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Hsa-miR-485-3p Can Be Involved in the Progression of Diabetic Retinopathy by Targeting IL1RN
2025 Volume 267 Issue 2 Pages 255-262
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2025 Volume 267 Issue 2 Pages 255-262
This article aimed to study the molecular mechanism of diabetic retinopathy (DR) and find new targets for the treatment of DR. IL1RN and hsa-miR-485-3p were screened the key molecules using bioinformatics means. Western blot or RT-qPCR results showed a high expression of hsa-miR-485-3p and a low IL1RN expression in high glucose (HG)-treated human retinal endothelial cells (HRECs) and DR patients. The dual luciferase reporter assay verified the targeting relationship of hsa-miR-485-3p to IL1RN, and up-regulating hsa-miR-485-3p hindered IL1RN expression. The Annexin-V/PI and transepithelial electrical resistance (TEER) assay displayed that HG induced HREC apoptosis and broke the tight connection function between cells. These effects of HG on HRECs were exacerbated by hsa-miR-485-3p upregulation and were attenuated by IL1RN overexpression. The receiver operating characteristic (ROC) curve and binary logistic regression analysis showed that hsa-miR-485-3p was a risk for progression to DR in patients with type 2 diabetes mellitus (T2DM), while IL1RN was a potential protective factor. In a word, hsa-miR-485-3p was involved in the development of DR by regulating IL1RN, and they have certain diagnostic value, indicating that they may be novel biomarkers of DR.
Diabetic retinopathy (DR) is a vascular disease caused by the chronic effects of diabetes mellitus (DM) (Wu et al. 2015). It is a common vascular disease among eye diseases (Kgame et al. 2023). The main pathological changes of DR were the loss of pericytes and endothelial cells, the thickening of the basement membrane, and the outward expansion of capillary walls to form microaneurysms (Santiago et al. 2009). According to the severity of retinopathy, DR is divided into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) (Fung et al. 2022). In NPDR, there may be outward expansion of the capillary walls, formation of microaneurysms, multiple intraretinal hemorrhages, obvious venous beading, and definite intraretinal microvascular abnormalities (Lai and Lo 2013). The abnormal microvascular was telangiectatic capillaries adjacent to areas of capillary occlusion. They were visible on dilated ophthalmoscopy as telangiectasia. They were considered a classic sign of ischemia and a predictor of impending progression to PDR (Wang et al. 2020).
Changes such as vitreous or preretinal hemorrhage and extraretinal neovascularization may occur in PDR (Wei et al. 2023). Retinal vascular proliferation can be seen as a futile attempt to form new vessels in the retina and iris to compensate for ischemia (Leysen et al. 2017). Macular oedema is an important feature of DR, where oedema or exudate due to increased vascular permeability of the retina can rupture the macular structures and lead to vision loss (Udaondo et al. 2021). The risk of DR is higher in type 1 diabetes than type 2 diabetes (Kampik 2022). Sex also plays a role in DR. For example, pregnancy in females can lead to the progression of DR and females were twice as likely as males to become blind due to DR (Kollias and Ulbig 2010). Among people with DM for 15 years or more, about one-third will be affected by DR (Fernández-Gutiérrez et al. 2023). In 2020, DR has become the fifth leading cause of preventable blindness worldwide and the fifth most common cause of moderate and severe visual impairment in people aged 50 and over (Fernandes et al. 2022). Controlling blood sugar, blood pressure and blood lipids was still the basis for reducing the occurrence and development of DR (Agarwal et al. 2014). Laser treatment timely was still effective in improving the vision of patients with PDR and macular oedema (Li et al. 2022). Vitrectomy may be necessary in advanced DR (Su et al. 2024).
The pathogenesis of DR was multifactorial, but the molecular mechanism was unclear. In this study, we aimed to explore the potential role of hsa-miR-485-3p in the progression of DR by modulating IL1RN. The function of hsa-miR-485-3p in a variety of human diseases has been reported (Taherdangkoo et al. 2020; Koh et al. 2021; Zhou et al. 2021; Lin et al. 2022), but its role in DR is unknown. IL1RN is associated with DM and its complications (Margaryan et al. 2020; Zou et al. 2022; Duarte et al. 2024), but its function in DR has been poorly reported.
