Mini-αA Upregulates the miR-155-5p Target Gene CDK2 and Plays an Antiapoptotic Role in Retinal Pigment Epithelial Cells during Oxidative Stress

Background Age-related macular degeneration (AMD) is the leading cause of serious vision loss in the elderly. Regulating microRNA (miRNA) gene expression offers exciting new avenues for treating AMD. This study aimed to investigate whether miRNAs and their target genes play an antiapoptotic role during oxidative stress-induced apoptosis of retinal pigment epithelial (RPE) cells via mini-αA. Methods ARPE-19 cells were treated with 3.5 mM NaIO3 for 48 h to establish a retinal degeneration model. Cells were treated with mini-αA (10, 15, and 20 μM) for 4 h. miR-155-5p was knocked down and overexpressed. Cell viability and apoptosis were measured using the Cell Counting Kit-8 assay and flow cytometry, respectively. The reactive oxygen species level was detected by flow cytometry. miR-155-5p target genes were predicted via bioinformatics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed for miR-155-5p target genes. A quantitative real-time polymerase chain reaction was performed to detect miRNAs and cell cycle-related target genes. Western blotting was performed to measure the levels of apoptotic pathway genes encoding Bcl-2, Bax, cleaved caspase-3, and cyclin-dependent kinase 2 (CDK2). Dual-luciferase reporter gene assay was performed to verify the targeted binding relationship between miR-155-5p and CDK2. Results NaIO3 can induce oxidative damage and promote apoptosis. Conversely, mini-αA had inhibitory effects and could reverse the oxidative damage and apoptosis triggered by NaIO3 in the retinal degeneration model. The expression of miR-155-5p was upregulated in cells treated with NaIO3 and was downregulated after mini-αA treatment. Furthermore, miR-155-5p can target the following cell cycle-related and proliferation-related genes: CDK2, CDK4, CCND1, and CCND2. Moreover, our study indicated that miR-155-5p was involved in the antioxidative damage and antiapoptotic effects of mini-αA via CDK2 regulation. Conclusions miR-155-5p promotes the antioxidative damage and antiapoptotic effects of mini-αA during oxidative stress-induced apoptosis of RPE cells via CDK2 regulation. This study provides a new therapeutic target for AMD.


