miR-200-3p suppresses cell proliferation and reduces apoptosis in diabetic retinopathy via blocking the TGF-β2/Smad pathway

Abstract Increasing evidence has shown that microRNAs (miRNAs) play an important role in the pathogenesis of diabetic retinopathy (DR). However, the role and mechanism of miRNA in regulating high glucose (HG)-induced ARPE-19 cell injury are still not well understood. The present study aimed to investigate the effects of miR-200a-3p on DR progression and reveal the underlying mechanisms of their effects. In the present study, we observed that miR-200a-3p was significantly decreased, while transforming growth factor-β2 (TGF-β2) expression was up-regulated in ARPE-19 cells treated with HG and retina tissues of DR rats. Subsequently, overexpression of miR-200a-3p significantly promoted cell proliferation, reduced apoptosis, as well as inhibited the levels of inflammatory cytokines secreted, matrix metalloprotease 2/9 (MMP2/9), and vascular endothelial growth factor (VEGF) in HG-injured ARPE-19 cells. Moreover, miR-200a-3p was proved to target TGF-β2 mRNA by binding to its 3′ untranslated region (3′UTR) using a luciferase reporter assay. Mechanistically, overexpression of miR-200a-3p reduced HG-induced ARPE-19 cell injury and reduced inflammatory cytokines secreted, as well as down-regulated the expression of VEGF via inactivation of the TGF-β2/Smad pathway in vitro. In vivo experiments, up-regulation of miR-200a-3p ameliorated retinal neovascularization and inflammation of DR rats. In conclusion, our findings demonstrated that miR-200a-3p-elevated prevented DR progression by blocking the TGF-β2/Smad pathway, providing a new therapeutic biomarker for DR treatment in the clinic.


Introduction
Diabetic retinopathy (DR) is one of the most common and serious complications of diabetic mellitus [1]. The worldwide prevalence of DR from a pooled meta-analysis of population studies conducted in the United States, Australia, Europe, and Asia reported an overall prevalence of 35% for any type of DR [2]. In the coming years, the prevalence of DR will rapidly increase with the increasing number and lifespan of people with diabetes. It is caused by abnormal retinal blood vessels that are either proliferatively or functionally incompetent, leading fluid and lipid into the retina and causing visual impairment [3]. However, there are no treatment measures currently to effectively restrict DR progression and the mechanism of DR remains unknown. microRNA (miRNA) is a family of short (20)(21)(22)(23)(24) nucleotides in length) single-stranded RNA, which binds to partially complementary sites primarily found in the 3 untranslated region (3 UTR) of target mRNA and inhibits gene expression via induction of mRNA degradation and translational repression [4,5]. The regulatory functions of miRNAs were widespread, including cellular proliferation, apoptosis,

Materials and methods
Cell culture and transfection ARPE-19 cells were purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. These cells were cultured in 10% fetal bovine serum (HyClone, Logan, U.S.A.) and 1% PS (100 units/ml penicillin and 100 mg/ml streptomycin) medium with GlutaMAX (DMEM, Gibco, U.S.A.).
The day before cell transfection, ARPE-19 cells were seeded in a six-well plate at the concentration of 2 × 10 4 cells per well and cultured in an incubator at 37 • C with CO 2 . Then, the above cells were transfected with miR-200a-3p mimic (mimic), pcDNA-3.1-TGF-β2 (TGF-β2), or TGF-β2+ miR-200a-3p mimic (TGF-β2+mimic) with 30 μl Lipofectamine 3000 reagent (Invitrogen Life Technologies, U.S.A.) according to the manufacturer's protocols. Cells without any treatment were recognized as the control group (NC). These cells were initially transfected for 6 h, then the culture medium was replaced with DMEM/f-12 medium containing 30 mM HG. After incubation for 24 h, the cells were subjected to the following experiments.

Reverse transcription-quantitative polymerase chain reaction
The total RNA of clinical specimens and cell lines was extracted by using TRIzol reagent (QIAGEN, Germany) according to the manufacturer's instructions. For mRNA detection, RNA samples were revere-transcribed into cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Japan). And reverse transcription (RT)-quantitative polymerase chain reaction (RT-qPCR) was performed with GoTaq qPCR Master Mix (TaKaRa, Japan) using the CFX96 Sequence Detection System (Bio-Rad, U.S.A.). For miRNA detection, RNA samples were reverse transcribed using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Japan) using Bio-systems 7300 Real-Time PCR systems. Table 1 Names and sequences of the primers   Name  Primer sequences   miR-200a-3p  F: 5 -CGTAACACTGTCTGGTAACGATGT-3   R: 5 -TGGTGTCGTGGAGTCG-3   TGF-β2  F: 5 -TCCATCTGTGAGAAGCCACA- Abbreviations: F, forward primer; R, reverse primer.
The sequence of the involved primers was designed and listed in Table 1. U6 and β-actin as an endogenous reference for miRNA and mRNA, respectively. RT-qPCR was performed in triplicate.

