J-H Zhao1, B Wang, X-H Wang, C-W Xu. 1. Department of Neurology, First Affiliated Hospital of China Medical University, Shenyang, China. miller78127@163.com.
Abstract
OBJECTIVE: To observe the effect of long non-coding ribonucleic acid (lncRNA) growth arrest specific 5 (GAS5) knockdown on the apoptosis of neurons in rats with cerebral infarction (CI), and to explore the potential mechanism of lncRNA GAS5 in the pathogenesis of CI. MATERIALS AND METHODS: A total of 60 adult male Sprague Dawley (SD) rats aged 12-14 weeks old and weighing (267.14±6.49) g were randomly divided into three groups: Sham operation group (Sham group, n=20), CI group (n=20) and CI + lncRNA GAS5 knockdown group [CI + GAS5 small interfering RNA (siRNA) group, n=20]. The rat model of focal CI was constructed by carotid artery embolization. After the CI model was successfully induced, a certain amount of lncRNA GAS5 siRNAs was injected into the rat lateral ventricle in a stereotactic manner. At 24 h after operation, triphenyl tetrazolium chloride (TTC) method was used to detect the infarction area in brain tissues of rats in each group. At the same time, the pathological changes of neurons in the hippocampus and prefrontal cortex of rats in each group were observed via hematoxylin and eosin (H&E) staining. The expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX) were detected via Western blotting. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was adopted to detect the number of apoptotic neurons in brain tissues of rats in each group. Meanwhile, the expression level of Notch intracellular domain (NICD) proteins was measured using the Western blotting technique and immunohistochemical staining. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed that the lncRNA GAS5 expression in brain tissues of rats in CI group was significantly higher than that of rats in Sham group (p<0.05). TTC staining results revealed that lncRNA GAS5 knockdown could remarkably reduce the CI area of rats in CI group (p<0.05). In addition, inhibiting lncRNA GAS5 could also significantly reduce the level of pro-apoptotic gene BAX and increase the expression level of anti-apoptotic gene Bcl-2 (p<0.05). In the meantime, the number of apoptotic neurons in CI + GAS5 siRNA group was also evidently decreased (p<0.05). Finally, it was found that lncRNA GAS5 knockdown notably inhibited the expression of NICD proteins (p<0.05). CONCLUSIONS: The inhibitory effect of lncRNA GAS5 knockdown on the apoptosis of neurons in CI rats may be related to the activation of the Notch1 signaling pathway. LncRNA GAS5 may be a new target for clinical treatment of CI.
OBJECTIVE: To observe the effect of long non-coding ribonucleic acid (lncRNA) growth arrest specific 5 (GAS5) knockdown on the apoptosis of neurons in rats with cerebral infarction (CI), and to explore the potential mechanism of lncRNA GAS5 in the pathogenesis of CI. MATERIALS AND METHODS: A total of 60 adult male Sprague Dawley (SD) rats aged 12-14 weeks old and weighing (267.14±6.49) g were randomly divided into three groups: Sham operation group (Sham group, n=20), CI group (n=20) and CI + lncRNA GAS5 knockdown group [CI + GAS5 small interfering RNA (siRNA) group, n=20]. The rat model of focal CI was constructed by carotid artery embolization. After the CI model was successfully induced, a certain amount of lncRNA GAS5 siRNAs was injected into the rat lateral ventricle in a stereotactic manner. At 24 h after operation, triphenyl tetrazolium chloride (TTC) method was used to detect the infarction area in brain tissues of rats in each group. At the same time, the pathological changes of neurons in the hippocampus and prefrontal cortex of rats in each group were observed via hematoxylin and eosin (H&E) staining. The expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX) were detected via Western blotting. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was adopted to detect the number of apoptotic neurons in brain tissues of rats in each group. Meanwhile, the expression level of Notch intracellular domain (NICD) proteins was measured using the Western blotting technique and immunohistochemical staining. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed that the lncRNA GAS5 expression in brain tissues of rats in CI group was significantly higher than that of rats in Sham group (p<0.05). TTC staining results revealed that lncRNA GAS5 knockdown could remarkably reduce the CI area of rats in CI group (p<0.05). In addition, inhibiting lncRNA GAS5 could also significantly reduce the level of pro-apoptotic gene BAX and increase the expression level of anti-apoptotic gene Bcl-2 (p<0.05). In the meantime, the number of apoptotic neurons in CI + GAS5 siRNA group was also evidently decreased (p<0.05). Finally, it was found that lncRNA GAS5 knockdown notably inhibited the expression of NICD proteins (p<0.05). CONCLUSIONS: The inhibitory effect of lncRNA GAS5 knockdown on the apoptosis of neurons in CIrats may be related to the activation of the Notch1 signaling pathway. LncRNA GAS5 may be a new target for clinical treatment of CI.