和测序鉴定，成功构建过表达载体pcDNA3.1+ASH1L。过表达载体转染牛CCs后，细胞中ASH1L mRNA及其蛋白表达水平、H3K36me1/2甲基化水平均显著升高（P<0.05）。另外，ASH1L过表达显著提高了活细胞率（91.85±1.25）%vs.（87.39±1.71）%，降低了细胞凋亡率（8.08±1.21）%vs.（12.51±1.72）%，并显著下调了凋亡基因BAX和CASPASE-3 mRNA表达水平（P<0.05），上调抗凋亡基因BCL-2 mRNA表达水平（P<0.01）。ASH1L过表达24、36 h时，细胞增殖率、增殖相关基因PCNA、CCND2表达水平显著升高（P<0.05）。可见，ASH1L过表达抑制了牛CCs凋亡，诱导了细胞增殖及H3K36me1/2甲基化水平的升高，为进一步研究ASH1L对CCs生长和卵泡发育的调控提供技术和理论基础。

关键词: ASH1L甲基转移酶, 牛卵丘细胞, 过表达, 细胞增殖, 细胞凋亡, H3K36 me1/2

Abstract: The aim of this study was to investigate the effects of overexpression of absent, small, or homeotic 1-like (ASH1L) on the proliferation and apoptosis of bovine cumulus cells (CCs). The cDNA fragment of bovine ASH1L SET domain was amplified by PCR using CCs cDNA as templates and cloned into pcDNA3.1 in order to construct a bovine ASH1L overexpression vector. The bovine CCs transfected with pcDNA3.1+ASH1L vector were used as the overexpression group (ASH1L-OE) and the wild CCs were the control group (Control). The levels of ASH1L protein and H3K36me1/2/3 methylation in CCs of ASH1L-OE and the control group were detected by immunofluorescence staining. The apoptosis and proliferation of CCs in both groups were analyzed by flow cytometry. The mRNA expression levels of apoptosis-related and proliferation-related genes were detected by quantitative RT-PCR. A 2 487 bp DNA fragment of bovine ASH1L SET domain was obtained by PCR amplification using bovine CCs cDNA as template and ligated with pcDNA3.1 vector; the overexpression vector pcDNA3.1+ASH1L was successfully constructed after NheⅠ/NotⅠ double digestion and sequencing. Levels of ASH1L mRNA, protein and H3K36me1/2 methylation were significantly increased in CCs transfected with overexpression vector (P<0.05). Moreover, ASH1L overexpression in CCs significantly increased the percentage of live-cells (91.85±1.25)% vs. (87.39±1.71)% and decreased the apoptosis rate (8.08±1.21)% vs.(12.51±1.72)%. The mRNA expression levels of apoptosis-related genes BAX and CASPASE-3 were significantly downregulated (P<0.05) and expression level of anti-apoptotic gene BCL-2 was upregulated in CCs with ASH1L overexpression (P<0.01). The proliferation rate of cells and the expression levels of proliferation-related genes PCNA and CCND2 in CCs with ASH1L overexpression was significantly increased at 24 and 36 h after transfection (P<0.05). These results suggest that ASH1L overexpression inhibits CCs apoptosis, induces cell proliferation and H3K36me1/2 methylation in bovine CCs. The study provides a technical and theoretical basis for revealing the regulation of ASH1L on CCs growth and follicle development.

Key words: ASH1L methyltransferase, bovine cumulus cell, overexpression, cell proliferation, cell apoptosis, H3K36 me1/2

中图分类号:

S823.3

王婉洁, 陈南珠, 邹惠影, 周心仪, 郝海生, 庞云渭, 朱化彬, 赵学明, 余大为, 杜卫华. 过表达组蛋白甲基转移酶ASH1L对牛卵丘细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2023, 54(8): 3358-3368.

WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.

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