Flow cytometry
The Annexin V-FITC/Propidium Iodide (PI) apoptosis detection kit (Thermo Fisher, U.S.A.) was employed to determine cell apoptosis ratio according to the manufacturer's instructions. Briefly, after transfection and HG treatment, 2 × 10 4 cells were harvested, washed twice in PBS, and re-suspended in fixation fluid. And then, ARPE-19 cells were stained with 5 μl Annexin V-FITC and 2 μl PI for 15 min and incubated at room temperature. Cell apoptosis was then analyzed with flow cytometry using BD LSR II Flow Cytometry (BD Biosciences, U.S.A.).

Western blotting
The total proteins of the tissues and cells were extracted by using the RIPA lysis buffer purchased from Beyotime Biotechnology (Shanghai, China) according to the manufacturer's protocol. The BCA kit (Beyotime Biotechnology, China) was used to quantify the protein concentration. After that, the target proteins were separated by conduction the electrophoresis with 10% SDS/polyacrylamide gel and transferred to the polyvinylidene fluoride (PVDF) membranes produced by Millipore (MA, U.S.A.). The 5% skim milk was diluted by TBS containing 0.1% Tween-20 and incubated with the membranes for 1 h at 37 • C. The PVDF membranes were incubated overnight at 4 • C with the primary rabbit antibodies against human TGF-β2 (1:1000

Enzyme-linked immune sorbent assay
The expression levels of IL-6, IL-1β, and TNF-α in the retina of rats were detected according to the method of enzyme-linked immune sorbent assay (ELISA) kit (R&D Systems, U.S.A.).

Statistical analysis
All data were collected and presented as the mean + − standard deviation. The data were then analyzed using SPSS 22.0 software (IBM, U.S.A.). Student's t test and one-way ANOVA were used to evaluate statistical significance. P<0.05 was considered to indicate a statistically significant difference.

Effect of HG on the cell viability, apoptosis, and inflammation in ARPE-19 cells
Previous studies suggested that HG was used to trigger the DR model in ARPE-19 cells, the effect of different concentrations of HG on cell viability, apoptosis, and inflammation was examined. Primarily, MTT assay results showed that ARPE-19 cells treated with different concentrations of d-glucose for 24 h significantly inhibited cell viability ( Figure 1A). The IC 50 value of glucose was calculated as 29.10 and 30 mM glucose, acts as HG, was selected to induce ARPE-19 cell injury for further experiments. Meanwhile, the rate of apoptotic cells of ARPE-19 treated with HG was higher than that of the control group (P<0.001, Figure 1B). Similarly, Western blotting showed that HG treatment significantly promoted the expression levels of pro-apoptotic protein Bax and Caspase-3 (both P<0.01, Figure 1C,D), but decreased anti-apoptotic protein Bcl-2 expression (P<0.01, Figure 1C,D). In addition, the expression of VEGF protein was markedly enhanced in HG pretreated ARPE-19 cells compared with the control group (P<0.01, Figure  1C,D). Furthermore, we observed that the expression levels of IL-6, IL-1β and TNF-α, were higher in HG-induced ARPE-19 cells than the NC group (all P<0.01, Figure 1E). These results indicated that the HG-induced ARPE-19 cell injury model was successful.

Effect of miR-200a-3p on proliferation and apoptosis of HG pretreated ARPE-19 cells
Increasing evidence confirmed that miRNAs exerted an important role in the proliferation and apoptosis of ARPR-19 cells exposed to HG [23]. Herein, we examined the expression level of miR-200a-3p in ARPE-19 cells treated with HG. As shown in Figure Figure 2B). MTT assay showed that overexpression of miR-200a-3p notably alleviated the inhibitory effect of HG on the proliferation of ARPE-19 cells (P<0.01, Figure 2C). Flow cytometry analysis results showed that up-regulation of miR-200a-3p significantly decreased the apoptosis rate of ARPE-19 cells treated with HG compared with the HG alone group (P<0.01, Figure 2D,E). Meanwhile, Western blotting revealed that the expression levels of Bax, Caspase-3, and VEGF were decreased in the miR-200a-3p+HG group compared with the HG treatment alone group (all P<0.01, Figure 2F,G), while enhancing the expression of Bcl-2 (P<0.01). Furthermore, the mRNA expression levels of IL-6, IL-1β, TNF-α in the miR-200a-3p+HG group were lower than the HG alone group (P<0.01, P<0.001, Figure 2H). These results strongly implied that overexpression of miR-200a-3p attenuated HG-induced cell apoptosis and inhibited cell viability in ARPE-19 cells.

