MiR-122 Participates in Oxidative Stress and Apoptosis in STZ-Induced Pancreatic β Cells by Regulating PI3K/AKT Signaling Pathway

At present, there are few reports concerning the relationship between miR-122 and diabetes. In addition, the effect of miR-122 on streptozotocin- (STZ-) induced oxidative damage in INS-1 cells remains unclear. The present study aimed to investigate the role and modulatory mechanisms involving miR-122 in diabetes. STZ was used to induce INS-1 cell damage. Reverse transcription-quantitative PCR was used to investigate the expression of miR-122. A TUNEL cell apoptosis detection kit was used to detect apoptosis. Intracellular ROS levels were determined using dichlorofluorescein-diacetate. The activities of insulin secretion, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) were measured using ELISA kits. Western blotting was used to measure the expression levels of Bax, Bcl-2, PI3K, p-PI3K, caspase-3 and caspase-9, cleaved-caspase-3 and cleaved-caspase-9, AKT, and p-AKT. Then, LY294002 (LY, PI3K inhibitor) was used to treat INS-1 cells, and oxidative stress and apoptosis were measured. The results showed that STZ-induced inhibitory effects on insulin secretion were mitigated by miR-122 inhibitor, and the activities of SOD, CAT, and GSH-px were also increased. Furthermore, miR-122 inhibitor inhibited apoptosis and oxidative stress in STZ-induced INS-1 cells. Finally, the addition of LY increased insulin levels; reduced the activities of SOD, CAT, and GSH-px; and promoted apoptosis in STZ-induced INS-1 cells. In conclusion, interference with miR-122 can inhibit oxidative stress and apoptosis in STZ-induced INS-1 cells, involving a mechanism of action related to the PI3K/AKT pathway.


Introduction
MicroRNAs (miRNAs) are a type of noncoding singlestranded RNA comprising small molecules 18-25 nucleotides in length [1] that mainly promote the degradation of mRNA or inhibit target messenger RNAs (mRNAs) through complementary base pairing with downstream target gene mRNA [2]. miRNAs are of diverse types and have complex functions that are widely involved in regulating the proliferation and differentiation of various cells [3], and dysregulation of expression can promote or inhibit tumor development. miR-122 is a type of miRNA with tumor suppressor properties [4]. It has been confirmed that miR-122 exhibits low expression in liver cancer [5] and breast malignancies [6], whereas there are a few studies concerning relationships involving miR-122 and pancreatic β cells.
Diabetes mellitus (DM) is a chronic metabolic disease that currently affects > 400 million people worldwide [7]. By 2030, the number of diabetics is expected to surge to 439 million [8]. Type 2 diabetes mellitus (T2DM) accounts for 90% of all diabetes cases. e International Diabetes Federation announced that 336 million people worldwide currently suffer from T2DM and that the disease is responsible for 4.6 million deaths each year, or one death every seven seconds [9]. Pancreatic β-cells are responsible for insulin secretion and control of plasma glucose levels [10].
ere is increasing evidence of a correlation between cell dysfunction/death and the onset of diabetes [11][12][13]. In the prediabetes stage of T2DM, pancreatic β-cell function is already diminished [14]. As the course of the disease progresses, the long-term high-glucose environment further damages the β-cells, leading to a gradual decline in insulin secretion and cellular apoptosis [15]. Improving the function of pancreatic β-cells and inhibiting β-cell apoptosis may be important targets to inhibit the occurrence and development of T2DM.