Introduction
Retinal pigment epithelial (RPE) cells form a layer between the extracellular segments of the choroid and photoreceptors, constituting a key structure for maintaining normal retinal metabolism and visual function [1]. Te abnormal function and apoptosis of RPE cells caused by aging, injury, metabolism, and genetic abnormalities can lead to retinal degeneration, visual impairment, and even irreversible vision loss, representing an important class of blinding diseases, including age-related macular degeneration (AMD), retinitis, and Stargardt's macular dystrophy [2]. Te global prevalence of AMD has reached 8.7%, representing one of the most important causes of blindness among the elderly worldwide [3,4]. AMD is clinically divided into early (moderate retinal edema and retinal pigment changes) and late (neovascularization and atrophy) stages [5]. Te pathogenesis of AMD involves the interaction between genetic polymorphisms and environmental risk factors, many of which lead to an increased oxidative stress in the retina [6]. In recent years, although gene and stem cell therapies have brought hope to patients with such diseases, most patients, especially those in the early stage of AMD, cannot beneft from these novel therapies owing to ethical problems, treatment risks, timing, efcacy, and high cost. RPE cell apoptosis is involved in the coinitiation mechanism of AMD and occurs throughout disease progression [7]. Terefore, it is of great clinical value to investigate the mechanism of maintaining the function and homeostasis of RPE cells and inhibiting apoptosis in these cells, leading to new therapeutic avenues.
Increasing evidence suggests that regulating microRNA (miRNA) expression provides exciting new avenues for the research and treatment of AMD [8]. Dysregulation of miR-17, miR-125b, and miR-155 has been reported in various mouse models of AMD as well as in the plasma and retina of individuals with AMD [9]. SanGiovanni et al. reported that miR-155-5p expression was signifcantly upregulated in the advanced AMD retina [10]. In addition, dysregulation of miR-9, miR-34a, and miR-155 has been reported in the serum of patients with AMD [11]. Terefore, miRNAs are potential biomarkers and novel pharmacological targets for AMD. Hou et al. showed that miRNA-34a inhibited RPE cell proliferation and migration by downregulating its target cyclin-dependent kinases (CDK) 2 and 6 and other cell cycle-related molecules [12]. Trough bioinformatics analysis, we revealed that miR-155-5p potentially targets the cell cycle-and proliferation-related genes encoding CDK2, CDK4, cyclin D1 (CCND1), and cyclin D2 (CCND2). Tis emphasizes the need to further explore the role of miR-155-5p and its downstream target genes in the development and progression of AMD.
α-Crystallins (αA and αB) and their derivatives have received increasing attention due to their great potential in preventing cell death [13]. Recent studies have reported that the expression of αA-and αB-crystallin is signifcantly upregulated in the cytosol and mitochondria of RPE cells in light-induced injury, retinal trauma, and other models of acute retinal degeneration. Moreover, the administration of human αA-or αB-crystallin protects RPE cells from oxidative and endoplasmic reticulum stress-induced apoptosis [14]. Several studies have confrmed that decreased α-crystallin expression can increase oxidative stress-induced cell death sensitivity, whereas increased α-crystallin expression exerts a protective efect [15][16][17]. A previous study revealed that the antiapoptotic efect exerted by αAcrystallin is associated with its molecular chaperone activity [14]. mini-αA is a functional fragment of αA-crystallin with molecular chaperone activity [18] and inhibits caspase-3 activation, thus protecting RPE cells from oxidative stressinduced apoptosis [19]. A previous study revealed that mini-αA can reduce apoptosis induced by NaIO 3 in RPE cells, thus exerting protective efects during retinal degeneration [20]. However, its specifc mechanism of action remains unclear. Terefore, identifying novel regulators mediated by mini-αA may help understand the molecular mechanisms of the antiapoptotic efects of mini-αA in RPE cells.
Tis study used mini-αA to treat a NaIO 3 -induced retinal degeneration model and evaluate its therapeutic efects. Trough bioinformatics prediction and validation, we further revealed the antiapoptotic efects of mini-αA on oxidative stress-induced apoptosis in RPE cells, ultimately providing a new therapeutic target for AMD.

Reactive Oxygen Species (ROS) Detection.
Te ROS level was measured using an ROS assay kit (S0033S; Beyotime). 2′,7′-Dichlorodihydrofuorescein diacetate (DCFH-DA; stock concentration, 10 mM) was diluted at 1 : 1000 in a serum-free medium to a fnal concentration of 10 μM. Te treated cells (mentioned above) were removed from the cell culture medium, and DCFH-DA was added to cover the cells. Subsequently, the cells were again incubated at 37°C for 20 min. Te samples were collected by trypsinization and fow cytometry (A00-1-1102; Beckman, USA) detection was performed.
2.5. Cell Apoptosis Assay. ARPE-19 cells were digested with trypsin (without EDTA). Te trypsinized cells were then washed twice with phosphate-bufered saline and centrifuged at 2000 rpm for 5 min. Next, 500 μL of binding bufer was added to the cells in suspension, followed by the addition and thorough mixing of 5 μL of annexin Vallophycocyanin (KGA1022; KeyGen, China) and 5 μL of 7-AAD (00-6993-50; Invitrogen, USA). After incubation for 15 min at room temperature under dark conditions, the apoptosis rate was measured using fow cytometry within 1 h (A00-1-1102; Beckman).

Statistical
Analysis. Statistical analysis was performed using GraphPad version 8.0. Experimental data were expressed as the mean ± standard deviation with at least three replicates. Diferences between two or more groups were analyzed using student's t-test or one-way analysis of variance. A P value of <0.05 was considered to indicate a statistically signifcant diference.

