microRNA-367-3p regulation of GPRC5A is suppressed in ischemic stroke

Abstract

Ischemic stroke is a major cause of mortality and long-term disability with limited treatment options, and a greater understanding of the gene regulatory mechanisms underlying ischemic stroke-associated neuroinflammation is required for new therapies. To study ischemic stroke in vivo, mice were subjected to sustained ischemia by intraluminal filament-induced middle cerebral artery occlusion (MCAo) for 24 h without reperfusion or transient ischemia for 30 min followed by 23.5 h reperfusion, and brain miRNA and mRNA expression changes were quantified by TaqMan OpenArrays and gene (mRNA) expression arrays, respectively. Sustained ischemia resulted in 18 significantly altered miRNAs and 392 altered mRNAs in mouse brains compared to Sham controls; however, the transient ischemic condition was found to impact only 6 miRNAs and 126 mRNAs. miR-367-3p was found to be significantly decreased in brain homogenates with sustained ischemia. G protein-coupled receptor, family C, group 5, member A (Gprc5a), a miR-367-3p target gene, was found to be significantly increased with sustained ischemia. In primary neurons, inhibition of endogenous miR-367-3p resulted in a significant increase in Gprc5a expression. Moreover, miR-367-3p was found to be co-expressed with GPRC5A in human neurons. Results suggest that loss of miR-367-3p suppression of GPRC5A may contribute to neuroinflammation associated with ischemic stroke.

Keywords: Microrna; brain; ischemia; neuroinflammation; neurons; stroke.

Figure 1.

Study design and mouse model of ischemic stroke. (a) Study design. (b) Diagram representing the mouse intraluminal filament-induced middle cerebral artery occlusion (MCAo) model. InoR: ischemia no reperfusion; IR: ischemia-reperfusion. (c) Regional cerebral blood flow (rCBF) recorded during and after 1 h of MCAo (n = 17–27). (d) Hanging wire tests (time). (n = 26–27). (e) Neurological deficit at 24 h (n = 26–27). Data presented as mean ± SD.

Figure 2.

Ischemic stroke alters brain miRNAs. (a–c) Volcano plots of significant differentially expressed miRNAs. Red, significantly increased; Blue, significantly decreased miRNAs (≥1.5-absolute fold change, p < 0.05; n = 5). InoR: ischemia no reperfusion; IR: ischemia-reperfusion. (a) InoR/Sham. (b) IR/Sham. (c) IR/InoR. (D) Real-time PCR quantification of candidate miRNAs (n = 7–8). One-way ANOVA Tukey’s multiple comparisons tests. Data presented as mean ± SD (*p < 0.05). Groups indicated by * without accompanied bar linking two groups indicates that the group with the * was compared to the sham control group.

Figure 3.

Ischemic stroke alter brain gene (mRNA) expression. (a) Principal Coordinate Analysis (PCoA) of mouse brain transcriptomes (n = 5). InoR: Ischemia no reperfusion (blue); IR: ischemia-reperfusion (red); and sham control (green). NMDS: Non-metric multidimensional Scaling. (b, c) Volcano plots of significant differentially expressed mRNAs. Red, significantly increased; Blue, significantly decreased miRNAs (≥1.5- absolute fold change, Benjamini–Hochberg-corrected p < 0.05; n = 5). Presented as Log2 fold change vs. −Log10 corrected p-values. (b) InoR/Sham (n = 5). (c) IR/Sham (n = 5). (d) Venn diagram of overlapping gene (mRNA) expression changes in response to sustained (InoR) and transient (IR) ischemia in the brain.

Figure 4.

miRNA regulatory modules are significantly altered in ischemic stroke. (a, b) Circos plots linking significantly altered miRNAs and their predicted inversely altered mRNA targets. Purple, miRNAs increased and mRNA decreased. Blue, miRNAs decreased and mRNAs increased. Expression signal (fold change) represented by gray bars in inner Circos ring. InoR: Ischemia no Reperfusion; IR: ischemia-reperfusion; and Sham control. (a) InoR/Sham (n = 5). (B) IR/Sham (n = 5). (C) Validation of candidate gene (mRNA) expression by real-time PCR for Tnf, Ccl3, Atf3, Cxcl2, Cxcl1 Socs3, Ccrl2, Gprc5a, Zfp36, Ptgs2, Fosb, Jun, Bcl2, Rarres2, Serpinb2, and Fos (n = 8). One-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD (*p < 0.05). Groups indicated by * without accompanied bar linking two groups indicates that the group with the * was compared to the sham control group.

Figure 5.

miR-367-3p regulates GPRC5A in neurons. (a) Conserved putative miR-367-3p target sites in the 3′ untranslated regions (3′ UTR) of mouse and human GPRC5A mRNAs. Black, putative seed region for miR-367-3p. (b, c) Real-time PCR quantification of (b) miR-367-3p (n = 6) and (c) GPRC5A mRNA (n = 6) in human brain cells: HCN-2 (cortical neurons), M059K (glial cells), CCF-STTG1 (astrocytes) and THP-1 (PMA-stimulated macrophages). RQV: relative quantitative value. (d, e) Real-time PCR quantification of (d) Gprc5a and (E) Tnf mRNA levels in mouse primary neurons transfected with locked-nucleic acid (LNA) miRNA inhibitors against scrambled control sequence (LNA-Scr) or miR-367-3p (LNA-367) (n = 9–11). Mann–Whitney non-parametric tests. Data are presented as mean ± SD (*p < 0.05).