miR-25 and miR-92b regulate insulin biosynthesis and pancreatic β-cell apoptosis

Pancreatic β-cell failure is a central hallmark of the pathogenesis of diabetes mellitus; however, the molecular basis underlying chronic inflammation-caused β-cell failure remains unclear. This study reported here specifically assessed the association between miR-25/miR-92b family and β-cell failure in diabetes. IL-1β and two additional ER stress activators, palmitate and tunicamycin were applied to evaluate the expression level miR-25 by Taqman® RT-PCR. Glucose- and potassium-stimulated insulin secretion assays were performed to assess β-cell function. Dual-luciferase activity, and western blotting assays were utilized for miR-25 target gene verification. CCK-8 and TUNEL staining were used to evaluate β-cell viability and apoptosis. miRNA ChIP identified the increased level of miR-25 in INS-1 cells by IL-1β treatment. Expression levels of miR-25 were significantly upregulated with the treatment of IL-1β, palmitate or tunicamycin in both INS-1 cells and human islets. Ectopic elevation of miR-25 recapitulated most featured β-cell defects caused by IL-1β, including inhibition of insulin biosynthesis and increased β-cell apoptosis. These detrimental effects of miR-25 relied on its seed sequence recognition and repressed expression of its target genes Neurod1 and Mcl1. The miR-25/NEUROD1 axis reduced insulin biosynthesis via transcriptional regulation of β-cell specific genes. The miR-25/MCL1 axis caused β-cell apoptosis in a CASPASE-3/PARP1-dependent manner. Comparable impairments were generated by miR-92b and miR-25, emphasizing the redundant biological roles of miRNA family members with the same seed sequence. MiR-25/miR-92b family plays a major role in β-cell failure occurring under inflammation and diabetes states.


Introduction
Pancreatic β cells play a pivotal role in maintaining glucose homeostasis, and their ability to regulate and control insulin levels largely determines the progression of diabetes [1,2]. The realization of glucose homeostasis requires both the recovery of β-cell numbers and the improvement of insulin synthesis and secretion. Pancreatic β-cell failure is therefore a crucial event in the pathogenesis of diabetes. The molecular mechanism that leads to islet β-cell damage, including endoplasmic reticulum stress, oxidative stress and pro-inflammatory cytokines, are being intensively investigated [3,4].
The observation that chronic inflammation has a deleterious impact on β-cell function and survival strongly suggests a role for cytokines in β-cell deterioration. Pro-inflammatory cytokines are released by activated immune cells, including macrophages and T-cells, activating IRE1 and JUN N-terminal kinase (JNK) phosphorylation to trigger β-cell secretory dysfunction and apoptosis [1]. The regulation of β-cell failure appears to involve a complex regulatory network. However, the current theories neglect the possible involvement of another type of gene regulator, namely microRNA (miRNA), although several studies have now indicated that miRNAs also play a critical role in β cell failure [5,6].
The miRNAs are small, non-coding single-stranded RNA molecules that function in the post-transcriptional regulation of gene expression. These molecules mainly promote the degradation of target mRNA or inhibit translation by binding to the 3′UTR region of their target mRNAs [7,8]. Much evidence has shown that miRNAs play an indispensable regulatory role in β-cell biology [9,10]. In the present study, we detected global variations in miRNA expression in rat insulinoma INS-1 cells incubated with IL-1β. Among the altered miRNAs we detected, we investigated the role of miR-25 in IL-1β-induced β-cell damage. We demonstrated that overexpression of miR-25 suppresses insulin biosynthesis and increases β-cell apoptosis by repression of NEUROD1 and MCL1 protein expression. We further verified that miR-92b has a comparable function to that of miR-25, based on their same seed sequence. These findings reveal the mechanism of miRNAmediated β-cell failure during the pathophysiology of diabetes and provide a potential therapeutic target for protection against diabetic β-cell failure.

Human primary islet culture and treatment
Human primary islets were provided from the First Affiliated Hospital of Nanjing Medical University. The research ethics committee of Nanjing Medical University approved the use of human islets. The research ethics committee of the First Affiliated Hospital of Nanjing Medical University approved the use of human islets. Human islets were cultured in RPMI-1640 medium (glucose: 11.1 mmol/L) containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin and cultured in 37°C and 5% carbon dioxide atmosphere for 3 h. For palmitate or tunicamycin treatment, human islets were incubated in the modified medium with 0.5% (weight for volume) BSA, various concentrations of palmitate (0.25 mmol/L or 0.5 mmol/L) or tunicamycin (10 μg/ml).

