Up‐regulation of miR‐192‐5p inhibits the ELAVL1/PI3Kδ axis and attenuates microvascular endothelial cell proliferation, migration and angiogenesis in diabetic retinopathy

Diabetic retinopathy (DR) is a common complication of diabetes mellitus that poses a threat to adults. MicroRNAs (miRNAs) play a key role in DR progression. However, the role and mechanism of miR‐192‐5p in DR remain unclear. We aimed to investigate the effect of miR‐192‐5p on cell proliferation, migration and angiogenesis in DR.


| INTRODUCTION
Diabetic retinopathy (DR) is one of the most serious clinical microvascular complications of diabetes mellitus, manifesting as vision loss and even blindness. 1 The incidence and severity of DR vary widely, and DR tends to be asymptomatic at the early stage. With the prolonged course of DR, it eventually leads to irreversible damage to vision. 2 DR is a disease with multifactorial aetiologies, including oxidative and hypoxic stress, retinal inflammation, which can cause the proliferation and migration of retinal microvascular endothelial cells and the formation of capillaries. 3 The clinical treatment of DR is mainly divided into surgical therapy, retinal laser therapy, intravitreal injection therapy and systemic therapy. 4 However, the curative effect is poor, and the symptoms are often progressively aggravated, which seriously affects the quality of life. 5 Therefore, more molecular mechanisms and new diagnostic markers for the treatment of DR are urgently needed.
MicroRNAs (miRNAs) are a class of single-stranded noncoding RNAs of approximately 22 nucleotides that are associated with various cellular processes, such as proliferation, apoptosis and angiogenesis. 6 Importantly, miRNAs are involved in DR. 7 For example, miR-126 was decreased in the retina of DR patients. 8 Dysregulation of miR-374a regulated HRMECs proliferation and migration. 9 Notably, miR-192-5p expression was decreased in diabetes, and its overexpression could reduce blood glucose levels in HepG2 cells. 10 Moreover, miR-192-5p was decreased in the diabetic retina. 11 Nevertheless, the exact mechanism of miR-192-5p in DR remains poorly understood.
ELAV-like RNA binding protein 1 (ELAVL1) is an RNAbinding protein that binds to a variety of proteins and maintains mRNA stability. 12 Literature found that ELAVL1 was up-regulated in the retina and HRECs of diabetic rats, and combined with VEGF mRNA to promote VEGF expression and angiogenesis. 13,14 However, the mechanism requires further exploration. We recently found that miR-192-5p could bind to ELAVL1 via starBase prediction. Herein, we speculated that reduced miR-192-5p could promote ELAVL1 expression and contribute to DR development.
Phosphoinositide 3-kinase delta (PI3Kδ) is a class IA catalytic isoform of PI3K encoded by the PIK3CD gene (p110δ) and is an important member of the lipid kinase family. 15 Currently, it has been reported that the expression of PI3Kδ was increased in high glucoseinduced human retinal microvascular endothelial cells (HRMECs). Furthermore, PI3Kδ regulated the activation of Akt signalling pathway in vascular endothelial cells and promoted cell proliferation, migration and angiogenesis, whereas the inactivation of PI3Kδ attenuated pathological retinal angiogenesis. 16 Nevertheless, the specific contribution of PI3Kδ to DR is largely unknown. According to the prediction of starBase, ELAVL1 has a binding site for PIK3CD. Therefore, we hypothesized that ELAVL1 might play a role in DR by binding to PIK3CD.
Herein, we aimed to investigate the role and mechanism of miR-192-5p in regulating DR pathogenesis. We hypothesized that in DR, up-regulation of miR-192-5p could suppress the ELAVL1/PI3Kδ axis, thereby attenuating microvascular endothelial cell proliferation, migration and angiogenesis.

| Clinical samples
Retinal fibrovascular membrane (FVM) samples were obtained from 20 DR patients who were admitted to Hainan West Central Hospital from September 2020 to September 2021. Vitreomacular interface disorder in non-diabetic patients was assigned to the control group. All specimens were stored at −80°C until being used. Informed consent was provided by all patients. Approval was obtained from the Ethics Committee of Hainan West Central Hospital (No. LLKY-2022-9). The clinical data of participants are shown in Table 1.