Mini-αA Inhibits Oxidative Stress-Induced Apoptosis of ARPE-19 Cells.
To determine the role of mini-αA during oxidative stress-induced apoptosis, ARPE-19 cells were treated with mini-αA. Te CCK-8 assay revealed that compared with the control group, cell viability decreased in the NaIO 3 group and increased in the NaIO 3 + mini-αA group (Figure 1(a)), suggesting that mini-αA has a protective efect on the NaIO 3 -induced retinal degeneration model, with 10 μM mini-αA exhibiting the most protective efect. Terefore, 10 μM mini-αA was selected for subsequent experiments. Additionally, the ROS levels signifcantly increased in ARPE-19 cells treated with NaIO 3 for 48 h (Figure 1(b)), which got signifcantly reduced following the treatment with 10 μM mini-αA for 48 h, indicating that mini-αA protects ARPE-19 cells from NaIO 3 -induced oxidative damage. Furthermore, fow cytometry analysis revealed that compared with the control group, apoptosis increased in the NaIO 3 group and decreased in the NaIO 3 + mini-αA group (Figure 1(c)). Western blotting further confrmed that compared with the control group, Bcl-2 expression decreased and Bax and cleaved caspase-3 expression increased in the NaIO 3 group, whereas Bcl2 expression increased and Bax and cleaved caspase-3 expression decreased in the NaIO 3 + mini-αA group (Figure 1(d) and 1(e)). Tese results indicate that NaIO 3 induces oxidative damage and promotes apoptosis in ARPE-19 cells, which were reversed by mini-αA.

Mini-αA Plays a Protective Role during Oxidative Damage and Apoptosis Induced by NAIO 3 by Downregulating miR-155-5p.
To determine whether miR-155-5p was involved in the therapeutic efect of mini-αA on oxidative damage, miR-155-5p interference and overexpression were performed. Figure 3(a) shows the successful transfection of miR-155-5p constructs. Compared with the NaIO 3 + NC inhibitor group, cell viability increased and ROS levels and apoptosis rates decreased in the NaIO 3 + miR-155-5p inhibitor group. Compared with the NaIO 3 + NC mimics group, cell viability increased and ROS levels and apoptosis rates signifcantly Journal of Ophthalmology decreased in the NaIO 3 + mini-αA + NC mimics group. Moreover, cell viability in the NaIO 3 + mini-αA + miR-155-5p mimics group signifcantly decreased, with a signifcant increase in the ROS levels and apoptosis rates (Figures 3(b)-3(d)), suggesting that miR-155-5p is involved in the therapeutic efect of mini-αA on oxidative damage and apoptosis.