Plasmid construction
Public sources (miRanda, TargetScan, and PicTar) provided all the sequences of miR-25 response elements. The wildtype (wt) and mutant (mt) 3′UTR-luciferase constructs were generated by annealing and cloning the short sequences of rat Neurod1 and Mcl1 into pMIR-REPORT Luciferase miRNA Expression Reporter Vector (Ambion) between the SpeI and HindIII sites. To construct MCL1 expression plasmids, the coding sequence for MCL1 was amplified by PCR from the mouse full-length cDNA and then cloned into pCMV5-myc vector between the EcoR1 and Kpn1 sites. We verified the validity of plasmids by sequencing.
Transient transfection and Luciferase report assay miRNA duplex mimics and negative controls were obtained from GenePharma (Shanghai, China). For transient transfection, Lipofectamine 2000 reagent (Invitrogen) was mixed with miRNA mimics or overexpression/reporter plasmids as previously described [12]. Luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI) on a TD-20/20 Luminometer (Turner BioSystems, Sunnyvale, CA) according to the manufacturer's protocols as previously described [12].

TUNEL staining
The In Situ Cell Apoptosis Detection Kit III (fluorescein isothiocyanate) (Boster Biological Technology, Wuhan, Hubei, China) was used in accordance with the manufacturer's protocol to carry out TUNEL Staining [14]. Observation of cells was performed under laser scanning confocal microscope (FV1200; Olympus, Tokyo, Japan).

Hoechst staining
Hoechst 33342 staining was used to stain nuclei. Fluorescence images were obtained using Leica LAS Image Analysis software (Leica). ImageJ 1.49 v software (National Institutes of Health, USA) was used to define a threshold to remove non-specific signals, and apoptotic cells were counted.

Statistical analysis
At least three independent experiments were performed and data are expressed as mean ± SEM. Two tailed Student's t test was applied to make comparisons between pairs of groups, or ANOVA in multiple groups. A p < 0.05 was considered statistically significant.

Diabetes pathogenic factors increase miR-25 in human islets and INS-1 cells
Our first approach was to evaluate the change of miRNAs levels under inflammatory conditions; therefore, we treated INS-1 cells with IL-1β for different durations and then conducted global miRNA expression profiling. We noted a We used a qRT-PCR assay to verify the altered expression levels of miR-25 by diabetes pathogenic factors like the proinflammatory cytokine IL-1β. We found a significant increase of miR-25 expression in INS-1 cells at 12 h, and a return to the basal level at 24 h post IL-1β treatment (Fig. 1A). We also introduced two classic ER stress activators, palmitate and tunicamycin. We observed a dose-dependent upregulation of miR-25 expression in palmitate-treated INS-1 cells (Fig. 1B). We further confirmed that palmitate also increased miR-25 expression in human primary islets (Fig. 1C). Treatment with tunicamycin triggered a more than two-fold increase in miR-25 expression in human islets (Fig. 1D). These results strongly suggested that diabetes pathogenic factors, like IL-1β, upregulated miR-25 expression through ER stress pathways.

