Effect of Beclin-1 shRNA on hypoxia-induced autophagy in SH-SY5Y cells.
10.12047/j.cjap.5661.2018.105
- Author:
Na LU
1
;
Cheng-Zhang LI
1
;
Xiao-Qiu LUO
1
;
Kun-Li YANG
1
Author Information
1. Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, China.
- Publication Type:Journal Article
- Keywords:
Beclin-1;
SH-SY5Y;
autophagy;
shRNA
- MeSH:
Apoptosis;
Autophagy;
Beclin-1;
Cell Hypoxia;
Cell Line, Tumor;
Humans;
RNA, Small Interfering
- From:
Chinese Journal of Applied Physiology
2018;34(5):464-469
- CountryChina
- Language:Chinese
-
Abstract:
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.
