Targeting upregulated RNA binding protein RCAN1.1: a promising strategy for neuroprotection in acute ischemic stroke

Abstract

Aims: To explore the expression changes and roles of the RNA-binding protein RCAN1.1 in acute ischemic stroke (AIS), and to preliminarily confirm the medicinal value of the RNA aptamer R1SR13 in AIS by targeting RCAN1.1.

Methods: Two mouse AIS models of middle cerebral artery occlusion (MCAO) and right common carotid artery ligation (R-CCAL) and oxygen glucose deprivation (OGD) model of AIS in primary neurons and SH-SY5Y were performed. The expression pattern of RCAN1.1 was assessed using real-time quantitative PCR (RT-qPCR) and western blotting (WB) in vivo and in vitro. The underlying mechanism for the elevation of RCAN1.1 in the upstream was investigated. Lentiviruses were administrated and the effect of RCAN1.1 in AIS was assessed by ATP level, caspase 3/7 assay, TUNEL and WB. The protective function of R1SR13 in AIS was evaluated both in vivo and in vitro.

Results: In two mouse models of AIS, RCAN1.1 mRNA and RCAN1.1 L protein were significantly upregulated in the ischemic brain tissue. The same results were detected in the OGD model of primary neurons and SH-SY5Y. The mechanistic analysis proved that hypoxia-inducible factor-1α (HIF1α) could specifically activate the RCAN1.1 gene promoter through combining with the functional hypoxia-responsive element (HRE) site (-325 to -322 bp). The increased expression of RCAN1.1 L markedly depleted ATP production and aggravated neuronal apoptosis under OGD condition. R1SR13, an antagonizing RNA aptamer of RCAN1.1, was demonstrated to reduce neuronal apoptosis caused by the elevated RCAN1.1 L in the cellular and animal models of AIS.

Conclusion: RCAN1.1 is a novel target gene of HIF1α and the functional HRE in the RCAN1.1 promoter region is -325 to -322 bp. The marked upregulation of RCAN1.1 in AIS promoted neuronal apoptosis, an effect that could be reversed by its RNA aptamer R1SR13 in vivo and in vitro. Thus, R1SR13 represents a promising strategy for neuroprotection in AIS and our study lays a theoretical foundation for it to become a clinically targeted drug.

Keywords: HIF1α; R1SR13; RBP; RCAN1; acute ischemic stroke; apoptosis.