MiR-124-3p Suppresses the Dysfunction of High Glucose-Stimulated Endothelial Cells by Targeting G3BP2

Background: Diabetic retinopathy (DR) is the most important manifestation of diabetic microangiopathy. MicroRNAs (miRNAs), members of non-coding RNAs, have been frequently reported to regulate various diseases including DR. MiR-124-3p is involved in DR based on bioinformatics. The current study aimed to investigate the role of miR-124-3p in high glucose (HG)-treated human retinal microvascular endothelial cells (HRMECs), an in vitro model of DR. Methods: Bioinformatics analysis was applied to reveal the targets downstream miR-124-3p. A series of assays including CCK-8, luciferase reporter, western blot, and tube formation assays were used to explore the function and mechanism of miR-124-3p in HG-stimulated HRMECs. Results: We found out that miR-124-3p was downregulated in HG-stimulated HRMECs. Functionally, miR-124-3p overexpression restrained the HG-induced cell injury of HRMECs. Mechanistically, we predicted 5 potential target mRNAs of miR-124-3p. G3BP stress granule assembly factor 2 (G3BP2) was validated to bind with miR-124-3p. Rescue assays showed that miR-124-3p suppressed cell injury of HG-stimulated HRMECs through G3BP2. In addition, miR-124-3p regulated the p38MAPK signaling pathway by G3BP2, and G3BP2 promoted injury of HG-treated HRMECs through the activation of the p38MAPK signaling pathway. Conclusion: MiR-124-3p suppressed the dysfunctions of HG-treated HRMECs by targeting G3BP2 and activating the p38MAPK signaling. This new discovery provided a potential biomarker for DR treatment.


INTRODUCTION
Diabetic retinopathy (DR) is a common microvascular complication of diabetes (Cheung et al., 2010;Henriques et al., 2015;Diallo et al., 2019). Early features of DR include blood-retina barrier (BRB) breakdown, capillary acellularity and pericyte loss and it has been confirmed that the main factor contributing to the progression of DR is chronic hyperglycemia (Stitt et al., 2016;Powers et al., 2017). In the last decade, significant advances in the diagnosis and treatment of DR have been made (Jenkins et al., 2015;Ebneter and Zinkernagel, 2016). However, DR is still the main cause of vision loss in the world (Ebneter and Zinkernagel, 2016;Horton et al., 2016). Therefore, it is of great significance to deepen our understanding of DR development.
In the early stage of DR, human retinal microvascular endothelial cells (HRMECs), components of the BRB, are impaired by the adverse impact of high glucose (HG), resulting in BRB dysfunction and accelerating DR progression (Strauss, 2005;Miyamoto et al., 2007). In many previous studies, HG-stimulated HRMEC was employed as an in vitro model of DR to explore the influences of specific genes on DR (Abu El-Asrar et al., 2016;Gu et al., 2019;Zhu et al., 2019). Similarly, HRMEC was employed as an in vitro model of DR in this study.
MicroRNAs (miRNAs) are small and short non-coding RNAs, binding to the 3 -untranslated regions (3 -UTRs) of messenger RNAs (mRNAs) to participate in the regulation of various diseases (Bentwich, 2008;Erson-Bensan, 2014;Mohr and Mott, 2015;Armand-Labit and Pradines, 2017;Lu and Rothenberg, 2018). For example, miR-22 overexpression restrains oxidative stress injury in diabetic cardiomyopathy by targeting Sirt 1 . MiR-27a-3p attenuates brain injury and blood-brain barrier dysfunction by targeting endothelial AQP11 after intracerebral hemorrhage (Xi et al., 2018).  facilitates cardiomyocyte hypertrophy via targeting MFN2 and FBXW7 (Wang et al., 2019). Specially, it has been reported that miRNAs are vital players in the regulation of DR (Mastropasqua et al., 2014;Martinez and Peplow, 2019;Satari et al., 2019;Shafabakhsh et al., 2019). Previous studies have demonstrated that miR-124-3p is closely related to several diseases including tuberous sclerosis complex angiomyolipoma and brain injury Vuokila et al., 2018;Liang et al., 2019). Importantly, it has been reported that MALAT1 exerts essential effect on DR partly through the regulation of Sag and Guca1a via miR-124-3p (You et al., 2018). Driven by it, we detected the expression of miR-124-3p in HG-stimulated HRMECs and found that HG induced the downregulation of miR-124-3p in HRMECs, which encourages us to further explore its functions in HG-stimulated dysfunctions of HRMECs.
G3BP stress granule assembly factor 2 (G3BP2) has been reported to participate in the regulation of several diseases including cardiac hypertrophy, foot-and-mouth disease, and cancers (Wei et al., 2015;Hong H.Q. et al., 2018;Visser et al., 2019). Particularly, G3BP2 was identified to aggravate the development of diabetic nephropathy (Carney, 2016;Zhao et al., 2016). Based on bioinformatics analysis, G3BP2 is targeted by miR-124-3p. However, no study has been conducted on the role of G3BP2 in DR.
We hypothesized that miR-124-3p can suppress the HGinduced injury in HRMECs and conducted functional assays to confirm it. Moreover, the association between G3BP2 and miR-124-3p was explored. Our research may shed some light on the pathology of DR, which may help develop more effective therapeutic methods to conquer this disease.

Cells and Cell Culture
The human retinal microvascular endothelial cells (HRMECs; Chinese Academy of Sciences Cell Bank, shanghai, China) were cultured in the Dulbecco's Modified Eagle Medium (DMEM; Gibco, United States) added with 10% fetal bovine plasma (FBS, Gibco). The HRMECs were incubated in a humidified atmosphere at 37 • C containing 5% CO 2 .

Cell Treatment
Human retinal microvascular endothelial cells (HRMECs) in control (Con) group were treated with 5 mM glucose. In mannitol group, cells were treated with 5 mM glucose plus 30 mM mannitol (osmotic control). In high glucose (HG) group, cells were treated with 15 mM, 30 mM, 45 mM glucose. Cells were treated with mannitol and/or glucose for 24 h. HRMECs treated with 30 mM glucose were cultured for 0 h, 12 h, 24 h, 48 h, and 72 h for the detection of cell viability. SB203580 (50 mM; a specific inhibitor of p38 MAPK) was used to treat HRMECs for 60 min as previously described Chen et al., 2020). SB203580 was bought from Sigma (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO, Sigma).

Western Blot
Cell lysate was collected using RIPA lysis buffer.

Luciferase Reporter Assay
The 3'UTR of G3BP2 containing miR-124-3p binding site was predicted from the starBase online database. The wild-type (Wt) or the mutant (Mut) 3'UTR of G3BP2 was inserted into pmirGLO vectors (Promega, Madison, MI) to construct the pmirGLO-G3BP2-Wt or Mut vectors. These vectors were then co-transfected with miR-124-3p mimics or NC mimics into HRMECs by Lipofectamine 3000 (Invitrogen). The luciferase activities were detected 48 h after transfection with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States). The firefly luciferase activity was normalized to Renilla luciferase activity.

Tube Formation Assay
The tube formation ability was determined by performing tube formation assay. Growth Factor Reduced Matrigel matrix (Corning) (300 µL) was put on the bottom of a 24-well plate, and HRMECs (2 × 10 4 cells/per well) were seeded into wells.
After 20 h, capillary-like structures were visualized with a Nikon Eclipse Ti inverted microscope (Nikon). For each well, at least six different fields were randomly chosen for observation. Finally, meshes and branch length of the capillary-like structures were evaluated employing ImageJ software (version1.49p; NIH, Bethesda, MD, United States).

Statistical Analysis
All experiments were performed three times. Statistical analysis was conducted by SPSS 13.0. The data were shown as the mean ± SD. Differences between two groups were analyzed employing the two-tailed unpaired t-test. Multiple comparisons were calculated using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