The genes related to DR were collected in the OMIM database (https://omim.org/), which were for the protein-protein interaction (PPI) network construct in the STRING database (https://cn.string-db.org/). The PPI network data was then imported into Cytoscape software to visualize, and the top 10 scoring genes were screened by using the cytohubba-MCC algorithm. The GSE185011 and GSE114477 datasets were from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/). By analyzing GSE185011 dataset, the differentially expressed genes in peripheral blood mononuclear cells of patients with DR and type 2 diabetes mellitus (T2DM) were obtained. The GSE114477 dataset contains the differentially expressed miRNAs in diabetic kidney disease (DKD) patients and healthy individuals. The upstream miRNAs of IL1RN were predicted by the miRDB database (https://mirdb.org/custom.html).
Cell culture and treatmentHRECs were purchased from Ophthalmology Laboratory of Harbin Medical University and cultured in a complete culture medium (San Diego, CA, USA) containing 10% fetal calf serum. Change the culture medium every two days. Subculture was performed when the cell concentration reached above 85%. Then the cells were cultured in a HG medium containing 30 mM anhydrous glucose for 48 hours. Meanwhile, cells cultured under the same conditions using a normal medium containing 5 mM glucose were used as controls.
RT-qPCR assayTotal RNA was extracted using TRIzol reagent (Ambion, Carlsbad, CA), and the purity and concentration of the extracted RNA were measured. Total RNA was reverse transcribed into cDNA using the miScript® II RT kit (QIAGEN) according to the manufacturer’s instructions. RT-qPCR analyses were performed using the miScript® SYBR Green PCR Kit (QIAGEN) on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA). Use 2-ΔΔt to analyze the data. The primer sequences were displayed in Supplementary Table S1.
Cell transfectionEmpty plasmid and overexpression plasmid were purchased from Huifeng Biological Technology Co., Ltd. (Changsha, China). Transfection was performed using Lipofectamine 3000 reagent (Invitrogen, USA).
Dual luciferase reporterThe wild-type/mutant-IL1RN reporter vectors (wt/mut-IL1RN) were co-transfected with hsa-miR-485-3p mimic or mimic negative control (NC) into HRECs by Lipofectamine 3000 (Invitrogen). The fluorescence intensity was measured using a dual luciferase system (Beyotime, Shanghai, China).
Western blot assayCells were lysed in RIPA Lysis Buffer containing protease inhibitors (Roche, USA), and then quantified using a BCA protein assay kit (Beyotime). Proteins were separated using SDS-PAGE and then transferred to a PVDF membrane. After incubation with skim milk, the membranes were incubated with the primary (Proteintech, China) and secondary (Sigma-Aldrich, USA) antibodies, successively. Then, the protein bands were developed using an ECL chemiluminescence kit (Beyotime) and were quantified using Image Lab 6.1 software.
Flow cytometry detectionApoptotic cells were quantitatively detected using an Annexin-V/PI apoptosis detection kit (Miltenyi Biotec, DER). Briefly, processed cells were collected and processed according to the manufacturer’s protocol. The obtained samples were subjected to flow cytometry on a BD FACS Calibur system (USA). The resulting data were analyzed using CellQuesecPro software.
Measurement of TEERCells were seeded on Transwell inserts (24-well format, 0.4 mm pore size, Corning, USA) and grown for a while. TEER was measured using an EVOM2 volt-ohmmeter (World Precision Instruments, Sarasota, FL, USA) at room temperature. The volt-ohmmeter was “zeroed” before measurement according to the manufacturer’s instructions. One Transwell insert was left empty as a control to determine the original resistance of the experimental setup. The net resistance value was calculated by subtracting the control wells from the experimental wells. The resulting transepithelial resistance value was the product of the resulting net resistance value and the experimental well area.
Case collectionThe peripheral blood samples from T2DM, NPDR, and PDR patients were collected for clinical analysis. Patient information was detailed in Supplementary Table S2. The Ethics Committee of Army Center of PLA approved this study. Written informed consent was obtained from all participants before the study began.
Statistical analysisEach set of experiments was repeated at least three times. SPSS and GraphPad software were used for ROC curve and data analysis. The correlation analysis between hsa-miR-485-3p and IL1RN expression was evaluated by Pearson method. Experimental data between two groups were analyzed using student’s t-test, and data from three or more groups were analyzed using one-way ANOVA. p < 0.05 was considered statistically significant.
