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于志君,袁琼,金志刚,向梓非,龙萍,朱明,杨越旺,胡霞敏.基于 RIP-Seq 技术检测 MRTF-A 结合 RNA 在小鼠 MCAO/ R 模型中表达[J].中国实验动物学报,2020,28(6):765~772.

基于 RIP-Seq 技术检测 MRTF-A 结合 RNA 在小鼠 MCAO/ R 模型中表达

Detection of MRTF-A-binding gene expression in the MCAO / R mouse model via RIP-Seq

投稿时间：2020-08-11

DOI：10. 3969 / j.issn.1005-4847. 2020. 06. 005

英文关键词:myocardin-related transcription factor A RNA-binding protein immunoprecipitation-high throughput sequencing cerebral ischemia / reperfusion injury differential gene analysis

基金项目:

作者	单位	E-mail
于志君	武汉科技大学医学院,新药创制研究所,职业危害识别与控制湖北省重点实验室,武汉 430065	yuzhijun@ wust.edu.cn
袁琼	武汉科技大学医学院,新药创制研究所,职业危害识别与控制湖北省重点实验室,武汉 430065
金志刚	华润武钢总医院,武汉 430080
向梓非	武汉科技大学医学院,新药创制研究所,职业危害识别与控制湖北省重点实验室,武汉 430065
龙萍	华润武钢总医院,武汉 430080
朱明	华润武钢总医院,武汉 430080
杨越旺	上海健康医学院,上海 200030
胡霞敏	上海健康医学院,上海 200030	huxm@ sumhs.edu.cn

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中文摘要:

目的应用 RIP-Seq 测序技术检测小鼠 MCAO/ R 模型中心肌素相关转录因子 A(MRTF-A) 结合 RNA 的表达差异,探讨 MRTF-A 潜在的作用机制。方法将 C57BL/ 6 小鼠随机分成假手术组及 I/ R 组,线栓法构建 MCAO 模型,再灌注 24 h 后,提取脑组织总蛋白,利用 MRTF-A 抗体免疫共沉淀后,进行 RNA 高通量测序,获得 MRTF-A 结合 RNA 的表达谱,继而对差异 RNA 进行 GO、KEGG 分析。结果与假手术组相比,I/ R 组有 429 个 RNA 发生差异表达(上调 203,下调 226),并且在功能元件和染色体分布上均具有显著差异。 GO 分子功能分析显示,差异表达 RNA 主要富集 RNA 结合、Poly(A)RNA 结合等注释。 KEGG 通路分析显示 10 条通路被显著富集,其中雌激素信号通路富集最显著。结论在脑 I/ R 损伤时 MRTF-A 结合 RNA 表达谱发生差异改变,为深入探讨 MRTF-A 的分子机制提供理论依据。

英文摘要:

Objective To detect the expression of RNA-bound myocardin-related transcription factor A (MRTF- A)-binding genes using RIP-Seq technology in a mouse middle cerebral artery occlusion (MCAO) / reperfusion model and explore the potential mechanism of action of MRTF-A. Methods C57BL/ 6 mice were randomly divided into the sham and cerebral ischemia / reperfusion (I/ R) injury groups. The model of focal MCAO was constructed using the suture method. After 24 h of reperfusion, total brain tissue protein was extracted. Furthermore, the expression profile of MRTF-A-binding genes was detected using immunoprecipitation plus high-throughput sequencing. Additionally, the differentially expressed genes were analyzed using GO and KEGG. Results Compared with the findings in the sham group, 429 genes were differentially expressed (203 upregulated and 226 downregulated genes) in the cerebral I/ R group. GO molecular function analysis revealed that the differentially expressed genes were mainly enriched in RNA binding and Poly(A) RNA binding. KEGG pathway analysis illustrated that 10 pathways were significantly enriched, among which the estrogen signaling pathway was most enriched. Conclusions The expression profile of MRTF-A-binding genes was significantly altered by cerebral I/ R injury, which provides a theoretical basis for in-depth exploration of the molecular mechanism of MRTF-A.

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