| Cell culture and DR cell model
Human retinal microvascular endothelial cells (HRMECs) were provided by Procell, which were later cultivated What's new?
• Diabetic retinopathy (DR) is a common complication of diabetes, characterized by inflammation, angiogenesis, microaneurysms and irregular blodd vessels, which can induce visual impairment. It is of great importance to inhibit retinal microvascular dysfunction for DR therapy. • In this study, we found that miR-192-5p could damage phosphoinositide 3-kinase delta (PI3Kδ) mRNA stability by directly targeting ELAV-like RNA bingding protein 1 (ELAVL1), thereby inhibiting attenuates microvascular endothelial cell proliferation, migration and angiogenesis. • The investigation of miR-192-5p in regulating the pathogenesis of DR may provide a novel target for DR treatment.
within endothelial cell medium (ECM, Gibco) based on the concentration of 10% fetal bovine serum (FBS, Gibco) under the conditions of 5% CO 2 and 37°C. To construct DR cell model, HRMECs were exposed to different glucose concentrations for 48 h, namely, normal-glucose (5.5 mM glucose, Normal), high-glucose (HG, 30 mM glucose) or osmotic control group (Mannitol group, 5.5 mM glucose supplemented with 24.5 mM mannitol).

| Western blotting assay
RIPA buffer (Beyotime) was utilized for extracting total cellular proteins. BCA method (Keygen Biotech) was applied in determining protein concentrations. After separating protein aliquots through SDS-PAGE, protein samples were transferred on PVDF membranes (Millipore). Later, 5% defatted milk was utilized to block membranes for a 1-h period. Blots were cultivated with anti-ELAVL1 antibody (#12582, 1:1000, Cell Signaling Technology) as well as PI3Kδ antibody (ab1678, 1:1000, Abcam) overnight under the temperature of 4°C. Subsequently, secondary antibody (Cell Signaling Technology) was used for additional 1-h incubation at room temperature. Signals were detected by enhanced chemiluminescence (Millipore). βactin was used as an internal reference.

| Cell viability assay
The detection of HRMECs cell viability was made by applying Cell Counting Kit-8 (CCK-8, Dojindo). After transfection, cells were inoculated in the 96-well plates (5000 cells/well). Twenty-four hours after seeding, the supplementation of each well was made with 10 μL CCK-8 solution. At 2-h post-incubation, we evaluated cell viability by measuring the absorbance (OD) (Bio Rad Laboratories, Inc.) at 450 nm.

| Transwell assay
3 × 10 3 cells/well were added into top Transwell chambers (8 μm, Millipore) using serum-free medium, whereas medium that contained 10% FBS was added to bottom chambers. At 24-h post-cultivation, 4% paraformaldehyde (PFA) was utilized to fix migrating cells, followed by staining using the 0.1% crystal violet. Olympus microscope was adopted for taking cell images. At the same time, the cell number was calculated from five random fields.

| Tube formation assay
Matrigel (50 μL/well, BD Biosciences) was added to 96-well plates and allowed to polymerize for 60 min at 37°C before seeding HRMECs on Matrigel-coated wells (7 × 10 3 /well). Eight hours after seeding, capillary-like structures were captured using microscopy (Olympus). Tube formation was quantified by selecting five different fields randomly using ImageJ software (NIH).

| Dual-luciferase reporter assay
This work cloned mutant (MUT) or wild-type (WT) 3′UTR in ELAVL1 to pmirGLO vector (Promega). Then, HRMECs were subjected to co-transfection using NC mimic or miR-192-5p mimic and vectors carrying ELAVL1-WT or ELAVL1-MUT with Lipofectamine 3000 (Invitrogen). Two days later, the dual-luciferase reporter assay system (Promega) was used for making the measurement of luciferase activities.

| RNA immunoprecipitation (RIP)
EZ-Magna RIP kit (Millipore) was utilized for RIP assay. Transfected cells were lysed using RIP lysis buffer. The extract was added to magnetic beads and exposed to ELAVL1 antibody, Ago2 antibody (Abcam) or anti-IgG (Sigma) at 4°C overnight. Finally, the relative enrichment of genes was calculated by RT-qPCR.