The PPI network was generated by input of DR-related genes into the STRING database and visualized using Cytoscape software (Supplementary Fig. S1 and Fig. 1A). The cytohubba-MCC algorithm was performed on the PPI network to obtain the top ten scoring genes (Fig. 1B). A total of 959 genes differentially expressed in DR and T2DM patients were obtained in the GSE185011 dataset. IL1RN was identified as a key gene by making the VENN diagram (Fig. 1C).
Screening of key genes for DR.
A. The visual PPI network after removing the edge nodes. B. The top ten scoring genes based on Cytohubba-MCC algorithm. C. VENN diagram to obtain key gene.
The expression level of IL1RN was down-regulated in the peripheral blood of DR patients based on the GSE185011 dataset (Fig. 2A). Our results showed that IL1RN expression was reduced in HRECs after HG induction (Fig. 2B). Then, the overexpression plasmid of IL1RN (op-IL1RN) was constructed, which effectively enhanced IL1RN expression level (Fig. 2C). After IL1RN was overexpressed in HG-treated HRECs, the ratio of Bcl-2-associated x protein and B-cell lymphoma-2 (Bax/Bcl-2) was down-regulated, and the number of apoptotic cells was also reduced accordingly (Fig. 2D,E). In HG-treated HRECs, the expression level of the tight junction marker vascular endothelial-cadherin (VE-cadherin) and the TEER value increased after IL1RN overexpression (Fig. 2F,G).
Effect of IL1RN on HG-induced HRECs.
A. Expression map of IL1RN in the GSE185011 dataset. B. The expression of IL1RN in HG-induced HRECs. C. Working efficiency of IL1RN overexpression plasmid. PC, plasmid control; op, overexpressed plasmid. D,E. Effect of IL1RN on Bax/Bcl-2 expression and the number of apoptotic cells in HG-induced HRECs. F,G. Effect of IL1RN on VE-cadherin expression and TEER value in HG-induced HRECs. *p < 0.05, **p < 0.01, ***p < 0.001.
Hsa-miR-485-3p was predicted to be a potential upstream miRNA of IL1RN, and they had a predicted binding sequence (Supplementary Fig. S2 and Fig. 3A). The working efficiency of hsa-miR-485-3p mimics was shown in Fig. 3B, whose transfection reduced the expression level of IL1RN (Fig. 3C). A dual-luciferase reporter experiment was performed to verify the binding relationship between hsa-miR-485-3p and IL1RN. The results showed that hsa-miR-485-3p mimics reduced the fluorescence intensity of cells with wt-IL1RN transfection, but did not influence the cells with mut-IL1RN (Fig. 3D). Hsa-miR-485-3p was highly expressed in HG-induced HRECs (Fig. 3E). When HRECs were transfected with the hsa-miR-485-3p mimic, the expression level of Bax/Bcl-2 increased; notably, hsa-miR-485-3p offset the inhibitory effects of IL1RN overexpression on Bax/Bcl-2 (Fig. 3F). The number of apoptotic cells also increased when hsa-miR-485-3p was up-regulated, and hsa-miR-485-3p mimic offset the inhibitory effect of IL1RN overexpression on cell apoptosis (Fig. 3G). Up-regulation of hsa-miR-485-3p reduced VE-cadherin expression and the TEER value of HRECs, which also offset the protective effect of IL1RN overexpression on the cell tight junction function (Fig. 3H,I).
Upregulation of hsa-miR-485-3p affected the role of IL1RN in HRECs.
A. Binding sequence of hsa-miR-485-3p and IL1RN 3’UTR. B. Working efficiency of hsa-miR-485-3p mimic (mim-485-3p). C. Regulation of IL1RN expression by hsa-miR-485-3p. D. Targeting effect of hsa-miR-485-3p on IL1RN. E. The expression of hsa-miR-485-3p in HG-induced HRECs. F,G. Effect of hsa-miR-485-3p on Bax/Bcl-2 expression and cell apoptosis in HG-induced HRECs. H,I. Effect of hsa-miR-485-3p on VE-cadherin expression and TEER value in HG-induced HRECs. *p < 0.05, **p < 0.01, ***p < 0.001.
