siRNA抑制牛子宫内膜上皮细胞Nrf2基因的表达研究

doi:10.16431/j.cnki.1671-7236.2019.06.001

摘要/Abstract

摘要：

为探讨核因子E2相关因子（nuclear factor E2 related factor，Nrf2）基因沉默及激活后对牛子宫内膜上皮细胞的影响，本试验利用小分子干扰（siRNA）技术及Nrf2的激动剂叔丁基对苯二酚（tBHQ），分别从Nrf2基因的下调和上调表达来研究Nrf2对牛子宫内膜上皮细胞中血红素加氧酶-1（HO-1）和Homebox A10（HOXA10）基因表达的影响。结果显示，经实时荧光定量PCR方法检测，在设计的2条siRNA序列（siRNA-1209和siRNA-1672）中，siRNA-1672在终浓度为75 nmol/L、作用时间24 h时抑制效果较好，抑制效率在80%以上；经Western blotting方法检测，Nrf2蛋白表达水平在转染后96 h极显著下降（P<0.01），而HO-1和HOXA10的mRNA表达量分别下降了60%和70%（P<0.01），蛋白表达量在96 h后极显著或显著下降（P<0.01；P<0.05）。此外，经CCK8方法检测，Nrf2基因表达沉默后，牛子宫内膜上皮细胞增殖能力减弱。而在tBHQ激活Nrf2试验中，经Western blotting方法检测，tBHQ终浓度为30 μmol/L时Nrf2蛋白表达量最高，极显著高于对照组（P<0.01），且HO-1和HOXA10的蛋白表达量与对照组相比也明显上升。结果表明，本试验设计的siRNA-1672能特异性地抑制牛子宫内膜上皮细胞Nrf2的表达，而抑制Nrf2的表达会导致牛子宫内膜上皮细胞增殖能力下降，且Nrf2对牛子宫内膜上皮细胞HO-1和HOXA10基因表达存在调控作用。

关键词: 牛; RNA干扰; 子宫内膜上皮细胞; Nrf2基因; HO-1基因; HOXA10基因

Abstract:

In order to investigate the effect of nuclear factor E2 related factor (Nrf2) gene on bovine endometrial epithelial cells,this experiment used small interference RNA (siRNA) technology and tert-butylhydroquinone(tBHQ) as Nrf2 activator to study the effects of Nrf2 on the expression of hemeoxygenase-1 (HO-1) and Homebox A10 (HOXA10) in bovine endometrial epithelial cells.The results showed that in the designed two siRNA sequences(siRNA-1209 and siRNA-1672),the siRNA-1672 had a better inhibitory effect at a final concentration of 75 nmol/L and a response time of 24 h by Real-time quantitative PCR,the inhibition efficiency was over 80%.Western blotting results showed that the protein expression level of Nrf2 extremely significantly decreased at 96 h after transfection (P<0.01);While the mRNA expression levels of HO-1 and HOXA10 extremely significantly decreased by 60% and 70% (P<0.01),respectively,and the protein expression of HO-1 and HOXA10 extremely significantly or significantly decreased after 96 h (P<0.01;P<0.05).In addition,CCK8 method was used in this test,it found that the proliferation ability of bovine endometrial epithelial cells was weakened.In the tBHQ-activated the expression of Nrf2 assay,the protein expression of Nrf2 was the highest in the final concentration of tBHQ at 30 μmol/L,which was extremely significantly higher than that of control group (P<0.01),and the protein expression levels of HO-1 and HOXA10 were also higher than that of control group.This experiment results indicated that the designed siRNA-1672 could specifically inhibit the expression of Nrf2 in bovine endometrial epithelial cells,while the inhibition of Nrf2 expression led to a decrease in the proliferation ability of bovine endometrial epithelial cells,and Nrf2 regulated the expression of HO-1 and HOXA10 genes in bovine endometrial epithelial cells.

Key words: bovine; RNAi; endometrial epithelial cell; Nrf2 gene; HO-1 gene; HOXA10 gene

中图分类号:

S814.8

胡修忠, 程蕾, 余婕, 向敏, 夏瑜, 陶弼菲, 周源, 王定发, 熊海谦. siRNA抑制牛子宫内膜上皮细胞Nrf2基因的表达研究[J]. 《中国畜牧兽医》, 2019, 46(6): 1567-1575.

HU Xiuzhong, CHENG Lei, YU Jie, XIANG Min, XIA Yu, TAO Bifei, ZHOU Yuan, WANG Dingfa, XIONG Haiqian. Study on Inhibition of Nrf2 Gene Expression in Bovine Endometrial Epithelial Cells by siRNA[J]. , 2019, 46(6): 1567-1575.

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