DEPTOR gene silencing promotes β-cell insulin secretion
10.3969/j.issn.2095-4344.0278
- VernacularTitle:沉默DEPTOR表达促进胰岛细胞释放胰岛素
- Author:
Hong QIU
1
;
Shu-Chang LAI
;
Dao-Yan PAN
;
Xiao WANG
;
Jie SHEN
Author Information
1. 南方医科大学第三附属医院
- From:
Chinese Journal of Tissue Engineering Research
2018;22(16):2577-2582
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Mammalian target of rapamycin (mTOR) complexes are a key regulator of pancreatic beta cells mass and function. DEP-domain containing mTOR-interacting protein (DEPTOR) is a common part of mTOR complexes and whether DEPTOR loss in islet β cells affects insulin-secreting function has never been identified. OBJECTIVE: To assess the alternation of insulin secretion by silencing DEPTOR gene in pancreatic β cells NIT-1 and to explore the underlying mechanism. METHODS: Three siRNA sequences for silencing DEPTOR gene were designed and constructed, which were transfected with lipofectamine into NIT-1 cells. There were six groups: blank transfection group (NIT-1 cells plus Lipofectamin), negative control group (NC-FAM), positive control group (GAPDH), siRNA deptor 1 group (siRNA deptor385), siRNA deptor 2 group (siRNA deptor766), and siRNA deptor 3 group (siRNA deptor1275). The transfection efficiency was determined by fluorescence microscope. The relative expression level of DEPTOR mRNA was detected by quantitative-PCR. Insulin secretion in the cell conditioned medium was determined by insulin ELISA kit. The expression level of DEPTOR downstream key protein was detected by western blot assay. RESULTS AND CONCLUSION: Specific green fluorescence accumulated in a punctated pattern under fluorescence microscope, indicating that the effectiveness of transfection was eligible. Quantitative-PCR results showed two (siDEPTOR385 and siDEPTOR766) of the three siRNA sequences could significantly disrupt the expression of DEPTOR mRNA, which had significant difference with negative control group (P＜ 0.05). The ELISA results showed that the total amount of insulin secretion in the effective transfected groups was significantly increased (P＜ 0.05). Western blot assay results showed the grey levels of p-s6 and p-4EBP-1 proteins were significantly elevated, while p-AKT of those former was slightly decreased. These findings suggest that siRNA technology can effectively silence the DEPTOR gene in NIT-1 cells, which improves β-cell insulin secretion in a manner of mTORC1 activation.