Overexpression of miR-25 suppresses insulin biosynthesis
We also examined the influence of miR-25 overexpression on β-cell function. GSIS and KSIS tests showed a decreased insulin content at 48 h after transfection (Fig. 2C, D), with a more marked effect apparent in the INS-1 cells than in the MIN6 cells (Fig. 2C, D). However, neither basal insulin secretion nor GSIS was affected by miR-25 overexpression ( Fig. 2A, B), nor was KSIS affected. These results indicate that miR-25 exerts its effects on β-cells by inhibiting insulin biosynthesis, rather than affecting β-cell insulin secretion. This contradicts the known inhibitory effects of IL-1β on both insulin biosynthesis and secretion, suggesting that miR-25 functions in a different way than IL-1β.
Previous research has demonstrated that β-cell maturation genes have a significant effect on insulin biosynthesis [15,16]. Transcription factors of insulin, including PDX1, MAFA, and NEUROD1, play a critical role in maturation. We further examined the volatility of gene expression levels under miR-25 regulation as a way to identify a possible regulation path by analyzing related genes from the aspect of RNA transcription.
Transfection with miR-25 led to a marked decrease in mRNA levels of Ins2, Pdx1, Glut2, and Gck, and a slightly smaller drop in Ins1 and Mafa mRNA levels (Fig. 2E). Ins2 is the only insulin gene in human beings; therefore, repression of Ins2 transcription by miR-25 overexpression may be an important driver of the diabetes process. By contrast, the Neurod1 mRNA levels remained unchanged. Further western blotting analysis revealed that overexpression of miR-25 reduced the levels of PDX1, MAFA, and NEUROD1 proteins (Fig. 2F, G). This finding suggested that the effect of miR-25 on NEUROD1 is mainly by inhibiting its translation. Taken together, upregulation of miR-25 may suppress insulin biosynthesis by inhibiting β-cell maturation genes. This mechanism differs in part from the mechanism by which IL-1β impairs β-cell function.
Overexpression of miR-25 increases ERstress-induced β-cell apoptosis β-cell dysfunction caused by diabetes pathogenic factors is usually accompanied by increased apoptosis. We therefore tested the influence of miR-25 overexpression on β-cell apoptosis by transfecting MIN6 and INS-1 cells  (Fig. 3B, C). These findings led us to assume that miR-25 induced β-cell apoptosis through a compensatory pathway.
We also confirmed the increase in cell apoptosis at the morphological and molecular levels. Hoechst staining for apoptotic cells in both cell lines showed significant increases in cells with overexpression of miR-25 (Fig. 3E, F). Western blotting analysis also revealed that upregulation of miR-25 increased the cleaved PARP1 and cleaved CASPASE-3 (cleaved CASP-3) levels and decreased the complete PARP levels (Fig. 3E, F). Cleaved CASP-3 is the active form of CASPASE-3 and cleaves PARP1, resulting in cell apoptosis. In other words, miR-25 promotes cell apoptosis through a CASPASE-3/PARP-1-dependent pathway.

Validation of the miR-25 target genes
We then investigated the potential mechanisms by which upregulation of miR-25 expression might trigger β-cell death. Target genes were predicted, and gene ontology analysis identified several potential apoptosis-related target genes. We recognized that miR-25-3p and miR-92b-3p have the same seed sequence, so we simultaneously assessed the conservation of miR-25-3p and miR-92b-3p binding sites. Ultimately, MCL1 and NEU-ROD1 were selected as suitable candidates.
We demonstrated our prediction of the target gene binding sites by cotransfecting INS-1cells, as described in the drawing note. As anticipated, the relative luciferase activity declined in response to miR-25 overexpression (Fig. 4C, D), and this effect was reversed by mutations of the miR-25 binding sites in the 3′UTR sequence of MCL1 or NEUROD1. Western blotting analysis also revealed a decrease in MCL1 and NEUROD1 in response to miR-25 overexpression in INS-1 cells (Fig. 4E), as well as in MIN6 cells (Fig. 4F). Measurements of the protein levels of PDX1 and MAFA also confirmed a similar decrease in response to  (E) or MIN6 cells (F) were transfected with NC or miR-25 or miR-92b duplexes for 48 h, and then protein levels of MCL1, NEUROD1, PDX1, MAFA, and Cleaved PARP1 were measured by western blotting. The percentages of individual protein remaining in miR-25/ miR-92 transfection group vs. NC group were calculated and listed on the right. α-tubulin was used as an internal standard. Data were presented by Mean ± SEM. * or # p < 0.05, ** or ## p < 0.01. Fig. 4E and F).