Discussion
AMD is a common irreversible eye disease characterized by visual impairment in the elderly [24]. RPE cell death caused by oxidative stress plays a vital role in retinal degeneration pathology and is associated with AMD [25]. Terefore, the protection of oxidative stress-induced RPE cells through antioxidative damage and antiapoptotic efects play a crucial role in treating AMD. In this study, we found that inhibiting the expression of miR-155-5p promoted the antiapoptotic efect of mini-αA on oxidative stress-induced RPE cell apoptosis via CDK2 regulation. For this purpose, an in vitro NaIO 3 -induced retinal degeneration model was established and treated with mini-αA, followed by bioinformatics prediction and functional verifcation. NaIO 3 is an oxidative toxic agent and its selective RPE cell damage makes it reproducible in in vitro and in vivo models of AMD [26]. Although NaIO 3 is not involved in AMD pathology, it can be utilized to understand the mechanism of RPE cell degeneration [27]. Oxidative stressinduced RPE cell apoptosis is an important pathogenic marker of AMD [28]. Oxidative stress afects the lipid-rich retinal outer segment structure and light processing in the macula [29]. In RPE cells, NaIO 3 -induced oxidative stress coordinates with multiple pathways to induce cell death. For example, kaempferol protects ARPE-19 cells from H 2 O 2 - (c) Densitometric quantitation of proteins is probed in Figure 4(b). (d) Te binding site between 3′UTR of CDK2 and miR-155-5p was predicted by miRanda, and dual-luciferase reporter assay was performed to detect the luciferase activity in wild-type (WT) and mutant (mut) of 293T cells after transfection with NC mimics and miR-155-5p mimics. ns, not signifcant; * P < 0.05 and * * P < 0.01.
induced oxidative damage and apoptosis through Bax/Bcl-2 and caspase-3 signaling pathways [30]. α-crystallin protects cells from oxidative stress-induced apoptosis [31]. mini-αA is derived from a highly conserved region of the human lens protein αA-crystallin and exerts anti-infammatory efects [32]. A previous study reported that mini-αA can protect RPE cells from apoptosis induced by NaIO 3 [20]. Consistently, this study revealed that mini-αA can reverse the oxidative damage and apoptosis induced by NaIO 3 in the retinal degeneration model. Many regulatory miRNAs have been implicated in AMD pathology and function [33,34]. Various miRNAs have been proven to be associated with AMD caused by oxidative stress [35,36]. In this study, eight miRNAs were selected for verifcation based on the literature to determine their role in AMD [9][10][11]. Among them, the expression of miR-155-5p was upregulated in cells treated with NaIO 3 and downregulated in those treated with mini-αA. Tis suggested that miR-155-5p played a signifcant role in the NaIO 3 -induced RPE cell retinal degeneration model. Further bioinformatics analysis revealed that miR-155-5p can target the following cell cycle-related and proliferation-related genes: CDK2, CDK4, CCND1, and CCND2. Terefore, genes involved in the miR-155-5p-mRNA network can help understand the onset and development of AMD, which warrants further exploration in future studies.
Several studies have reported the role of miR-155-5p in eye-related diseases. For example, toxoplasmosis is associated with miR-155-5p upregulation [37]. During corneal wound healing, miR-155-5p reduces corneal epithelial permeability by reshaping tight epithelial junctions [38]. In diabetic macular edema, the inhibition of miR-155-5p expression downregulates cell proliferation, angiogenesis, and vascular endothelial growth factor levels [39]. Tese studies demonstrate that miR-155-5p can potentially be used as a biomarker for eye-related diseases. A previous study revealed that decreased miR-1246 expression enhanced the antiapoptotic efect of mini-αA on RPE cells during oxidative stress [40]. Moreover, the expression of miR-155-5p was upregulated in the retina of individuals with advanced AMD [10]. Terefore, we interfered and overexpressed miR-155-5p to determine the mechanism of miR-155-5p in the therapeutic efect of mini-αA during oxidative damage.
CDK2 belongs to the CDK serine/threonine kinase family and is an important regulator of G 1 /S-phase conversion. Bevacizumab signifcantly reduces CDK2, CDK4, and CDK6 as well as cyclin D and E expression and has a preventive efect on AMD by blocking G 1 /S progression in ARPE-19 cells [41]. In addition, miR-34a inhibits RPE cell proliferation and migration by downregulating its target CDK2 and other cell cycle-related molecules [12]. Tis suggests that CDK2 plays a signifcant role in AMD. Based on bioinformatics prediction and functional validation, we revealed that miR-155-5p may be associated with the antioxidative and apoptotic efect of mini-αA via CDK2 regulation. Terefore, miR-155-5p-mediated CDK2 regulation might play a vital role in AMD and could be utilized as a novel molecular biomarker for AMD. However, this study has some limitations, and further studies are warranted to verify the identifed miRNA/mRNA role in AMD pathogenesis.

Conclusion
AMD is a degenerative disease of RPE cells; therefore, determining the role of RPE cells in the disease progression has great clinical signifcance. NAIO 3 can induce the degeneration of RPE cells. Our study revealed that mini-αA can attenuate the NaIO 3 -induced apoptosis and ROS level elevation in RPE cells and can inhibit NaIO 3 -induced upregulation of miR-155-5p. Interference of miR-155-5p expression in NaIO 3 -induced retinal degeneration cell model reduced cell apoptosis and intracellular ROS levels; moreover, miR-155-5p could target CDK2. In conclusion, miR-155-5p promotes the antiapoptotic role of mini-αA in oxidative stress-induced RPE cell apoptosis via CDK2 regulation. Tis study provides a basis for AMD clinical treatment and prognosis and a novel target for treating AMD.

Data Availability
Te datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.