Dichlorofluorescein Staining to Determine Cell ROS Levels.
INS-1 cells were seeded at a density of 2 × 10 5 cells/well in a 6-well plate. e control group is divided into Group Control (normal INS-1 cells), Group STZ (cells treated with STZ), Group STZ+inhibitor-NC (cells treated with STZ and inhibitor-NC), and Group STZ+miR-122 inhibitor (cells treated with STZ and miR-122 inhibitor). After 24 h, the kit (Reactive Oxygen Species Assay Kit, Shanghai Yisheng Bio-Technology Co., Ltd. China) was used according to the instructions. e culture medium was discarded and cells were washed once with PBS (Beyotime Institute of Biotechnology). Dichlorofluorescein-diacetate was diluted in a serum-free culture medium (Gibco; ermo Fisher Scientific) at a ratio of 1 : 1000 to a final concentration of 10 μM. Cells were added to a 6-well plate (1000 μL/well) and incubated with cells for 30 min (37°C, 5% CO 2 ). e culture medium was discarded and cells were washed with serumfree culture medium 3 times. Observed images (×400) were acquired under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling
Assay. INS-1 cells were seeded at a density of 2 × 10 5 cells/well in a 6-well plate. e cells were divided into the following groups: control group (normal INS-1 cells), STZ (cells treated with STZ), STZ + inhibitor-NC (cells treated with STZ and inhibitor-NC), STZ + miR-122 inhibitor (cells treated with STZ and miR-122 inhibitor), and STZ + LY (a PI3K inhibitor, Beyotime Institute of Biotechnology) + miR-122 inhibitor (cells treated with STZ, LY294002, and miR-122 inhibitor). After 24 h, the culture medium was discarded, and cells were washed once with PBS (Beyotime Institute of Biotechnology). Cells were fixed with immunostaining fixative (Beyotime Institute of Biotechnology) at 25°C for 30 min and washed with PBS once. After adding the immunostaining permeabilization solution, incubation was conducted for 5 min at room temperature, and the cells were washed twice with PBS. TUNEL detection solution was added to cells and incubated for 60 min at 37°C in the dark. Cells were washed with PBS 3 times and nuclei counterstained with DAPI (Beyotime). e stained slides were observed under a light microscope (Olympus Corporation, ×100).

Statistical
Analysis. Data are presented as mean-± standard deviation (SD) and were analyzed using SPSS v. 19.0 and GraphPad Prism 6. Comparisons between groups were performed using Student's t-test. For multiple comparisons, one-way ANOVA followed by Tukey's post hoc test was carried out to analyze differences. P < 0.05 indicated statistically significant differences.

miR-122 Participates in Oxidative Stress in STZ-Induced
Pancreatic β Cells. To study whether miR-122 plays a role in STZ-induced oxidative stress in pancreatic β cells, the expression levels of miR-122 at 12 and 24 h were detected by RT-qPCR. e results are shown in Figure 1(a). Compared with the control group, the expression of miR-122 increased with time. Compared with the control group, insulin levels in the model group were downregulated (1-C), and ROS levels were upregulated (1-D, E). SOD, CAT, and CAH-px activities were downregulated (1-F), indicating that STZinduced oxidative stress in pancreatic β cells. After adding miR-122 inhibitor (1-B), compared with the control group, the insulin content increased; ROS levels decreased; and SOD, CAT, and CAH-px activities increased, indicating that inhibition of miR-122 can reduce STZ-induced INS-1 cell oxidative stress.

Inhibition of miR-122 Decreases Cell Apoptosis.
In the control group, there was less green fluorescence (Figure 2(a)), high Bcl-2 expression, low Bax, and C-caspase-3, and C-caspase-9 expression, indicating less apoptosis in the control group, while the model group exhibited higher green fluorescence and low Bcl-2 expression. e expression of Bax, and C-caspase-3, and C-caspase-9 was upregulated, indicating STZ-induced apoptosis in INS-1 cells (Figure 2(b)). After adding the miR-122 inhibitor, the green fluorescence was decreased, the expression of Bcl-2 was upregulated, and the expression of Bax, C-caspase-3, and C-caspase-9 was downregulated. is showed that miR-122 inhibitor can reduce cell apoptosis.

Interference with miR-122 Activates PI3K/AKT Signaling Pathway in Pancreatic β Cells Induced by STZ.
Western blotting was used to detect the expression levels of p-PI3K, PI3K, p-AKT, and AKT (Figure 3(a)). e expression of p-PI3K and p-AKT in the control group was upregulated, and the expression of p-PI3K and p-AKT in the model group was downregulated. Compared with the model group, the expression of p-PI3K and p-AKT was upregulated after adding miR-122 inhibitor, indicating that miR-122 is related to the PI3K/AKT signaling pathway.

e Expression Level of PI3K/AKT Protein Was
Downregulated after LY294002 Treatment. Western blotting was used to detect the expression levels of PI3K/AKT signaling pathway-related proteins after adding LY (Figure 3). Compared with the control group, the expression of p-PI3K and p-AKT was higher in the miR-122 inhibitor group. Compared with the miR-122 inhibitor group, the expression was downregulated after the addition of LY294002 (LY), indicating that interference with the PI3K/AKT signaling pathway was successful.

Cell Oxidative Stress Levels Increase after Adding LY294002.
e control group showed less green fluorescence, and the model group showed more green fluorescence. Green fluorescence increased after adding STZ, but instead decreased upon miR-122 inhibitor treatment, and again enhanced in the presence of LY294002 cotreatment. ROS levels are shown in Figure 3(d), which indicated that inhibiting the PI3K/AKT signaling pathway increased ROS abundance. In Figure 3(f ), compared with the miR-122 inhibitor group, the activities of SOD, CAT, and GAH-px decreased after adding LY, indicating that inhibiting the PI3K/AKT signaling pathway could enhance cellular oxidative stress.    e control group exhibited less green fluorescence, and the model group showed more green fluorescence (Figure 4(a)). STZ treatment obviously increased Tunel-positive cells, which decreased by miR-122 inhibitor and enhanced by LY294002, indicating that inhibiting the PI3K/AKT signaling pathway could increase cell apoptosis. Compared with the miR-122 inhibitor group, after adding LY, the expression of Bcl-2 was   International Journal of Endocrinology downregulated, and the expression of Bax, C-caspase-3, and C-caspase-9 was upregulated (Figure 4(b)), indicating that the inhibition of the PI3K/AKT signaling pathway increased INS-1 cell apoptosis.

Discussion
Diabetes is an endocrine and metabolic disease characterized by high blood glucose levels [16]. At present, it is believed that the pathogenesis of diabetes is mainly caused by the apoptosis and dysfunction of β cells, which leads to decreased insulin secretion [17]. Expression levels of the apoptosis-related proteins Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, procaspase-3, and procaspase-9 were analyzed using western blotting. * P < 0.05, * * P < 0.01, * * * P < 0.001 versus control group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus STZ group. e experiment was repeated three times. which leads to body damage [18]. ROS is a metabolite of redox reactions in the body, which has the effect of scavenging free radicals [19]. When the body is in pathological states, such as diabetes, the functions of mitochondria are diminished, and the structural integrity of the cell membrane is abolished, with a large amount of ROS produced [20].
As shown in Figure 1, the control group showed less green fluorescence, indicating a low ROS content, while the STZ-induced model group showed more green fluorescence, indicating higher ROS levels. ROS content in the miR-122 inhibitor group decreased, indicating that miR-122 could inhibit ROS levels. SOD, GSH-P x, and CAT are antioxidant enzymes. When their content in the body is reduced, the free radical clearance rate is reduced, which leads to oxidative stress [21]. As shown in Figure 1, the levels of SOD, CAT, and GSH-Px were higher in the control group, while the contents in the model group were significantly reduced. Increased activity was identified in the STZ + miR-122 inhibitor group, indicating that miR-122 could enhance the activity of SOD, CAT, and GSH-Px. erefore, our experiments have shown that miR-122 inhibitor can inhibit cellular oxidative stress.
Apoptosis is a specific mode of cell death with a characteristic pattern of morphological, biochemical, and molecular changes [22]. Apoptosis is closely regulated by the B-cell lymphoma 2 (Bcl-2) family and by the caspase family of intracellular proteins [23]. e caspase family is a central component of the machinery responsible for apoptosis [24].
e Bcl-2 family regulates both pro-and antiapoptotic proteins by mediating the permeabilization of the mitochondrial membrane, and Bcl-2 proteins serve as an "apoptotic switch" [25]. Our TUNEL experiments showed that miR-122 had an inhibitory effect on apoptosis ( Figure 2). Western blotting experiments revealed that, in the STZ + miR-122 inhibitor group, the expression of Bax, C-caspase-3, and C-caspase-9 was downregulated, and the expression of Bcl-2 was increased, indicating that miR-122 could inhibit cell apoptosis.
Oxidative stress and apoptosis of INS-1 cells were detected after the addition of LY294002 (LY). As shown in Figure 4, the ROS content of the STZ + miR-122 inhibitor group was increased, while the activities of SOD, CAT, and GSH-Px decreased, indicating that miR-122 regulates oxidative stress in INS-1 cells through the PI3K/AKT signaling pathway. Figures 3 and 4 show that after the addition of LY, compared with the STZ + miR-122 inhibitor group, apoptosis was increased, and increased apoptotic protein contents indicated that miR-122 regulated oxidative stress in INS-1 cells through the PI3K/AKT signaling pathway.

Conclusions
In summary, miR-122 inhibits oxidative stress and apoptosis of INS-1 cells, and its regulation is related to the PI3K/AKT signaling pathway. However, there are limitations to the present study. First, the study was an in vitro one, and no in vivo experiments were performed. Second, the molecular mechanisms underlying the effects of miR-122 inhibition by the PI3K/AKT signaling pathway on INS-1 cell function were not fully investigated. ese issues require further indepth investigations and will be addressed in future studies.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.