OBJECTIVE: To observe the effect of hypoxia on autophagy in Beclin-1-knockdown SH-SY5Y cells by constructing a stable transfected SH-SY5Y cell lines of silencing Beclin-1 gene. METHODS: Beclin-1shRNA lentiviral vector and negative control lentiviral vector were constructed; the vector was transfected into SH-SY5Y cells; then the expression of Beclin-1 mRNA was detected by RT-PCR, the level of Beclin-1 protein was detected by Western blot. CCK-8 method was used to determine the effect of Beclin-1 knockdown on the viability of SH-SY5Y cells. Next, the blank control, negative control and transfected cells were cultured under 21% normoxia and 5% hypoxia conditions. The expression of LC3 protein in each group was detected by Western blot and the autophagic bodies were observed by electron microscopy. RESULTS: Beclin-1 shRNA significantly inhibited the expression of Beclin-1 mRNA and protein in SH-SY5Y cells; after silencing Beclin 1 gene, the survival rate of Beclin-1 shRNA group cells was no different from that of negative control (NC) group. After 5% hypoxia treatment, compared with NC group, the ratio of LC3Ⅱ/LC3Ⅰand the number of autophagy bodies were all decreased in Beclin-1 shRNA group. CONCLUSIONS: Beclin-1 knockdown SH-SY5Y cell lines and negative control cell lines were successfully established. Lentivirus-mediated Beclin-1 shRNA has no effect on the viability of SH-SY5Y cells, but can inhibit hypoxia-induced autophagy.
OBJECTIVE: To observe the effect of hypoxia on autophagy in Beclin-1-knockdown SH-SY5Y cells by constructing a stable transfected SH-SY5Y cell lines of silencing Beclin-1 gene. METHODS: Beclin-1shRNA lentiviral vector and negative control lentiviral vector were constructed; the vector was transfected into SH-SY5Y cells; then the expression of Beclin-1 mRNA was detected by RT-PCR, the level of Beclin-1 protein was detected by Western blot. CCK-8 method was used to determine the effect of Beclin-1 knockdown on the viability of SH-SY5Y cells. Next, the blank control, negative control and transfected cells were cultured under 21% normoxia and 5% hypoxia conditions. The expression of LC3 protein in each group was detected by Western blot and the autophagic bodies were observed by electron microscopy. RESULTS:Beclin-1 shRNA significantly inhibited the expression of Beclin-1 mRNA and protein in SH-SY5Y cells; after silencing Beclin 1 gene, the survival rate of Beclin-1 shRNA group cells was no different from that of negative control (NC) group. After 5% hypoxia treatment, compared with NC group, the ratio of LC3Ⅱ/LC3Ⅰand the number of autophagy bodies were all decreased in Beclin-1 shRNA group. CONCLUSIONS:Beclin-1 knockdown SH-SY5Y cell lines and negative control cell lines were successfully established. Lentivirus-mediated Beclin-1 shRNA has no effect on the viability of SH-SY5Y cells, but can inhibit hypoxia-induced autophagy.