The β cell line of the pancreas (MIN6) was obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and incubated in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. In the present experiment, the normal glucose control (5 mM glucose, NG group) was set as a control group, the mannitol group (MA) was set up to exclude the osmotic pressure effects of HG on cells. The MIN6 cells were incubated in complete medium containing 25 mM glucose (final concentration in the medium) for 24 h, which was called the HG group (16). The HG + mimic-NC group was transfected with a mimic-NC and then induced with 25 mM glucose. The HG + miR-532-5p mimic group was transfected with the miR-532-5p mimic and then induced with 25 mM glucose. The HG + miR-532-5p mimic + pcDNA-NC group were transfected with miR-532-5p mimic and the pcDNA-NC plasmid, and were then induced with 25 mM glucose. The HG + miR-532-5p mimic + pcDNA-CCND1 group was transfected with miR-532-5p mimic and the pcDNA-CCND plasmid, and were then induced with 25 mM glucose.

StarBase (http://starbase.sysu.edu.cn/) was used to identify the binding sites of miR-532-5p and CCND1.

Cells were seeded at a density of 1×10⁴ cells/well in 96-well plates. Following treatment of the cells, 20 µl MTT solution (5 mg/ml; Gen-view Scientific, Inc.) was added to each well and cells were incubated at 37°C with 5% CO₂ for 4 h. Subsequently, 100 µl DMSO was added to dissolve the formazan crystals at 37°C for 10 min. The optical density at 490 nm was measured the following day to determine the quantities of formazan formed by cleaving of MTT in living cells.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then reverse transcribed into cDNA using the First-Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). The SuperScript™ III Platinum™ SYBR Green One-Step qRT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used according to the manufacturer's protocols as follows: 95°C for 10 min, 40 cycles of 95°C for 10 sec, 55°C for 10 sec and 72°C for 30 sec. Relative expression levels were calculated according to the 2^−ΔΔCq method (17). The following primers were used: miR-532-5p forward, 5′-GCGCGCATGCCTTGAGTGTAG-3′ and reverse, 5′-ATCCAGTGCAGGGTCCGAGG-3′; CCND1 forward, 5′-ACGAAGGTCTGCGCGTGTT-3′ and reverse, 5′-CCGCTGGCCATGAACTACCT-3′; p53 forward, 5′-GGCCCACTTCACCGTACTAA-3′ and reverse, 5′-GTGGTTTCAAGGCCAGATGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; Insulin1 forward, 5′-TAGTGACCAGCTATAATCAGAG-3′ and reverse, 5′-ACGCCAAGGTCTGAAGGTCC-3′; Insulin2 forward, 5′-CCCTGCTGGCCCTGCTCTT3-3′ and reverse, 5′-AGGTCTGAAGGTCACCTGCT-3′; and GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. Insulin1 and Insulin2 were detected as transcripts of the insulin gene, which are markers of pancreatic β cells (18). GAPDH was used as the housekeeping gene for CCND1, Insulin1, Insulin2 and p53. U6 was used as the housekeeping gene for miR-532-5p.

Cells (1×10⁵ cells/well) were seeded into 6-well plates and cultured for 24 h at 37°C with 5% CO₂. Cell transfection was performed when the cells reached 80% confluency. The miR-532-5p mimic and mimic-negative control (mimic-NC; Invitrogen; Thermo Fisher Scientific, Inc.) were transfected directly into cells. For overexpression of miR-532-5p, the cells were transfected with mimic at a final concentration of 25 nM for 48 h at 37°C. For pcDNA-NC (empty vector, Invitrogen; Thermo Fisher Scientific, Inc.) and pcDNA-CCND1, full length transcript of CCND1 was amplified from cDNA obtained from 293T cells by PCR using PrimeSTAR^® HS DNA polymerase (Takara Bio, Inc.), and was transfected at a final concentration of 500 ng for 48 h at 37°C. The PCR amplification product was inserted into the KpnI and BamHI sites of the pcDNA vector (Invitrogen; Thermo Fisher Scientific, Inc.), which was termed pcDNA-CCND1. Transfection was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Transfection efficiency was determined by RT-qPCR 48 h after transfection.

Cytokine concentration normalized to total insulin was detected using ELISA kits (ELISA MAX™ Deluxe Set Human IGFALS; cat. no. 445904 BioLegend, Inc.) (16). After the cells were centrifuged (300 × g, 10 min, 37°C), cell-free supernatant was diluted to 100 µl (1:20) and incubated with the specific capture antibody and detection antibody. Measurements were made at an optical density of 450 nm.

