Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (16): 2577-2582.doi: 10.3969/j.issn.2095-4344.0278

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DEPTOR gene silencing promotes β-cell insulin secretion

Qiu Hong, Lai Shu-chang, Pan Dao-yan, Wang Xiao, Shen Jie   

  1. the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China
  • Received:2018-02-15 Online:2018-06-08 Published:2018-06-08
  • Contact: Shen Jie, Chief physician, Professor, Doctoral supervisor, the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China
  • About author:Qiu Hong, Master candidate, the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81770878; the Major Project of Guangzhou Science Technology and Innovation Commission, No. 201604020007

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.

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程

Key words: Insulin-Secreting Cells, Insulinoma, Gene Silencing

CLC Number: