Chen Ming, Ma Guiling, Yue Yin, Wei Yu, Li Qiang, Tong Zichuan, Zhang Lin, Miao Guobin, Zhang Jianjun
Heart Center, Beijing Chaoyang Hospital, Capital Medical University, No. 8 Gong-Ti South Road, Beijing 100020, PR China.
Heart Center, Beijing Chaoyang Hospital, Capital Medical University, No. 8 Gong-Ti South Road, Beijing 100020, PR China.
Int J Cardiol. 2014 Apr 15;173(1):65-73. doi: 10.1016/j.ijcard.2014.02.007. Epub 2014 Feb 20.
Endoplasmic reticulum (ER) stress is a common subcellular response to stresses and central to ER stress is increased expression of glucose-regulated protein 78 (GRP78). However, the mechanisms for GRP78 upregulation remained poorly understood. Our study goal was to shed light on this issue.
H2O2 was used to create cellular models of ER stress in neonatal rat ventricular cells (NRVCs) and rat aorta vascular smooth muscle cells (RAVSMCs). Molecular Biology techniques were used to quantify protein and mRNA levels. Luciferase reporter gene assay was employed to investigate miRNA targeting. MTT assay and ELISA were used to detect cell death.
MiRNAs belonging to the miR-30 family including miR-30a, b, c, d and e were all downregulated in ER stress induced by H2O2 in cardiovascular cells NRVCs and RAVSMCs, along with the upregulation of GRP78, cleaved ATF6, CHOP, and cleaved caspase-12. GRP78 was confirmed to be a target gene for miR-30. Artificial knockdown of miR-30 by antimiR-30 triggered the phenotypic ER stress with significant GRP78/ATF6/CHOP/caspase-12 upregulations and cell death, while miR-30 replacement mitigated ER stress. Knockdown of CHOP by siRNA regulated all members of the miR-30 family whereas sequestration of C/EBP transcription factor by its decoy downregulated miR-30 miRNAs.
Collectively, downregulation of the miR-30 family miRNAs contributes to the ER stress and the associated upregulation of GRP78 in the cardiovascular system. The participation of miR-30 creates a positive feedback loop in the ER stress signaling pathway. MiR-30 replacement may be a viable approach for alleviating disorders associated with ER stress.
内质网（ER）应激是细胞对各种应激的常见亚细胞反应，而葡萄糖调节蛋白78（GRP78）表达增加是内质网应激的核心。然而，GRP78上调的机制仍知之甚少。我们的研究目标是阐明这一问题。
使用过氧化氢（H2O2）建立新生大鼠心室细胞（NRVCs）和大鼠主动脉血管平滑肌细胞（RAVSMCs）的内质网应激细胞模型。采用分子生物学技术定量蛋白质和mRNA水平。利用荧光素酶报告基因检测法研究miRNA靶向作用。采用MTT法和ELISA法检测细胞死亡情况。
在H2O2诱导的心血管细胞NRVCs和RAVSMCs内质网应激中，属于miR-30家族的miRNAs，包括miR-30a、b、c、d和e均下调，同时GRP78、裂解的ATF6、CHOP和裂解的caspase-12上调。GRP78被证实是miR-30的靶基因。用抗miR-30人工敲低miR-30会引发内质网应激表型，导致GRP78/ATF6/CHOP/caspase-12显著上调和细胞死亡，而miR-30替代则减轻内质网应激。用siRNA敲低CHOP可调节miR-30家族的所有成员，而用其诱饵封闭C/EBP转录因子则下调miR-30 miRNAs。
总体而言，miR-30家族miRNAs的下调导致心血管系统中的内质网应激以及相关的GRP78上调。miR-30的参与在内质网应激信号通路中形成了一个正反馈环。miR-30替代可能是缓解与内质网应激相关疾病的一种可行方法。
