Diabetes mellitus is a metabolic disorder caused by insufficient insulin secretion. The expression of microRNA (miR)-532-5P is downregulated in diabetes, but its specific role in diabetes has not yet been elucidated. The present study aimed to investigate the specific mechanism underlying the effects of miR-532-5p on diabetes. Cell viability was determined using an MTT assay. The expression levels of miR-532-5P, cyclin D1 (CCND1), Insulin1 and Insulin2 were detected using reverse transcription-quantitative PCR. The expression of miR-532-5p and CCND1 were overexpressed in cells by cell transfection. ELISA was used to detect insulin secretion. 2′,7′-dichlorodihydrofluorescein diacetate was used to quantify reactive oxygen species levels in cells. Apoptosis was detected using a TUNEL assay. Western blotting was performed to detect the expression of apoptosis-related proteins, CCND1 and p53. A dual-luciferase reporter assay was conducted, and verified the targeted binding of miR-532-5p and CCND1. The expression of miR-532-5p was downregulated in high glucose (HG)-induced MIN6 cells. Overexpression of miR-532-5p could improve the HG-induced decline in insulin secretion and inhibit HG-induced oxidative stress and apoptosis in cells. miR-532-5p can target and regulate the expression of CCND1. Overexpression of miR-532-5p downregulated HG-induced cell insulin secretion, oxidative stress and apoptosis by downregulating CCND1, which is involved in regulating the expression of p53. To conclude, miR-532-5p regulated oxidative stress and insulin secretion damage in HG-induced pancreatic β cells by downregulating the expression of CCND1, which is involved in the upregulation of the expression of p53.
Diabetes is a chronic disease involving metabolic disorders of sugar, protein and fat that is primarily caused by insufficient insulin in the body. β cells are endocrine cells that secrete insulin. Insulin secretion by islet β cells is mainly influenced by blood glucose levels (
MicroRNAs (miRNAs/miRs) are an important regulator of numerous physiological and pathophysiological processes, and serve a key role in a number of biological processes, such as cell proliferation, differentiation, apoptosis and carcinogenesis (
By querying the StarBase website, it was identified that miR-532-5p could target cyclin D1 (CCND1). CCND1, a member of the cyclin family, is a regulator of cyclin-dependent kinase. CCND1 expression has been reported to be upregulated in diabetic islets (
The apoptotic activation gene p53 induces cell cycle stagnation at the G0 stage, as well as apoptosis. A previous study demonstrated that p53 serves an important role in the initiation of apoptosis under different physiological conditions (
The present study examined the regulatory effects of miR-532-5p on diabetes and explored the underlying mechanisms by inducing islet cells with HG. The present study provided a theoretical basis for the investigation of the underlying mechanisms of diabetes and potential drug targets.
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 (
StarBase (
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 (
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
Cytokine concentration normalized to total insulin was detected using ELISA kits (ELISA MAX™ Deluxe Set Human IGFALS; cat. no. 445904 BioLegend, Inc.) (
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
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 (
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 (
StarBase was used to identify that miR-532-5p could target the 3′UTR of CCND1 (
Compared with those in the pcDNA-NC group, the expression levels of CCND1 in the pcDNA-CCND1 group were significantly increased, indicating successful overexpression (
During the study, abnormal expression levels of p53 were observed in the cells (
miRNAs have been considered as potential biomarkers of tissue-specific origin, which affect the occurrence and development of diabetes by participating in processes such as collective oxidative stress and the inflammatory response (
At present, studies on miR-532-5p are concerned with its role in other diseases. miR-532-5p expression is downregulated in H9C2 cells exposed to hypoxia, and in the myocardium of rats with acute myocardial infarction, to reduce the apoptosis of H9C2 cells (
The targeted binding of miR-532-5p to CCND1 was identified using the StarBase website, and this targeting relationship was further verified in the present study using a luciferase reporter assay. Studies have reported that the expression of CCND1 in diabetes is upregulated (
During the experiment, it was revealed that overexpression of miR-532-5p regulated the expression levels of p53 in HG-induced MIN6 cells by downregulating CCND1 expression. A previous study reported that adaptive EGF expression sensitized pancreatic cancer cells to ionizing radiation by activating the CCND1/p53/PARP signaling pathway (
Due to the length of the article, only
In conclusion, the present study revealed that miR-532-5p regulated oxidative stress and insulin secretion in HG-induced pancreatic β cells by downregulating the expression levels of CCND1, which was involved in the regulation of p53 expression.
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No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ZZ and WS made substantial contributions to the conception and design of the study, and the acquisition of data. HC made substantial contributions to analysis and interpretation of data. ZZ and WS confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Cell viability and miR-532-5p expression in HG-induced cells. (A) Cell Counting Kit-8 assay was performed to detect cell viability. (B) Reverse transcription-quantitative PCR was used to detect the expression of miR-532-5p. ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose.
Overexpression of miR-532-5p induces insulin secretion in HG-induced cells. (A) RT-qPCR was performed to detect the expression of miR-532-5p following transfection with miR mimic and NC. (B) ELISA assay was conducted to detect the levels of insulin secretion. The expression levels of (C) Insulin1 and (D) Insulin2 were detected using RT-qPCR. **P<0.01, ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.
Overexpression of miR-532-5p inhibits oxidative stress levels in HG-induced cells. (A) 2′,7′-dichlorodihydrofluorescein diacetate fluorescence probe was used to detect ROS expression. (B) Quantification of ROS expression. ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; NC, negative control; ROS, reactive oxygen species.
Overexpression of miR-532-5p inhibits apoptosis levels in HG-induced cells. (A) TUNEL assay was performed to detect the apoptosis rates of cells. (B) The expression levels of apoptosis-related proteins were detected using western blotting. **P<0.01, ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; NC, negative control; ROS, reactive oxygen species.
Overexpression of miR-532-5p downregulates CCND1 expression in HG-induced cells. (A) Targeted binding sites of miR-532-5p and CCND1. (B) Luciferase reporter gene assay was performed to verify the binding between miR-532-5p and CCND1. The expression of CCND1 was determined using (C) reverse transcription-quantitative PCR and (D) western blotting. **P<0.01, ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; NC, negative control; CCND1, cyclin D1; WT, wild-type; Mut, mutant.
Overexpression of miR-532-5p improves the impaired functions of secreted insulin in HG-induced cells by downregulating CCND1. (A) RT-qPCR and (B) western blotting were performed to detect the expression of CCND1. (C) ELISA assay was used to detect the levels of insulin secretion. RT-qPCR was conducted to detect the expression levels of (D) Insulin1 and (E) Insulin2. **P<0.01, ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; NC, negative control; CCND1, cyclin D1; RT-qPCR, reverse transcription-quantitative PCR.
Overexpression of miR-532-5p inhibits oxidative stress levels in HG-induced cells by downregulating CCND1. (A) 2′,7′-dichlorodihydrofluorescein diacetate fluorescence probe was used to detect ROS expression. (B) Quantification of ROS expression. (C) TUNEL assay was performed to detect the apoptosis rate of cells. (D) Quantification of apoptosis rates. (E) The expression levels of apoptosis-related proteins were determined using western blotting. *P<0.05, **P<0.01, ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; NC, negative control; CCND1, cyclin D1; ROS, reactive oxygen species.
Overexpression of miR-532-5p regulates the expression of p53 in HG-induced cells by downregulating CCND1. p53 expression was determined using (A) reverse transcription-quantitative PCR and (B) western blotting. *P<0.05, **P<0.01, ***P<0.001. HG, high glucose; miR, microRNA; MA, mannitol group; NG, normal glucose; NC, negative control; CCND1, cyclin D1.