| Statistical analysis
Results were represented by mean ± SD and explored with Graphpad Prism 8. Each experiment was conducted in six replicates. We used the Student's t test for making the comparison between two groups, while One-way anova among multiple groups. Repeated measures anova is used for not independent groups. In this study, p < 0.05 stood for statistical significance. expression was decreased in HRMECs with HG, compared with normal glucose group or mannitol group (Figure 1b). Therefore, miR-192-5p was reduced in DR patients and HG-treated HRMECs.

| MiR-192-5p up-regulation suppressed microvascular endothelial cell proliferation, migration and angiogenesis in DR cell model
To further explore the function of miR-192-5p in DR, HG-induced HRMECs were transfected with miR-192-5p mimic. RT-qPCR showed that compared to the control cells, miR-192-5p expression was inhibited after HG treatment, and in HG-treated HRMECs, the level of miR-192-5p was elevated with the transfection of miR-192-5p mimic, as compared with the NC mimic group (Figure 2a). Next, CCK8 experiments revealed that HRMECs viability increased after HG treatment, but cell viability decreased in HG cells with miR-192-5p upregulation ( Figure 2b). Meanwhile, HG-induced cell migration. Nevertheless, cell migration was diminished by overexpressed miR-192-5p (Figure 2c). Moreover, tube formation ability of HRMECs was induced after HG stimulation, but impaired in HG cells with miR-192-5p up-regulation ( Figure 2d). Thus, these results suggested that cell proliferation, migration and angiogenesis in HG-treated HRMECs were suppressed by miR-192-5p overexpression.

| MiR-192-5p directly bound to ELAVL1 and inhibited its expression
Further, the relationship between miR-192-5p and ELAVL1 was investigated. Luciferase reporter assay revealed that in HRMECs with miR-192-5p mimics, the fluorescence values decreased in the ELAVL1 wildtype group, while there was no significant change in the ELAVL1 mutant group (Figure 3a). Next, the results of RIP assay validated that both miR-192-5p and ELAVL1 were enriched in Ago2 antibody complex ( Figure 3b). Furthermore, we detected that miR-192-5p level was decreased in HG-induced HRMECs with miR-192-5p inhibitor compared to control cells (Figure 3c). Afterwards, ELAVL1 expression was elevated in HGtreated HRMECs, and overexpressed miR-192-5p reduced ELAVL1 expression, whereas miR-192-5p inhibitor elevated ELAVL1 level (Figure 3d,e). All in all, miR-192-5p could target ELAVL1 and suppress ELAVL1 expression.

| The reduction in microvascular endothelial cell proliferation, migration and angiogenesis caused by miR-192-5p up-regulation was reversed by ELAVL1 overexpression
We overexpressed ELAVL1 in HG-induced HRMECs and discovered that both ELAVL1 gene and protein expression were increased (Figure 4a). MiR-192-5p up-regulation repressed the proliferation and migration of HG cells. However, the decrease in HG cell proliferation and migration generated by miR-192-5p overexpression could be restored by ELAVL1 overexpression (Figure 4b,c). In addition, in HG-treated cells, the inhibition effect of miR-192-5p mimics on tube formation could also be overturned by co-transfection of oe-ELAVL1 (Figure 4d). Taken together, miR-192-5p modulated cell proliferation, migration and angiogenesis in HG-treated HRMECs through ELAVL1.

| ELAVL1 maintained PI3Kδ mRNA stability
We next characterized the interaction between ELAVL1 and PI3Kδ. RIP assay verified the binding of ELAVL1 to PI3Kδ in HRMECs (Figure 5a). Notably, the abundance of PI3Kδ mRNA in ELAVL1 immune precipitates was decreased with overexpressed miR-192-5p, and PI3Kδ mRNA abundance in ELAVL1 immune precipitates was increased after knockdown of miR-192-5p (Figure 5b). Additionally, ELAVL1 expression was diminished in HGtreated HRMECs transfected with sh-ELAVL1 ( Figure 5c). Meanwhile, the expression of PI3Kδ was significantly down-regulated after ELAVL1 knockdown, and the expression of PI3Kδ was up-regulated after ELAVL1 overexpression (Figure 5d). In the presence of actinomycin D, knockdown of ELAVL1 accelerated the decay of PI3Kδ mRNA, whereas overexpression of ELAVL1 slowed down the decay of PI3Kδ mRNA (Figure 5e). Altogether, the above data indicated that ELAVL1 could maintain PI3Kδ mRNA stability.