ROS levels of cells were detected using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (Sigma-Aldrich; Merck KGaA), which could be rapidly oxidized into the fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of intracellular ROS. Fluorescence was monitored with a laser scanning confocal microscope (Leica Microsystems GmbH) at 488 nm (magnification, ×200). The amount of ROS was quantified as the relative fluorescence intensity of DCF per cell in the scanned area.

A total of 3×10⁴ cells were seeded in 24-well plates and incubated overnight. The cells were treated after fusion. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then washed with PBS. Subsequently, 0.3% Triton X-100 in PBS was added and incubated for 5 min. The cells were collected and washed with PBS three times, and treated with 50 µl TUNEL assay solution (Roche Diagnostics GmbH) at 37°C in the dark for 60 min, followed by the addition of stop solution. Subsequently, cells were incubated with DAB solution and stained with hematoxylin and eosin for 5 min at room temperature according to the manufacturer's protocol. Stained apoptotic cells were visualized at ×20 magnification under an LSM 710 laser scanning confocal microscope (Carl Zeiss AG).

Cells were collected, lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) and incubated for 30 min on ice. Subsequently, proteins were detected using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). A total of 40 µg protein was loaded onto 10% SDS-polyacrylamide gels to separate proteins, which were subsequently transferred to PVDF membranes. The membranes were blocked with 10% skimmed milk for 2 h at room temperature, followed by incubation overnight at 4°C with the following primary antibodies: Anti-Bax (1:1,000; cat. no. 14796S; Cell Signaling Technology, Inc.), anti-caspase-3 (1:1,000; cat. no. 700182; Thermo Fisher Scientific, Inc.), anti-cleaved caspase-3 (1:1,000; cat. no. PA5-17913; Thermo Fisher Scientific, Inc.), anti-Bcl-2 (1:1,000; cat. no. 15071S; Cell Signaling Technology, Inc.), anti-CCND1 (1:1,000; cat. no. MA5-14512; Thermo Fisher Scientific, Inc.), anti-P53 (1:1,000; cat. no. MA5-12557; Thermo Fisher Scientific, Inc.) and anti-GAPDH (1:1,000; cat. no. 5174S; Cell Signaling Technology, Inc.). Subsequently, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibodies (1:5,000; cat. nos. A32731 and A11032; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The signals were detected using enhanced chemiluminescence reagent (GE Healthcare), and ImageJ software (version 146; National Institutes of Health) was used to analyze the fold changes of protein levels.

To validate the direct targeting of miR-532-5p, the 3′ untranslated region (3′UTR) of the putative target gene CCND1 was cloned into the psiCHECK2 vector (Promega Corporation), according to the manufacturer's instructions. The mutated or wild-type CCND1 cells were divided into mimic-NC + CCND1 group and miR-532-5p mimic + CCND1 group. Vectors containing the respective 3′UTRs were co-transfected with miRNA mimic (5′-CAUGCCUUGAGUGUAGGACCGU-3′) into cells (1×10⁶ cells) at a final concentration of 500 ng for 48 h at 37°C using Lipofectamine 2000 according to the manufacturer's protocol. Mutations in each of the predicted target sites in CCND1 3′UTRs were generated by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) according to the manufacturer's protocols. Subsequently, the cells were washed with PBS and lysed with cell lysis buffer (Beyotime Institute of Biotechnology) after transfection. The luciferase activity was measured using a plate reader (BD Biosciences) and was normalized to Renilla luciferase activity (pRL-TK) using the Luc-Screen™ Extended-Glow Luciferase Reporter Gene Assay system (cat. no. T1033; Thermo Fisher Scientific, Inc.). All procedures were conducted according to the manufacturers' instructions. Luciferase gene plasmid (containing WT and Mut UTRs) was constructed by Thermo Fisher Scientific, Inc.

