MicroRNA Regulation for Inflammasomes in High Glucose-Treated ARPE-19 Cells

Purpose This study aimed to evaluate the expression of microRNAs (miRNAs) and inflammasomes in diabetes-induced retinal cells and to determine their role in the pathogenesis of diabetic retinopathy (DR). Methods To establish diabetes-induced cell models, ARPE-19 cells were treated with high glucose. The expression levels of five miRNAs (miR-185, miR-17, miR-20a, miR-15a, and miR-15b) were measured in high glucose-treated ARPE-19 cells using real-time quantitative polymerase chain reaction. Western blotting was performed to measure inflammasome expression in cellular models. miR-17 was selected as the target miRNA, and inflammasome expression was measured following the transfection of an miR-17 mimic into high glucose-treated ARPE-19 cells. Results In high glucose-treated ARPE-19 cells, miRNA expression was substantially downregulated, whereas that of inflammasome components was significantly increased. Following the transfection of the miR-17 mimic into high glucose-treated ARPE-19 cells, the levels of inflammasome components were significantly decreased. Conclusions This study investigated the relationship between miRNAs and inflammasomes in diabetes-induced cells using high glucose-treated ARPE-19 cells. These findings suggested that miR-17 suppresses inflammasomes, thereby reducing the subsequent inflammatory response and indicating that miRNAs and inflammasomes could serve as new therapeutic targets for DR.


Introduction
Diabetic retinopathy (DR) is a common microvascular complication of diabetes and the leading cause of blindness among working middle-aged adults worldwide [1].Chronic hyperglycemia activates alternative glucose metabolism pathways, resulting in oxidative stress that induces retinal microvascular damage via infammation, vascular hyperpermeability, and neuroglial dysfunction [2].Although the pathogenesis of DR is relatively well known, the specifc mechanisms underlying the disease remain unknown.Terefore, substantial eforts have been made to identify the target molecules associated with DR.
MicroRNAs (miRNAs) are small noncoding RNAs that can regulate gene expression by binding to the 3′ untranslated region of target mRNA molecules and inhibiting their translation or promoting their degradation [3,4].Tey are also candidate substances that have been extensively studied regarding their involvement in the pathogenesis of DR [5].We already confrmed a decrease in the levels of fve miRNAs, including miR-185, miR-17, miR-20a, miR-15a, and miR-15b, by measuring miRNA levels in the aqueous humor of patients with diabetic macular edema (DME) [6].However, changes in the expression of these miRNAs have not yet been demonstrated in cells.Additionally, the specifc mechanisms by which these miRNAs afect DR pathogenesis remain unclear.
Using bioinformatics analysis in a preliminary study, we confrmed that these miRNAs are associated with vascular endothelial growth factor and infammatory eye disease.As a link between miRNA and infammation, we investigated infammasomes as an additional candidate implicated in the pathogenesis of DR.
Terefore, we hypothesized that miRNAs regulate the infammatory response in DR by regulating the infammasome pathways.To test this hypothesis, we measured the expression levels of fve miRNAs (miR-185, miR-17, miR-20a, miR-15a, and miR-15b) and infammasome components in a high glucose-treated human retinal pigment epithelial cell line (ARPE-19) and explored the pathological mechanisms of DR through their relationship.

