Nan fang yi ke da xue xue bao = Journal of Southern Medical University · 2021
[Effects of lentivirus-mediated RNA interference of HIF-1α and PTEN on oxygen-glucose deprivation injury in primary cultured rat neurons].
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Tang J, Yang Y, Chen J, Li T, Dai Y
Аннотация
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 (P < 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 (P < 0.05) without obviously affecting the expressions of HIF-1α, NR2A or VEGFa (P>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.
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 (P < 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 (P < 0.05) without obviously affecting the expressions of HIF-1α, NR2A or VEGFa (P>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.