[慢病毒介导的HIF-1α和PTEN基因RNA干扰对原代培养大鼠神经元氧糖剥夺损伤的影响]
[Effects of lentivirus-mediated RNA interference of HIF-1α and PTEN on oxygen-glucose deprivation injury in primary cultured rat neurons].
作者信息
Tang J, Yang Y, Chen J, Li T, Dai Y
机构信息
Department of Child Health Care, Children's Hospital of Chongqing Medical University//National Clinical Research Center for Child Health and Disorders//Ministry of Education Key Laboratory of Child Development and Disorders//Chongqing Key Laboratory of Child Health and Nutrition, Chongqing 40014, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Dec 20;41(12):1795-1800. doi: 10.12122/j.issn.1673-4254.2021.12.06.
OBJECTIVE
To observe the effects of lentivirus-mediated RNA interference (RNAi) of hypoxia-inducible factor 1α (HIF-1α) and phosphatase and tensin homolog on chromosome ten (PTEN) on oxygen-glucose deprivation (OGD) injury in primary cultured rat neurons.
METHODS
Primary cultures of neonatal SD rat neurons were infected by lentiviral vectors carrying short hairpin RNA (shRNA) targeting HIF-1α or PTEN followed 4 days later by hypoxic exposure, and the control neurons were infected with the empty virus only with or without subsequent hypoxic exposure. Twenty-four hours after hypoxia, the interference efficiency was assessed with qRT-PCR, and lactate dehydrogenase (LDH) assay and AnnexinV-FITC/ PI assay were performed to detect neuronal damage and apoptosis. The expressions of the related proteins were determined with Western blotting.
RESULTS
Lentivirus-mediated RNAi effectively silenced the mRNA expression of the target genes. HIF-1α silencing obviously aggravated the hypoxia-induced damage and apoptosis of the neurons, enhanced the expression of PTEN protein and significantly lowered the expressions of p-PTEN, p-AKT, NR2A and VEGFa ( < 0.05). PTEN silencing significantly alleviated hypoxia-induced damage and apoptosis of the neurons and increased the cellular expressions of p-PTEN and p-AKT ( < 0.05) without obviously affecting the expressions of HIF-1α, NR2A or VEGFa (>0.05).
CONCLUSION
An up-regulated expression of HIF-1α causes down-regulation of PTEN expression to protect primary cultured rat neurons against OGD injury.
目的
观察慢病毒介导的缺氧诱导因子1α（HIF-1α）和第10号染色体上的磷酸酶和张力蛋白同源物（PTEN）的RNA干扰（RNAi）对原代培养大鼠神经元氧糖剥夺（OGD）损伤的影响。
方法
用携带靶向HIF-1α或PTEN的短发夹RNA（shRNA）的慢病毒载体感染新生SD大鼠神经元原代培养物，4天后进行缺氧暴露，对照神经元仅感染空病毒，有无后续缺氧暴露均可。缺氧24小时后，用qRT-PCR评估干扰效率，进行乳酸脱氢酶（LDH）测定和AnnexinV-FITC/PI测定以检测神经元损伤和凋亡。用蛋白质印迹法测定相关蛋白的表达。
结果
慢病毒介导的RNAi有效沉默了靶基因的mRNA表达。沉默HIF-1α明显加重了缺氧诱导的神经元损伤和凋亡，增强了PTEN蛋白的表达，并显著降低了p-PTEN、p-AKT、NR2A和VEGFa的表达（<0.05）。沉默PTEN显著减轻了缺氧诱导的神经元损伤和凋亡，并增加了细胞中p-PTEN和p-AKT的表达（<0.05），而对HIF-1α、NR2A或VEGFa的表达无明显影响（>0.05）。
结论
HIF-1α表达上调导致PTEN表达下调，从而保护原代培养的大鼠神经元免受OGD损伤。
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