High Glucose
Treatment.ARPE-19 cells were starved for 24 hours and then treated with 30 mM D-glucose (Sigma, St. Louis, MO, USA) in 2% FBS media at 37 °C for 24 hours.Cells cultured in DMEM without glucose were used as controls.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR).
Total RNA was extracted from ARPE-19 cells using an miRNeasy Micro Kit (Qiagen, Valencia, CA, USA).cDNA was synthesized from the extracted RNA samples and amplifed using a Mir-X miRNA First-Strand Synthesis Kit (TAKARA, Shiga, Japan).Te PCR mixtures containing the Mir-X miRNA qRT-PCR TB Green Kit (TAKARA) were used for RT-qPCR, and the expression levels of the target genes were detected using the Applied Biosystems Quant-Studio 3 Real-Time PCR Instrument (Termo Fisher Scientifc, Cleveland, OH, USA).Five miRNAs (miR-185, miR-17, miR-20a, miR-15a, and miR-15b), which were signifcantly decreased in aqueous humor of patients with DME and associated with infammation in the bioinformatics analysis in our previous study, were selected for the RT-qPCR [6].Table 1 lists the primer sequences used in the RT-qPCR.U6 and mRQ 3′ Primer, which are components of the Mir-X miRNA First-Strand Synthesis Kit, were used as controls.Te RT-qPCR protocol comprised denaturation at 95 °C for 10 seconds, followed by PCR at 95 °C for 5 seconds and 60 °C for 20 seconds.A total of 40 cycles were performed, and the results were analyzed by the 2 −ΔΔCt method.

miRNA Regulation.
For the fve miRNAs analyzed via RT-qPCR, a PubMed search was conducted using keywords specifc to each miRNA and infammasome.Te search revealed that miR-17 expression has been extensively studied in relation to infammasomes.Terefore, miR-17 was selected as the fnal candidate miRNA regulator.AccuTarget Human miR-17 and negative control mimics were obtained from Bioneer (Daejeon, Korea) and transfected into ARPE-19 cells using the Lipofectamine RNAi-MAX transfection reagent (Invitrogen, Carlsbad, CA, USA) per the manufacturer's protocol.After transfection, the cells were starved for 24 hours and treated with 30 mM of glucose in 2% FBS for 20 hours.Western blotting was performed on the infammasome after cell transfection to see if the regulation of miR-17 could modulate the infammasome.β-actin was used as a control protein.

Statistical Analyses.
All data are expressed as the mean ± standard deviation of three or more independent experiments.Values were analyzed using Prism 8

Expression of miRNAs and Infammasome Components in ARPE-19 Cells Treated with High Glucose Concentrations.
Figure 1 shows the RT-qPCR results of the expression levels of the fve miRNAs in ARPE-19 cells treated with high glucose and cells in the control group.Te miR-185, miR-17, miR-20a, and miR-15b expression levels were signifcantly downregulated in the high glucose-treated group compared to those in the control group (all P < 0.05).Although miR-15a expression also decreased in high glucose-treated cells, the decrease was not statistically signifcant (P � 0.06).Figure 2 presents the western blot results for infammasome components in ARPE-19 cells treated with high glucose concentrations.Te levels of NLRP3, the main infammasome component, were signifcantly increased in high glucose-treated cells compared to those in the control group (P < 0.05).TXNIP, an upstream infammasome regulator, was also increased in cells treated with high glucose, and this increase was accompanied by a corresponding increase in IL-1β, the fnal infammasome pathway product (all P < 0.05).However, although the caspase-1 expression increased in high glucose-treated cells, this diference was not statistically signifcant.

Expression of Infammasome Components after Treating ARPE-19 Cells Treated with High Glucose with the miR-17
Mimic.miR-17 overexpression was confrmed by RT-qPCR after miR-17 mimic treatment in high glucose-treated ARPE-19 cells (Figure 3(a)).After the cells were treated with the miR-17 mimic, the TXNIP, NLRP3, and IL-1β levels decreased back to the levels before high glucose treatment, indicating that miR-17 overexpression inhibited the high glucose-induced increases in these infammasome components (Figures 3(b) and 3(c)).

