Downregulation of the miR-30 family microRNAs contributes to endoplasmic reticulum stress in cardiac muscle and vascular smooth muscle cells

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Cardiac cell; ER stress; GRP78; Oxidative stress; Vascular smooth muscle cells; miR-30a.