Data are presented as the mean ± standard deviation. Each experiment was repeated three times. SPSS version 19.0 software (IBM Corp.) was used to perform statistical analysis. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The MTT assay results demonstrated that compared with that of cells in the NG and MA groups, the survival rate of cells in the HG group was decreased (Fig. 1A), and this was accompanied by a decrease in the expression levels of miR-532-5p (Fig. 1B). This suggested that miR-532-5p served an important role in HG-induced cells. Subsequently, miR-532-5p was overexpressed using the cell transfection technique, and transfection efficiency was measured by RT-qPCR (Fig. 2A). Furthermore, cells were divided into NG, MA, HG, HG + mimic-NC and HG + miR-532-5p mimic groups. ELISA was used to detect the functions of secreted insulin. Compared with those in the NG and MA groups, the insulin secretion levels in the HG group were decreased. Compared with those of cells in the HG + mimic-NC group, the insulin secretion levels of cells in the HG + miR-532-5p mimic group were increased (Fig. 2B). In addition, RT-qPCR was used to detect the gene transcription levels of Insulin1 and Insulin2, and the trend was consistent with that of the total insulin level (Fig. 2C and D). The results demonstrated that overexpression of miR-532-5p improved the impaired functions of secreted insulin in HG-induced cells.

Subsequently, the levels of ROS were detected. Compared with those in the NG and MA groups, the ROS levels in the HG group were increased. Additionally, following overexpression of miR-532-5p, the ROS levels were decreased compared with the HG + mimic-NC group (Fig. 3A and B). TUNEL staining was subsequently used to detect apoptosis. Compared with the NG and MA groups, the HG group exhibited an increased apoptosis rate (Fig. 4A), and this was accompanied by increased expression levels of Bax and cleaved caspase-3, and decreased expression levels of Bcl-2 (Fig. 4B). Additionally, compared with that of cells in the HG + mimic-NC group, the apoptosis rate of cells in the HG + miR-532-5p mimic group exhibited a significant decline, and this was accompanied by decreased expression levels of Bax and cleaved caspase-3, and increased expression levels of Bcl-2. The results revealed that overexpression of miR-532-5p could inhibit oxidative stress levels in HG-induced cells.

StarBase was used to identify that miR-532-5p could target the 3′UTR of CCND1 (Fig. 5A). Additionally, a luciferase reporter assay was used to verify the targeted binding of miR-532-5p and CCND1. The results demonstrated that before CCND1 mutation, luciferase activity was significantly decreased in the miR-532-5p mimic + CCND1 group compared with that in the Vector + CCND1 group. After CCND1 mutation, the luciferase activity was not significantly altered between the Vector + CCND1 and miR-532-5p mimic + CCND1 groups (Fig. 5B). Expression levels of CCND1 in cells were detected following overexpression of miR-532-5p. As shown in Fig. 5C and D, compared with those in the NG and MA groups, the expression levels of CCND1 in the HG group were significantly increased. Compared with those in the HG + mimic-NC group, the expression levels of CCND1 in the HG + miR-532-5p mimic group were decreased.

Compared with those in the pcDNA-NC group, the expression levels of CCND1 in the pcDNA-CCND1 group were significantly increased, indicating successful overexpression (Fig. 6A and B). Subsequently, the cells were divided into NG, HG, HG + mimic-NC, HG + miR-532-5p mimic, HG + miR-532-5p mimic + pcDNA-NC and HG + miR-532-5p mimic + PCDNA-CCND1 groups. The secretion of insulin by the cells was detected using ELISA. Compared with those in the HG + miR-532-5p mimic + pcDNA-NC group, the total insulin levels in the HG + miR-532-5p mimic + pcDNA-CCND1 group were decreased (Fig. 6C), as were the levels of Insulin1 (Fig. 6D) and Insulin2 (Fig. 6E). Subsequently, ROS levels were detected, and the trend was the opposite of that detected for insulin (Fig. 7A and B). Apoptosis was detected using a TUNEL assay. Compared with that in the HG + miR-532-5p mimic + pcDNA-NC group, apoptosis in the HG + miR-532-5p mimic + pcDNA-CCND1 group increased (Fig. 7C and D), and this was accompanied by increased expression levels of Bax and cleaved caspase-3, and decreased expression levels of Bcl-2 (Fig. 7E). These results indicated that overexpression of miR-532-5p improved the impaired functions of secreted insulin and inhibited oxidative stress levels in HG-induced cells by downregulating CCND1 expression.

During the study, abnormal expression levels of p53 were observed in the cells (Fig. 8A and B). Compared with those in the NG group, the expression levels of p53 in the cells were increased following HG induction, whereas the expression levels of p53 in the cells were decreased following overexpression of miR-532-5p. Compared with that in the HG + miR-532-5p mimic + pcDNA-NC group, p53 expression in the HG + miR-532-5p mimic + pcDNA-CCND1 group was increased. Therefore, it was preliminarily concluded that overexpression of miR-532-5p regulated the expression levels of p53 in HG-induced cells by downregulating CCND1 expression.