Knockdown of PVT1 Exerts Neuroprotective Effects against Ischemic Stroke Injury through Regulation of miR-214/Gpx1 Axis

Abstract

Previous studies have reported that lncRNA PVT1 was closely related to ischemic stroke. Here, the role of PVT1 in ischemic stroke and the underlying mechanism were investigated. OGDR-stimulated PC12 cells were used to construct a cell model to mimic ischemic stroke. si-PVT1, miR-214 mimic, inhibitor, or the negative controls were transfected into PC12 cells prior to OGDR treatment. PVT1, miR-214, and Gpx1 expression was measured by qRT-PCR and western blotting assays. Cell proliferation and apoptosis were tested by CCK-8 assay and western blotting. The expression levels of inflammatory factors were determined by ELISA Kit. Results showed that PVT1 was increased significantly in OGDR PC12 cells. PVT1 knockdown significantly enhanced cell viability and attenuated cell apoptosis, ROS generation, and inflammation in OGDR PC12 cells. More importantly, PVT1 or Gpx1 was a target of miR-214. Mechanistically, PVT1 acted as a competing endogenous RNA of miR-214 to regulate the downstream gene Gpx1. In conclusion, PVT1 knockdown attenuated OGDR PC12 cell injury by modulating miR-214/Gpx1 axis. These findings offer a potential novel strategy for ischemic stroke therapy.

Figure 1

PVT1 was overexpressed in OGDR PC12 cells. (a) qRT-PCR analysis of PVT1 and (b) miR-214 mRNA level in OGDR PC12 cells. (c) Western blotting analysis of Gpx1 level in OGDR PC12 cells. ^∗∗p < 0.01. Data were shown as the mean ± SD based on three independent experiments.

Figure 2

PVT1 knockdown attenuated ischemic injury through miR-214/Gpx1 in OGDR PC12 cells. (a) PVT1 expression was measured by qRT-PCR in si-PVT1 PC12 cells. (b) CCK-8 analysis of si-PVT1 effect on PC12 cell viability. (c) qRT-PCR analysis of si-PVT1 role on PCNA expression. (d) Western blotting analysis of si-PVT1 effect on Bcl-2, Bax, and cleaved-caspase-3 levels in PC12 cells. (e) ELISA assay analysis of the effect of si-PVT1 on the productions of ROS and (f) IL-6, IL-1β, and TNF-α in OGDR PC12 cells. ^∗∗p < 0.01 and^∗∗∗p < 0.001; ^#p < 0.05 and^##p < 0.01. Data were shown as the mean ± SD based on three independent experiments.

Figure 3

PVT1 functions as a ceRNA of miR-214 in PC12 cells. (a) The binding sites of PVT1 and miR-214, as shown by Starbase. (b) Detection of miR-214 expression in PC12 cells. (c) Measurement of the luciferase activity of PVT1. (d) Measurement of PVT1 or miR-214 expression in Ago2, IgG, and Input groups. (e) Detection of PVT1 expression by qRT-PCR. (f) Observation of PVT1 effect on miR-214 expression by qRT-PCR. ^∗∗p < 0.01 and^∗∗∗p < 0.001. Data were shown as the mean ± SD based on three independent experiments.

Figure 4

miR-214 directly targeted Gpx1 in PC12 cells. (a) The binding sites of Gpx1 and miR-214, as shown by Starbase. (b) Measurement of the luciferase activity of Gpx1. (c) qRT-PCR analysis of Gpx1 mRNA expression. (d) Detection of Gpx1 protein level by western blotting. ^∗∗p < 0.01. Data were shown as the mean ± SD based on three independent experiments.

Figure 5

PVT1 knockdown attenuated ischemic injury through miR-214/Gpx1 in PC12 cells. PC12 OGDR cells were treated with si-PVT1, combined with miR-214 inhibitor or pcDNA-Gpx1. (a) Measurement of the mRNA expression or (b) protein level of Gpx1. (c) CCK-8 analysis of the cell viability. (d) QRT-PCR analysis of PCNA expression. (e) Western blotting analysis of Bcl-2, Bax, and cleaved-caspase-3 levels. (f) ELISA assay analysis of the levels of ROS and (g) IL-6, IL-1β, and TNF-α, respectively. ^∗∗p < 0.01;  ^#p < 0.05;  ^&p < 0.05. Data were shown as the mean ± SD based on three independent experiments.