miR-200a-3p negatively regulated TGF-β2 expression
The miR-200a-3p target gene was obtained from the bioinformatics database (TargetScan; http://www.targetscan. org/vert 71/). The bioinformatics analysis results showed that miR-200a-3p had a binding site with TGF-β2, as shown in Figure 3A. To further investigate the relationship between miR-200a-3p and TGF-β2 was checked by the dual-luciferase reporter gene system. The result showed that the co-transfection of miR-200a-3p mimic and TGF-β2-WT significantly decreased the luciferase activity (P<0.01, Figure 3B), but co-transfection of miR-200a-3p mimic and TGF-β2-MUT did not change the luciferase activity. Moreover, Western blotting showed that overexpression of miR-200a-3p decreased the expression of TGF-β2 protein (P<0.01; Figure 3C). Taken together, these results suggested that TGF-β2 was the direct target of miR-200a-3p which negatively regulated TGF-β2 expressions.

miR-200a-3p targets TGF-β2 to protect HG-induced ARPE-19 cells injury in vitro
To further determine whether miR-200a-3p regulates HG-induced cell injury though targeting TGF-β2 in ARPE-19 cells. Western blotting results showed that overexpression of TGF-β2 enhanced the expression of TGF-β2, Smad2 and Smad3 in HG treated ARPE-19 cells compared with the NC group or HG alone group ( Figure 4A,B), while no significant difference between the HG+TGF-β2+miR-200a-3p mimic group or HG+miR-200a-3p mimic+SRI group and HG treatment alone group. MTT analysis results showed that overexpression of TGF-β2 markedly decreased cell viability of HG pretreated ARPE-19 cells compared with the only HG treatment group (P<0.05, Figure 4C). Flow cytometry analysis results showed that up-regulation of TGF-β2 increased the percentage of apoptotic ARPE-19 cells treated with HG (P<0.05, Figure 4D,E). Moreover, the apoptosis-related proteins Bax, Bcl-2, and Caspase-3 detected by Western blotting were consistent with the result of the flow cytometry analysis ( Figure 4F). Furthermore, the VEGF, MMP-2, MMP-9, TNF-α, IL-6, and IL-1β levels in ARPE-19 cells by transfecting with pcDNA-TGF-β2 plus HG were significantly increased compared with the only HG treated group (all P<0.05, Figure 4G). However, co-transferred with miR-200a-3p mimic+SRI or miR-200a-3p mimic+pcDNA-TGF-β2 in HG pretreated ARPE-19 cells abolished the effect of TGF-β2 up-regulation on the cell viability and the inflammatory cytokines mRNA expression ( Figure  4). These results suggested that up-regulated miR-200a-3p alleviated HG-induced ARPE-19 cell injury by blocking the TGF-β2/Smad pathway.

Effect of miR-200a-3p on the progression of DR through regulating TGF-β2/Smad pathway in vivo
To further examine whether miR-200a-3p-elevated alleviated the progression of DR by blocking the TGF-β2/Smad pathway in the DR rats' model. First, the body weight and the levels of blood glucose in DR rats from normal and STZ-induced diabetic rats at 0, 2, 4, 6, 8, and 10 weeks were measured to evaluate whether the construction of the diabetic rats' model was successful or not. There was significant weight loss and blood glucose increased in DR rats compared with the age-matched control group (NC; Figure 5A,B). Next, immunohistochemical staining demonstrated that the positive expression of VEGF in the retina tissues of the DR model was higher than that of the NC group, as well as, overexpression of miR-200a-3p significantly decreased VEGF positive expression in retinal tissues ( Figure  5D). However, activation of TGF-β2/Smad pathway (SRI-011381, SRI) markedly down-regulated the inhibitory effect of miR-200a-3p on VEGF expression in DR rats ( Figure 5D). Moreover, ELISA results showed that the levels of inflammatory cytokines secreted in the DR+agomir group were lower than that in the DR group (all P<0.01, Figure 5C), but no significant difference was found between the DR+agomir+SRI group and DR group. Western blotting analysis results revealed that the expression levels of apoptotic-related proteins, TGF-β2, VEGF, MMP-2, and MMP-9 in retinal tissues of the DR model were significantly overexpressed compared with the NC group (all P<0.01, Figure 5E-G), while overexpression of miR-200a-3p significantly repressed these effects. However, inhibition of the TGF-β2/Smad pathway alleviated the protection effect of miR-200a-3p ( Figure 5E-G). Furthermore, RT-qPCR results showed that the expression of miR-200a-3p was down-regulated in the retinal tissues of the DR model compared with the NC group (P<0.001, Figure 5H), but overexpressed in the DR+agomir group (P<0.01). Taken together, these results collectively illustrated that overexpression of miR-200a-3p alleviated the progression of DR via blocking the TGF-β2/Smad pathway.