According to RT-qPCR, compared with individuals without DR, IL1RN was significantly deficient in the peripheral blood of DR patients, while hsa-miR-485-3p presented an overexpression. With the increasing severity of disease, the expression of IL1RN gradually decreased, and hsa-miR-485-3p level gradually increased (Fig. 4A,B). Moreover, the expression of hsa-miR-485-3p was negatively correlated with IL1RN (Fig. 4C). We used ROC curves to evaluate the diagnostic value of hsa-miR-485-3p and IL1RN for the progression of T2DM to DR. Among them, the area under curve (AUC) of IL1RN was 0.715 (Fig. 4D), and hsa-miR-485-3p was 0.734 (Fig. 4E). The AUC of their combination was 0.793, show a higher reliability (Fig. 4F). What’s more, they were classifiers for patients with NPDR and PDR. The predicted AUC of IL1RN and hsa-miR-485-3p was 0.732 and 0.709, respectively (Fig. 4G,H), and their combined AUC was 0.790 (Fig. 4I). These results indicate that hsa-miR-485-3p and IL1RN were reliable predictors of DR development.
Clinical value of hsa-miR-485-3p and IL1RN.
A,B. Expression of IL1RN and hsa-miR-485-3p in the peripheral blood of T2DM and DR patients. C. The correlation between the expression of hsa-miR-485-3p and IL1RN. D-F. The ROC curves of hsa-miR-485-3p and IL1RN individually and jointly in predicting the progression of T2DM to DR. G-I. The ROC curves of hsa-miR-485-3p and IL1RN individually and jointly in predicting the progression of NPDR to PDR.
The basic pathological changes of DR include selective loss of pericytes, thickening of the basement membrane, formation of microaneurysms, endothelial cell proliferation, and neovascularization (Xue et al. 2022). Long-term exposure of retinal endothelial cells to high concentrations of blood sugar can lead to damage to the blood-retinal barrier and blockage of retinal capillaries (Agard et al. 2022). It will accelerate the formation of macular oedema and pathological retinal neovascularization.
The pathogenesis of DR is very complex and involves many different mechanisms (Liu et al. 2022). Hyperglycemic damage affects the enzymatic machinery responsible for epigenetic modifications (Gong and Su 2017). These modifications alter gene expression without affecting the DNA sequence (Kowluru 2017). As a highly conserved single-stranded small non-coding RNA, miRNA can regulate more than 30% of genes in eukaryotic cells. It can specifically bind to the 3ʹ-UTR of targets and inhibit gene expression by interfering the stability of its mRNA. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can regulate the expression of multiple genes through one miRNA, or it can finely regulate the expression of a certain gene through a combination of several miRNAs (Su et al. 2021). Several studies have confirmed that multiple miRNAs were involved in the occurrence and development of DR. Overexpression of miR-200b improves the expression of vascular endothelial growth factor in the retina of diabetic rats (Pang et al. 2020). MiR-15b and miR-16 play a role in suppressing insulin resistance, thereby protecting HRECs from hyperglycemia-induced apoptosis (Ye and Steinle 2015). MiR-29a is reduced in diabetic retina, and it may prevent the occurrence of DR in rat models by down-regulating angiotensinogen (Zhang et al. 2017). We demonstrated that hsa-miR-485-3p mediated HG-induced apoptosis of HRECs by targeting IL1RN.
Blood-retinal barriers (BRBs) maintain retinal health by providing essential nutrients and removing metabolites and toxins (Kang and Yang 2020). During the pathogenesis of DR, various transcription factors were highly activated and began to regulate the transcription of multiple genes. The continuous triggering of hyperglycemia will initiate a series of signaling cascades, causing pericyte apoptosis (Kang and Yang 2020). The death of pericytes gradually changed the structure of retinal microvessels and led to the breakdown of BRBs. Tight junctions are closed chains of transmembrane junction proteins between adjacent cells (Wittchen et al. 2013). It can seal the gaps between cells and prevent extracellular substances from entering the tissue to ensure the stability of the internal environment. TEER refers to the electrical resistance across a cell monolayer. It reflects the degree of tightness between cells and the permeability of cells (Aibani et al. 2021). The higher the TEER value, the tighter the connections between cells, and the greater the restrictions on the exchange of information and materials through the cell layer (Gamboa and Leong 2013). Our study showed that up-regulation of hsa-miR-485-3p aggravated HREC tight junction function impairment induced by HG, and overexpression of IL1RN saved this impairment.
In this study, we found that IL1RN reduced the apoptosis level in HG-induced HRECs and improved the tight junction function of cells. Up-regulation of hsa-miR-485-3p blocked the protective effects of IL1RN on HG-induced HRECs. Together with the clinical data, we infer that hsa-miR-485-3p and IL1RN may be the novel biomarkers of DR.
The authors declare no conflict of interest.