| Overexpressed PI3Kδ overturned the inhibitory effects of miR-192-5p up-regulation on microvascular endothelial cell proliferation, migration and angiogenesis
Finally, we evaluated the function of PI3Kδ in miR-192-5p-regulated DR. Our results revealed increased PI3Kδ expression in HG-induced HRMECs transfected with oe-PI3Kδ compared to the control groups ( Figure 6a). Next, we discovered that miR-192-5p greatly reduced cell proliferation and migration in HG-treated cells, which was obviously abrogated by the co-transfection with oe-PI3Kδ (Figure 6b,c). Moreover, compared to HG groups, the tube formation ability of HG cells was effectively suppressed by miR-192-5p up-regulation, while overexpression of PI3Kδ could reverse this effect (Figure 6d). Error bars stand for mean ± SD from six replicate experiments. One-way anova followed by Tukey's post hoc test was performed for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

| DISCUSSION
Diabetes mellitus is a metabolic disease that easily causes damage to tissues and organs, leading to pathological changes. 17 DR is a common complication of type 1 and type 2 diabetes, and one of the leading causes of irreversible vision loss. 18 DR-induced visual impairment is characterized by inflammation, angiogenesis, microaneurysms and irregular blood vessels. 19 The formation of proliferative neovascularization in DR patients will lead to vitreous haemorrhage and complications such as tractional retinal detachment, resulting in irreversible vision loss. 20 Therefore, reversing retinal microvascular dysfunction is of great importance for DR treatment. Here, we demonstrated that miR-192-5p up-regulation inhibited cell proliferation, migration and angiogenesis in HG-treated HRMECs, which was mediated by negatively regulation of ELAVL1 and PI3Kδ expression. Various miRNAs have been reported to be involved in DR progression. For instance, miR-148a-3p resulted in increased cell viability and decreased cell apoptosis in HG-triggered DR through targeting TGFB2 and FGF2. 21 MiR-29b-3p suppressed HRMECs proliferation and angiogenesis via binding to VEGFA and PDGFB. 22 Recently, dysregulation of miR-192-5p was observed to be related to hyperglycaemia and DR. MiR-192-5p inhibition greatly elevated blood glucose content in HepG2 cells. 10 Additionally, methane exerted a protective role in DR through up-regulating retinal miR-192-5p expression. 11 In our project, miR-192-5p was greatly diminished in DR clinical samples and HRMECs induced by HG. Furthermore, we also found that under HG condition, miR-192-5p overexpression led to inhibition of microvascular endothelial cell proliferation, migration and angiogenesis. These findings imply that miR-192-5p is crucial for suppressing DR development.
MiR-192-5p has been reported to bind to targets in a variety of diseases. For example, miR-192-5p could target TRPM7, thereby inhibiting cervical cancer proliferation and invasion. 23 Overexpressed miR-192-5p rendered gastric cancer cells more sensitive to cisplatin. 24 In our project, miR-192-5p could target ELAVL1 and negatively F I G U R E 3 MiR-192-5p directly bound to ELAVL1 and inhibited its expression. (a) Luciferase activity of the wild-type or the mutant of ELAVL1 in HRMECs with miR-192-5p mimics. (b) RIP assay revealed binding of miR-192-5p to ELAVL1. (c) RT-qPCR analysis of miR-192-5p expression in cells transfected with miR-192-5p inhibitor or NC inhibitor. (d, e) Expression of ELAVL1 was evaluated using RT-qPCR and Western blot. All data are represented as the mean ± SD of six independent experiments. Student's t test (for a, b) and one-way anova followed by Tukey's post hoc test (for c-e) were performed for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. regulate its expression. Indeed, literatures indicated that ELAVL1 played a role part in diabetes. As an example, ELAVL1 depletion induced osteogenesis in a diabetic mouse model. 25 In addition, ELAVL1 was involved in renal tubular epithelial pyroptosis in diabetic nephropathy. 26 We further demonstrated that after overexpressing ELAVL1, the effects of miR-192-5p on HG-regulated endothelial cell proliferation, migration and angiogenesis were attenuated. Altogether, we proved that miR-192-5p reduced DR progression through binding to ELAVL1.