Discussion
As the incidence of DR is increasing all over the world, it is necessary to find the regulatory mechanism or biomarker in the occurrence and development of DR for clinical treatment and diagnosis. In the present study, we found that miR-200a-3p was down-regulated in ARPE-19 cells treated with HG and the retina tissues of DR rats, and overexpression of miR-200a-3p promoted cell viability and decreased the expression levels of MMP-2, MMP-9, TNF-α, IL-1β, and VEGF, which was reversed by up-regulating TGF-β2. The results illustrated that elevated miR-200a-3p alleviated HG-induced cell apoptosis and immunoreaction through down-regulating TGF-β2 in DR progression. Previous studies found that activation of the TGF-β2/Smad pathway contributed to the progression of DR [24]. TGF-β2 could modulate the regulation of cell differentiation, proliferation, apoptosis, epithelial-mesenchymal transition (EMT), and immune response through regulating its downstream Smad family proteins, and eventually promoted the development of DR [18,25]. Yang et al. proved that the TGF-β2/Smad pathway plays an important role in the proliferation, migration, and EMT of HG pretreated human lens epithelial cells [26]. Our studies confirmed that overexpression of TGF-β2 promoted HG-induced cell apoptosis, proinflammatory cytokines secretion, and VEGF expression in ARPE-19 cells. In addition, many studies have elucidated TGF-β receptor-induced responses were mediated through multiple signaling pathways, including ERK/p38/JNK [27], PI3K/Akt/mTOR [28], and NF-κB [29]. In the present study, our data confirmed that HG-induced cell apoptosis and VEGF elevated in ARPE-19 cells via activating the TGF-β2/Smad pathway. Moreover, the mechanism of TGF-β2 modulated migration and EMT of vascular endothelial cells to regulate DR progression will be investigated in our further study.
More and more studies reported that miR-200 families were found to be aberrantly expressed in malignant tumors [30], DR, and pulmonary fibrosis [31]. Herein, our results demonstrated that miR-200a-3p expression was decreased in HG pretreated ARPE-19 cells and retinal tissues of DR rats. Such as, Lo et al. reported that miR-200a-3p was lower expressed in retinal tissues of DR rats [32]. Recently, some miRNAs mediated the progression of DR by regulating the proliferation, apoptosis, inflammatory damage, and MMPs secretion of retinal pigment epithelial (RPE) cells. For example, Ding et al. found that miR-200b/c up-regulation suppressed the proliferation and migration of RPE cells [33]. Repression miR-34a could decrease HG-induced apoptosis and inflammation cytokines secretion of RPE cells treated with HG [32]. MMP-2 was significantly decreased in RPE cells through up-regulating miR-29a [34]. In addition, the miR-200 family mediated the development and progression of many diseases through regulating TGF-β [35,36]. For example, miR-200a [37], miR-200b [38], miR-200c [39]. Importantly, our results found that overexpression of miR-200a-3p targets TGF-β2 to suppress HG-induced ARPE-19 cells apoptosis and inflammation cytokines secretion in vitro.
In conclusion, our results confirmed that increased miR-200a-3p suppressed HG-induced cell apoptosis and immunoreaction by blocking the TGF-β2/Smad pathway in DR progression. Furthermore, this mechanism may provide new biomarkers and targets for the diagnosis and treatment of DR.

Data Availability
The datasets used and/or analyzed in the current study are available from the corresponding author on request. PI, propidium iodide; PVDF, polyvinylidene fluoride; RPE, retinal pigment epithelial; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Smad, sterile alpha motif domain containing; STZ, streptozotocin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; WT, wild type.