microRNA-802 is involved in palmitate-induced damage to pancreatic β cells through repression of sirtuin 6

Abstract

Free fatty acid (FFA)-induced pancreatic β-cell loss is implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). It has been documented that circulating microRNA (miR)-802 levels are significantly greater in T2DM patients than in healthy subjects. However, the role of miR-802 in FFA-induced damage to β cells is still unclear. In the present study, we measured the expression of miR-802 in the INS-1 rat insulinoma cell line after palmitate treatment for 48 h. Gain- and loss-of-function studies were conducted to determine the function of miR-802 in palmitate-induced apoptosis and reactive oxygen species (ROS) production. The target gene(s) of miR-802 was functionally characterized. Compared to control cells, palmitate treatment caused a time- and concentration-dependent induction of miR-802 in INS-1 cells. Knockdown of miR-802 significantly blocked palmitate-induced apoptosis and attenuated ROS formation. Moreover, miR-802 downregulation prevented the reduction of prosurvival proteins Mcl-1 and Bcl-xL by palmitate. In contrast, ectopic expression of miR-802 stimulated apoptosis and ROS generation in INS-1 cells. Sirtuin 6 (SIRT6) was identified to be a direct target gene of miR-802. Overexpression of miR-802 suppressed the expression of SIRT6. Enforced expression of SIRT6 abolished the induction of apoptosis and ROS production by miR-802. Taken together, miR-802 is required for palmitate-induced damage to β cells by targeting SIRT6 and represents a potential therapeutic target for T2DM.

Keywords: Diabetes; SIRT6; free fatty acid; miR-802.

Figure 1

Upregulation of miR-802 in INS-1 cells by palmitate. A. INS-1 cells were treated with indicated concentrations of palmitate for 24 h, and miR-802 levels were measured by real-time PCR analysis. B. INS-1 cells were treated with palmitate (1.0 mM) for indicated times and measured for miR-802 expression. *P < 0.05 vs. untreated control cells.

Figure 2

miR-802 overexpression causes oxidative damage to INS-1 cells. A. Cell viability assay. INS-1 cells were transfected with control miR or miR-802 mimic and tested for viability after 48 h. *P < 0.05 vs. nontransfected control cells. B. Apoptosis detected by annexin-V/PI staining. Representative dot plots of flow cytometry are shown. Numbers inserted indicate the results from three independent experiments. C. Measurement of ROS levels using the cell permeant reagent 2’,7’-dichlorofluorescein diacetate. *P < 0.05 vs. nontransfected control cells. D. INS-1 cells were pretreated with NAC or DMSO (as vehicle control) before transfection with miR-802 mimic and tested for apoptosis. *P < 0.05.

Figure 3

Depletion of miR-802 attenuates palmitate- induced apoptosis and oxidative stress. INS-1 cells were transfected with anti-miR-802 or control (Anti-miR) and treated with palmitate (1.0 mM) for 30 h. A. Apoptosis analysis by flow cytometry after annexin-V/PI staining. *P < 0.05. B. Measurement of ROS production in INS-1 cells. *P < 0.05. C. Western blot analysis of Mcl-1 and Bcl-xL protein levels. Numbers indicate fold change in protein levels relative to control.

Figure 4

SIRT6 serves as a direct target of miR-802. A. Bioinformatic analysis using miRDB software (http://mirdb.org/) showed that the 3’-UTR of SIRT6 mRNA carried a putative binding site for miR-802. Mutation of the binding site (underlined) was achieved with the QuikChange site-direct mutagenesis kit. B. Western blot analysis of SIRT6 protein levels in INS-1 cells transfected with control miR or miR-802 mimic. Numbers indicate fold change in protein levels relative to control. C. Luciferase reporter assay. HEK-293T cells were transfected with wild-type or mutant SIRT6 3’-UTR reporters (0.1 μg) together with pRL-TK (0.02 μg) as well as miR-802 mimic or control miR (50 nM). Luciferase activities were measured 48 h posttransfection. *P < 0.05.

Figure 5

Enforced expression of SIRT6 rescues INS-1 cells from miR-802-induced apoptosis. (A) Western blot analysis of SIRT6 protein levels in INS-1 cells transfected with indicated constructs. Numbers indicate fold change in SIRT6 protein levels. (B) INS-1 cells transfected with indicated constructs and tested for apoptosis 48 h after transfection. (C) Measurement of ROS production in INS-1 cells treated as in (A). *P < 0.05.