Discussion
We confrmed the downregulation of fve miRNAs (miR-185, miR-17, miR-20a, miR-15a, and miR-15b) in high glucose-treated ARPE-19 cells.In addition, increases in several components related to the infammasome pathway, such as NLRP3, TXNIP, and IL-1β, were confrmed in those cells.Using experiments with miR-17 mimic transfection, it was confrmed that miR-17 overexpression suppressed the increase in infammasomes caused by high glucose treatment.Tis study provides novel evidence of miRNA downregulation and infammasome overexpression in diabetic retinal cells, elucidating their interrelationship.
Properly designing cell models is crucial for understanding the pathogenesis of DR.Although the pathophysiological changes in DR predominate in the inner retina [16], the outermost RPE cells are also afected by high glucose levels, as demonstrated and extensively studied using ARPE-19 cells, a human RPE cell line.Recent studies have focused on elucidating the pathological mechanisms of DR via miRNA-related pathways, thereby leveraging the ease of transfection of ARPE-19 cells.Although a standardized method for treating ARPE-19 cells with high glucose is yet to be established, viability assays conducted using ARPE-19 cells treated with various glucose concentrations for 24 h revealed an IC50 value of approximately 30 mM glucose, prompting some studies to employ 30 mM as the glucose concentration for further investigations [17,18].Terefore, in this study, the same conditions were replicated by subjecting ARPE-19 cells to high glucose treatment.Our experiments provide substantial evidence that the cell models employed in this study exhibited changes characteristic of DR.Tus, we have sufcient grounds to apply the experimental fndings related to miRNAs and infammasomes to DR.
MiRNAs are small RNA molecules transcribed from the human genome that play important roles in gene expression regulation and modulation of infammatory responses [19,20].miRNA expression dysregulation has been linked to various infammatory diseases, including ocular infammatory and degenerative diseases [21,22].Additionally, miRNAs have been extensively studied in diabetes, where they are involved in regulating insulin secretion, sensitivity, and β cell function [23].Regarding DR, our previous study confrmed the downregulation of miR-185, miR-17, miR-20a, miR-15a, and miR-15b, which are associated with infammatory cytokines, in the aqueous humor of patients with DME [6].However, the exact involvement of these miRNAs in the pathogenesis of DR has not been fully elucidated.
Infammasomes, multicellular proteins involved in activating the infammatory response, are crucial because of their interaction with miRNAs that regulate infammation [10][11][12].In diabetes, the NLRP3 infammasome is associated with insulin resistance and the progression of type 2 diabetes [24].Te activation of the NLRP3 infammasome in immune cells, such as macrophages, can impair insulin signaling by releasing proinfammatory cytokines, contributing to insulin resistance [25].It may also contribute to β cell dysfunction and apoptosis, leading to decreased insulin production and the development of type 2 diabetes [26].However, few studies have been conducted on the role of the miRNArelated infammasome in DR, and to the best of our knowledge, no studies have demonstrated this pathway in human RPE cell lines.
Among the fve miRNAs selected in our study, miR-17 and miR-20a suppress infammasome expression by regulating TXNIP [27,28].TXNIP, a key player in glucose metabolism, is involved in high glucose-induced reactive oxygen species generation and mitochondrial pathway apoptosis in pancreatic β cells, which has been linked to the development and progression of type 2 diabetes [29,30].Te inhibition of the TXNIP/NLRP3 infammasome pathway by miR-17 was confrmed in β cells of diabetic rats [27,31], brains of hypoxic-ischemic injured rats [32], and retinal Müller glial cells of mice after induction of high fat dietinduced insulin resistance [33].Te same results were obtained in our study using high glucose-treated ARPE-19 cells.Similarly, miR-20a acts as a negative regulator of the infammatory response in rheumatoid arthritis fbroblast-like synoviocytes by targeting TXNIP [28].While the involvement of miR-20a in the infammasome pathway in diabetes has not been reported, its inclusion in the miR-17-92 cluster alongside miR-17 suggests a potential similar role in DR through the regulation of TXNIP expression.Te remaining three miRNAs and their association with the infammasome have been reported relatively recently.MiR-185 has been reported to inhibit the infammasome pathway by targeting MyD88 and CXCR4 in neuropathic pain [34].
Our study has several strengths.First, we confrmed the expression of various miRNAs in ARPE-19 cells.Given that these miRNAs are signifcantly reduced in patients with DME in clinical practice, the results of this study suggest the potential of these miRNAs as therapeutic targets for DR, including in patients with DME.Furthermore, we demonstrated the role of miRNAs in regulating infammasomes in DR pathogenesis, specifcally via miR-17 regulation.Although the role of miRNAs in infammasome regulation in β cells has been previously recognized, our study is the frst to provide evidence of their relationship in DR.Lastly, most studies investigating miR-17 and infammasomes have utilized rodent cells.In contrast, our study holds greater clinical relevance by employing a human RPE cell line.
However, this study has several limitations.First, this study was conducted solely on fve miRNAs identifed in our previous study, potentially overlooking other signifcant miRNAs that could be related to infammasomes.We selected these fve miRNAs based on their fold change greater than −50 log 2 value and their relevance to DR and angiogenesis.Other miRNAs that showed a signifcant decrease in DME patients was not included in our study.Secondly, among the fve miRNAs, miRNA regulation focused exclusively on miR-17, given its well-known role in regulating infammasomes via TXNIP.Although miR-20a shares a similar mechanism with miR-17, miR-185, miR-15a, and miR-15b were excluded because of insufcient evidence of their association with the infammasome in DR.Incorporating miRNA regulation of these additional miRNAs could enhance the identifcation of their causal relationship with the infammasome.Tirdly, we used the ARPE-19 cell line to model DR.DR primarily afects the inner retina, specifcally the neural retina, where the blood-retina barrier is compromised due to hyperglycemia.However, we used the ARPE-19 cell line for several reasons.Te frst is the availability and robustness of the ARPE-19 cell line.ARPE-19 cells are well characterized, easy to maintain, and widely available, making them a convenient model for in vitro studies.Teir use allows for reproducible and controlled experiments.Te second reason is the efect of infammation and oxidative stress on RPE cells.Te RPE is a signifcant source of infammatory cytokines and reactive oxygen [37].ARPE-19 cells are used to study these infammatory and oxidative responses, which are critical components of DR pathogenesis.Nevertheless, if experiments are conducted using cell lines originating from the inner retina or animal models, it is expected that the current results could be more broadly applicable.Finally, the small sample size might have contributed to the inability to derive statistical signifcance for certain measures.