Beta-cell apoptosis induced by miR-25 is reversed by overexpression of MCL1
The regulatory effect of miR-25 on MCL1 has been described previously, but a direct relationship between the reduction in MCL1 and the effect of miR-25 on β-cell apoptosis has not been demonstrated. Both Hoechst staining and TUNEL staining showed a significant increase in apoptosis in β-cells transfected with miR-25 mimics. However, this situation was reversed by overexpression of MCL1 (Fig. 5B, C) and was reflected by the partial reversal of the increase in pyknotic cells and TUNEL-positive β-cells that had resulted from transfection of miR-25 (Fig. 5D, E). These findings indicated that at least part of the detrimental impact of miR-25 expression is mediated by a reduced expression of MCL1, a crucial antiapoptotic protein.
Western blotting analysis also revealed a decrease in cleaved PARP and cleaved CASP-3 and an increase in MAFA following overexpression of MCL1 in β-cells transfected with miR-25 mimics (Fig. 5A). The observed reduction in apoptosis markers agreed with the Hoechst and TUNEL staining results and further established MCL1 as an important mediator in miR-25 regulated β-cell apoptosis in an inflammatory environment. However, one notable observation was that MCL1 overexpression did not restore the levels of NEUROD1 protein.

Discussion
In this study, we identified that diabetic pathogenic factors, such as IL-1β and palmitate, upregulated miR-25 expression in human islets and rat insulinoma INS-1 cells. The increased amounts of miR-25 bind to the 3′-UTR of Neu-rod1 and Mcl1 and suppress the amounts of NEUROD1 and MCL1 proteins. The miR-25/NEUROD1 axis then inhibits insulin biosynthesis by transcriptional regulation of β-cell specific genes, while the miR-25/MCL1 axis triggers β-cell apoptosis in a CASPASE-3/PARP1-dependent manner. The regulatory role of miR-25 is conserved among species, and we confirmed this in both INS-1 and MIN6 cells. Thus, miR-25 instigates a stepwise impairment of β-cell function during the pathogenesis of diabetes mellitus.
The increased miR-25 expression in β cells treated with proinflammatory IL-1β was discovered using an Affymetrix miRNA ChIP. Previous research has demonstrated that both IL-1β and palmitate induce PERK phosphorylation, which then phosphorylates eIF2α to induce cell apoptosis by an ER stress-dependent mechanism [17][18][19]. We further confirmed that palmitate and tunicamycin, two classic ER stress activators, further enhanced the miR-25 level in human islets, indicating a broad regulatory effect of miR-25 under ER stress conditions. The molecular mechanisms upregulating miR-25 expression require further investigation. Currently, some theories have been put forward for the mechanism of miR-25 regulation. For example, inhibition of the expression of DNA methyltransferases (DNMTs), and TET1 protein- dependent pathway [20]. Both ectopic overexpression of miR-25 and treatment with IL-1β result in defective insulin biosynthesis and increased β-cell apoptosis. However, IL-1β also inhibits GSIS due to enhanced NO production and dampened mitochondrial ATP production [21,22]. GSIS represents an early damage event in β cells, but GSIS defects were barely affected by increased miR-25 expression. We also tested KSIS, which causes insulin release by membrane depolarization. The lack of any alteration in GSIS and KSIS abilities by miR-25 obviously excludes the involvement of insulin secretion due to soluble Nethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and ATP production from mitochondrial bioenergy processes.
We also observed a further inhibitory effect on cell viability by miR-25 elevation by IL-1β treatment of INS-1 cells. This hints that miR-25 and IL-1β may trigger β-cell apoptosis through different pathways. The differences largely involve the activation of NF-κB/iNOS/COX2 pathway by IL-1β but not by miR-25 (data not shown). As a downstream molecule of IL-1β, miR-25 may function in a compensatory pathway. Both miR-34a and miR-153 inhibit insulin secretion by targeting several SNAREs to inhibit insulin granule docking and fusion, and they promote β-cell apoptosis by targeting BCL2 [23,24]. Alternatively, miR-146a, which is upregulated by IL-1β/NF-κB, causes either impairment or negative feedback protective effects through different targets, including Irak1 and Traf6 [25]. Here, we show that miR-25, unlike other IL-1β-regulated miRNAs, specifically targets NEUROD1 and MCL1 to function as a unique supplement pathway to β-cell dysfunction during diabetes progression.
