穹窿主体蛋白通过上调干扰素调节因子2抑制动脉内皮细胞凋亡
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南京医科大学心血管疾病转化医学协同创新中心

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国家自然科学基金项目(面上项目,重点项目,重大项目)


Major vault protein inhibits apoptosis of aortic endothelial cells through upregulating interferon regulatory factor 2
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The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)

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    摘要:

    目的:探究穹窿主体蛋白(major vault protein,MVP)对动脉内皮细胞(endothelial cell,EC)增殖的作用,揭示MVP对动脉EC发挥保护作用的潜在机制。方法:以人主动脉EC(Human aortic EC,HAEC)为细胞模型,感染慢病毒以抑制或过表达MVP。使用CCK-8实验和流式细胞术检测细胞增殖活性和死亡。使用凋亡抑制剂Z-VAD和坏死性凋亡抑制剂Nec-1确定细胞死亡方式。以流式细胞术检测Annexin V结合阳性率和Caspase 3活性,以Western Blot检测Caspase剪切体蛋白表达以评价细胞凋亡。以荧光定量PCR和Western Blot技术鉴定靶分子,并明确MVP与靶分子之间的调控关系。结果:敲降MVP抑制HAEC增殖,促进HAEC死亡,过表达MVP结果则相反。Z-VAD逆转MVP敲降引起的死亡,而Nec-1无此作用。过表达MVP抑制TNF-α诱导的HAEC凋亡,敲降MVP时作用相反。MVP通过上调干扰素调节因子2(interferon regulatory factor 2,IRF2)促进凋亡抑制蛋白1(cellular inhibitor of apoptosis protein 1,cIAP1)的转录表达增多。敲降IRF2逆转MVP过表达引起的cIAP1表达增多和凋亡抑制。结论:MVP通过上调IRF2蛋白促进cIAP1转录表达从而抑制TNF-α诱导的HAEC凋亡,发挥对EC的保护作用。

    Abstract:

    Objective: To investigate the effects and the underlying mechanism of major vault protein (MVP) on the proliferation of arterial endothelial cells. Methods: Human aortic endothelial cell (HAEC) were infected with lentivirus to inhibit or overexpress MVP. Cell proliferation and death were detected with CCK-8 assay and flow cytometry. Apoptosis inhibitor Z-VAD and necroptosis inhibitor Nec-1 were used to distinguish the mode of cell death. Annexin V binding and Caspase 3 activity were detected by flow cytometry, and cleaved Caspase was examined by Western Blot. Real time PCR and Western Blot were performed to investigate target molecules and the regulatory relationship between target molecules and MVP. Results: Knockdown of MVP inhibited the proliferation activity of HAEC and promoted the HAEC cell death. Overexpression of MVP resulted in the opposite results. Treatment with Z-VAD reversed HAEC death caused by MVP knockdown, while Nec-1 did not. Consistently, TNF-α-induced HAEC apoptosis was inhibited by MVP overexpression and exaggerated by MVP knocked down. MVP promoted the transcriptional expression of cellular inhibitor of apoptosis proteins1 (cIAP1) by up-regulating interferon regulatory factor 2 (IRF2) protein expression. IRF2 knockdown reversed the increase in cIAP1 expression and decrease in apoptosis caused by MVP overexpression. Conclusion: MVP promoted cIAP1 transcription by up-regulating IRF2 protein expression, thereby inhibiting TNF-α-induced apoptosis and promoting the proliferation of arterial EC.

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  • 收稿日期:2022-12-12
  • 最后修改日期:2023-02-10
  • 录用日期:2023-08-09
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