Conclusions
Despite the limitations mentioned above, this study successfully established diabetes-induced cell models using high glucose-treated ARPE-19 cells and investigated the association between miRNA and infammasome expression.Notably, our fndings indicated that miR-17 suppresses TXNIP expression and reduces infammasome expression, leading to a reduction in infammatory cytokines in ARPE-19 cells treated with high glucose treatment.Tese results ofer valuable insights into miRNA and infammasome interactions and have several clinical implications.Tey suggest miR-17 or its mimics as potential therapeutic targets for DR propose new biomarkers for monitoring disease progression and could inspire novel treatments, including drugs or gene therapies.Furthermore, these fndings support personalized medicine approaches, enhancing treatment based on individual molecular profles.Overall, miR-17 shows promise as a key target for developing more efective interventions for DR.

Figure 1 :
Figure 1: RT-qPCR of fve miRNAs in high glucose-treated ARPE-19 cells.Data are expressed as the mean ± standard deviation.Data are combined from three samples per group in each miRNA experiment.Te results, analyzed using the unpaired t-test, are shown as the mean ± standard deviation.* P < 0.05 and * * P < 0.01.

Figure 2 :Figure 3 :
Figure 2: Representative blots (a) and quantitative analysis (b) of western blot for infammasome components in high glucose-treated ARPE-19 cells.Data are combined from fve samples per group in each protein experiment except for four samples in the normal group for the NLRP3 experiment.Te results, analyzed using an unpaired t-test, are shown as the mean ± standard deviation.* P < 0.05.

Table 1 :
Sequence of the polymerase chain reaction primers used in the experiments.
(GraphPad Software, San Diego, CA, USA).Multiple comparisons were conducted using one-or two-way analysis of variance (ANOVA).Te mean values of two independent groups were compared using an unpaired t-test.Statistical signifcance was set at P < 0.05.