NEUROD1 binds to the E-box element in the promoter region of genes and regulates comprehensive gene expressions that play important roles in both pancreatic endocrine and nervous system development. Deletion of Neurod1 results in defective pancreatic islet morphogenesis and early development of diabetes [26]. Indeed, NEUROD1, in combination with PDX1 and MAFA, determines the development and functional maturation of β cells by regulating insulin secretion genes, such as Glut2 and Gck [26]. We previously reported that diabetes pathogenic factors upregulate miR-24 to inhibit insulin secretion and β-cell growth via targeting Neurod1 and Hnf1a [12]. However, the elevation of miR-24 does not induce β-cell apoptosis but instead guides β-cell dedifferentiation to avoid ER-stress insults [27]. The regulatory role between miR-24/ NEU-ROD1 might explain the pro-apoptotic effect of NEUROD1 under ER-stress conditions. MCL1, as a BCl-2 homolog, is known to function as an anti-apoptotic protein by sequestering the multi-BH domain pro-apoptotic BAX/BAK proteins and the pro-apoptotic BH3-only proteins, such as BIM and PUMA [28]. Some reports have shown that MCL1 is decreased by proinflammatory cytokines and ER stressors and that its decrease leads to β-cell apoptosis in both rodent and human islets. Importantly, relatively low levels of cytokines were able to reduce MCL1 protein levels and promote apoptosis in the human EndoCβH1 β-cell line, suggesting that MCL1 reduction is an early event in the development of diabetes. Indeed, β-cell-specific MCL1 knockout (βMCL1 KO) mice are prone to multiple low-dose streptozotocin-induced islet inflammation [29].
Palmitate and ER stressors inhibit MCL1 translation through eIF2α phosphorylation, whereas cytokines reduce MCL1 protein amounts through a combination of translation inhibition and JNK pathway which targets MCL1 for degradation [18]. Cytokines also upregulate miR-29 expression to repress MCL1 translation, leading to β-cell demise in a type 1 diabetic mouse model [18]. Here, we also found that miR-25 could decrease MCL1 protein levels. Furthermore, recovery of MCL1 expression rescued miR-25-induced β-cell apoptosis, indicating that MCL1 also contributes to the pathogenesis of type 2 diabetes. Decreased MCL1 leads to proapoptotic Bax translocation from the cytoplasm to the mitochondria and gives rise to mitochondrial outer membrane permeabilization, followed by cytochrome c release, CASPASE-3 cleavage, PARP1 cleavage, and, ultimately, cell apoptosis, consistent with our current data [18,30].
Our study also complements previous research that demonstrated that a lack of miRNA-25 in β-cells could hasten cell apoptosis and loss of cell function, although the effect was mild. This suggests that both excessive and absent expression of miRNAs can cause damage to cells [31]. Compensatory elevation of other members in the same miRNA "family" may be the explanation. We also confirmed that miR-92b could regulate the expression of miR-25 target genes due to their common seed sequences. The miR-106b-25 family is actually quite important in normal endocrine function, as losses may cause endocrine failure [32]. For example, a severe defect in β-cell division was observed in miR-17-92/miR-106b-25 double knockout mice [32]. Studies by Munish Kumar et al. have also revealed that miR-25 expression repressed the transcription and expression of the p53 gene, a famous tumor suppressor gene, along with transactivation genes, such as p21, Bax, Puma, and Gadd45α, thereby hindering cell death. Overall, the available research suggests that β cells might have more complex regulatory networks that partly rely on different environmental stimulation signals, but this possibility requires further correlational research.
The establishment of commercial human pancreas β-cell lines has not yet been completely successful, despite many efforts invested over the past few decades. Therefore, access to functional human islets or derived β-cell lines is limited. Rodent insulinoma cell lines are often used for research on insulin production and cell survival related to miRNA studies. We restricted our study to the INS-1 and MIN6 β-cell lines due to their relatively wide acceptance. One clinical research report involving two T1DM cohorts has shown that T1DM children have significantly increased levels of miR-25 in their serum, and that this level negatively correlates with residual β-cell function [33]. Given the diversity in different races and species, more animal and human experiments remain to be done in the future.
In summary, our study reveals that miR-25 can be induced by pathogenic factors of diabetes, and that it drives β-cell failure by directly repressing NEUROD1 and MCL1 production. The findings further elaborate on the regulatory networks involving miRNAs in control of islet β-cell survival and function. The results also broaden our understanding of the pathophysiological development of diabetes and point to new strategic directions for the prevention of the loss of β-cell mass during the development of diabetes mellitus.