PI3Kδ is an essential component of the downstream signalling mechanisms of multiple immune receptors, such as T cell receptor and B cell receptor. 27 Meanwhile, PI3Kδ is responsible for the production of PIP3 and the activation of AKT. 28 Recently, one study reported that PI3Kδ promoted Akt activation, cell proliferation, migration and tube formation in vascular endothelial cells of DR. 16 Moreover, PI3Kδ induced vascular contractility in type 1 diabetes mice. 29 In this study, we confirmed that ELAVL1 could bind to PI3Kδ mRNA and F I G U R E 4 The reduction in microvascular endothelial cell proliferation, migration and angiogenesis caused by miR-192-5p upregulation was reversed by ELAVL1 overexpression. (a) HG-induced HRMECs were transfected with oe-ELAVL1 or oe-NC. RT-qPCR and Western blot were performed to test ELAVL1 level. (b) CCK8 assay was used to determine cell viability in different groups. (c) Transwell assay for analysis of cell migration in different groups. (d) Tube formation experiment was conducted to assess angiogenesis ability of HRMECs in different groups. Values were expressed as mean ± SD of six separate determinations. One-way anova followed by Tukey's post hoc test was performed for statistical analysis. *p < 0.05, **p < 0.01. maintain its stability. Additionally, the suppressive effects of miR-192-5p up-regulation on microvascular endothelial cell proliferation, migration and angiogenesis could be attenuated by overexpressed PI3Kδ. Therefore, miR-192-5p can modulate angiogenesis in DR through PI3Kδ.
In summary, the research revealed that miR-192-5p was markedly decreased in DR. Overexpressed miR-192-5p could inhibit microvascular endothelial cell proliferation, migration and angiogenesis. Moreover, miR-192-5p exerted protective effects by negatively regulating the ELAVL1/PI3Kδ pathway. Our findings indicate miR-192-5p may act as one novel therapeutic target to inhibit DR progression. In the future, potential anti-angiogenic effect of miR-192-5p in DR needs to be further investigated using animal models. Moreover, our research should be confirmed by large-scale clinical studies and more indepth mechanistic studies. F I G U R E 5 ELAVL1 maintained PI3Kδ mRNA stability. (a) RIP detection of the binding of ELAVL1 to PI3Kδ. Student's t test. (b) The abundance of PI3Kδ mRNA in ELAVL1 immune precipitates was detected by RIP. Student's t test. (c) RT-qPCR and Western blot were utilized to analyse ELAVL1 level. One-way anova followed by Tukey's post hoc test. (d) RT-qPCR and western blot were used to evaluate PI3Kδ expression. One-way anova followed by Tukey's post hoc test. (e) PI3Kδ mRNA abundance treated with actinomycin D was evaluated using RT-qPCR after knockdown or overexpression of ELAVL1. Repeated measures anova. Data are presented as mean ± SD of six independent experiments. *p < 0.05, **p < 0.01.

CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
All data generated or analysed during this study are included in this article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Samples of retinal fibrovascular membrane (FVM) were obtained from 20 DR patients who were admitted to Hainan West Central Hospital from September of 2020 to September of 2021. Informed consent was provided by all patients. Approval was obtained F I G U R E 6 Overexpressed PI3Kδ overturned inhibitory effects of miR-192-5p up-regulation on microvascular endothelial cell proliferation, migration and angiogenesis. (a) HG-induced HRMECs were transfected with oe-PI3Kδ or oe-NC. RT-qPCR and Western blot were performed to assess PI3Kδ level. (b) CCK8 assay was used to determine cell viability in different groups. (c) Transwell assay for analysis of cell migration in different groups. (d) Tube formation experiment was conducted to assess angiogenesis ability of HRMECs in different groups. Error bars represent SD of the mean from six separate determinations. One-way anova followed by Tukey's post hoc test was performed for statistical analysis. *p < 0.05, **p < 0.01. from the Ethics Committee of Hainan West Central Hospital.

CONSENT FOR PUBLICATION
The informed consent was obtained from study participants.