Yes-associated protein (Yap), a regulator of cellular apoptosis, has been demonstrated to be involved in cerebral ischemia-reperfusion (IR) injury through poorly defined mechanisms. The present study aimed to explore the role of Yap in regulating cerebral IR injury in vitro, with a focus on mitochondrial fission and ROCK1/F-actin pathways. Our data demonstrated that Yap was actually downregulated in N2a cells after cerebral hypoxia-reoxygenation (HR) injury, and that lower expression of Yap was closely associated with increased cell death. However, the reintroduction of Yap was able to suppress the HR-mediated N2a cells death via blocking the mitochondria-related apoptotic signal. At the molecular levels, Yap overexpression sustained mitochondrial potential, normalized the mitochondrial respiratory function, reduced ROS overproduction, limited HtrA2/Omi release from mitochondria into the nucleus, and suppressed pro-apoptotic proteins activation. Subsequently, functional studies have further illustrated that HR-mediated mitochondrial apoptosis was highly regulated by mitochondrial fission, whereas Yap overexpression was able to attenuate HR-mediated mitochondrial fission and, thus, promote N2a cell survival in the context of HR injury. At last, we demonstrated that Yap handled mitochondrial fission via closing ROCK1/F-actin signaling pathways. Activation of ROCK1/F-actin pathways abrogated the protective role of Yap overexpression on mitochondrial homeostasis and N2a cell survival in the setting of HR injury. Altogether, our data identified Yap as the endogenous defender to relieve HR-mediated nerve damage via antagonizing ROCK1/F-actin/mitochondrial fission pathways.

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In the present study, the aim was to investigate the function of microRNA-323 (miR-323) in cerebral infarction and its underlying mechanism. A rat model of cerebral infarction was established and hippocampal tissues were analyzed. In addition, to further understand the role of miR-323, PC12 cells were transfected with miR-323 mimics or inhibitors and subjected to hypoxia to model cerebral infarction. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of miR-323. A luciferase reporter assay was conducted to analyze miR-323 target sites the partial sequence of the 3'-untranslated region of SMAD3 mRNA in vitro. Western blot analysis was also used to analyze transforming growth factor-β1 (TGF-β1) and SMAD3 protein expression levels. It was observed that miR-323 expression was significantly upregulated in rats with cerebral infarction compared with rats in the sham-control group. In addition, overexpression of miR-323 induced nerve cell toxicity and reduced nerve cell growth in an in vitro model of cerebral infarction, whereas downregulation of miR-323 caused the opposite effects on nerve cell toxicity and growth in this model. In addition, overexpression of miR-323 directly targeted and suppressed SMAD3 expression in the in vitro model of cerebral infarction, while inhibition of miR-323 induced SMAD3 expression. The use of a SMAD3 inhibitor suppressed the effect of anti-miR-323 on nerve cell toxicity in the in vitro model of cerebral infarction. Collectively, these findings suggested that miR-323 suppresses nerve cell apoptosis in cerebral infarction via the TGF-β1/SMAD3 signaling pathway.