MiR-124-3p Was Downregulated in HG-Stimulated HRMECs
First, we detected the viability of HRMECs in the medium containing glucose of different concentrations (15 mM, 30 mM, and 45 mM). The results demonstrated that the viability of HRMECs was enhanced by HG treatment for 24 h, and 30 mM HG achieved the best effects ( Figure 1A). We employed 30 mM HG to treat HRMECs in the following experiments. Next, we observed that the viability of HRMECs was significantly enhanced by the increasing time of HG (30 mM) treatment and the viability reached the highest at 48 h ( Figure 1B), so we treated cells with HG for 48 h in the following assays. In addition, we detected the expression of miR-124-3p by increased concentrations of glucose, and found that miR-124-3p level in HRMECs was gradually decreased by increased concentrations of glucose ( Figure 1C). Moreover, miR-124-3p level in HRMECs was also time-dependently downregulated by HG stimulation (Figure 1D). The present study is based on the bioinformaticsbased results of a previous study (You et al., 2018), which showed that miR-155-5p, miR-1-3p, miR-122-5p, miR-223-3p, miR-125b-5p, and miR-124-3p are potential links between MALAT1 and five visual perception-related genes (PDE6G, GUCA1A, RHO, SAG, and PRPH2) in DR. Expression of abovementioned molecules in the in vitro model of DR needs validation. We found that miR-155-5p, miR-122-5p, miR-223-3p, miR-125b-5p were all downregulated in HG-treated HRMECs, while expression of miR-1-3p showed no significant difference between control and HG groups (Supplementary Figure 1A). Expression of MALAT1, PDE6G, GUCA1A, RHO, SAG, and PRPH2 in HRMECs was increased by HG stimulation (Supplementary Figure 1B), which was consistent with the previous study.

MiR-124-3p Inhibited the p38MAPK Signaling Pathway Through G3BP2
A previous study has demonstrated that G3BP2 is involved in the regulation of the p38MAPK signaling pathway, and p53 is a downstream target of p38 mitogen-activated protein kinase (p38MAPK) signaling (Zhao et al., 2016). Accordingly, we hypothesized that miR-124-3p regulated p38MAPK signaling pathway through G3BP2 in HG-stimulated HRMECs.
With western blot analysis, we observed that miR-124-3p overexpression reduced the protein levels of phosphorylated p53 and p38, which were then partially recovered by overexpressed G3BP2, suggesting that miR-124-3p regulated the p38MAPK signaling pathway through G3BP2 (Figure 5).

G3BP2 Promoted Cell Injury of HG-Treated HRMECs Through the p38MAPK Signaling Pathway
To investigate whether G3BP2 regulates cell injury through the p38MAPK signaling pathway in HG-stimulated HRMECs, we performed rescue assays. First, we found that G3BP2 overexpression-induced increase of the protein levels of phosphorylated p53 and p38 was counteracted by the introduction of SB203580 in HG-stimulated HRMECs ( Figure 6A). Next, we observed that the introduction of SB203580 offset the promotive effect of G3BP2 overexpression on the viability of HG-stimulated HRMECs ( Figure 6B). G3BP2 overexpression-induced elevation of mesh number and branch length in tube formation of HG-stimulated HRMECs was offset by SB203580 ( Figure 6C). Meanwhile, G3BP2 overexpressioninduced increase in the levels of proteins associated with angiogenesis (VEGFA, TGFB1 and Angiopoietin-1) was rescued by the treatment of SB203580 in HG-stimulated HRMECs ( Figure 6D). The decrease in the levels of proteins associated with blood-ocular barrier (ZO-1, Occludin and Claudin-5) caused by upregulated G3BP2 was partially restored by SB203580 in HG-stimulated HRMECs ( Figure 6E). In summary, G3BP2 contributed to HG-stimulated injury of HRMECs through the activation of the p38MAPK signaling pathway.

DISCUSSION
Since HRMECs are considered as the major targets of hyperglycemic injury , the exploration of the role of miRNAs that regulate HRMECs is of great importance for the understanding of DR. The current study aimed to explore the role of miR-124-3p in HG-stimulated HRMECs, an in vitro model of DR. Our data exhibited the vital role of miR-124-3p in suppressing the dysfunctions of HG-stimulated HRMECs. First, our findings demonstrated that miR-124-3p was downregulated in HG-stimulated HRMECs. MiRNAs are major regulators in a variety of diseases (Kaul and Krams, 2015;Trionfini and Benigni, 2017). Specially, it has been confirmed that miRNAs exert pivotal functions in the initiation and development of DR. For example, miR-138-5p exerts a protective role the early DR by regulating NOVA1 (Bao and Cao, 2019). Serum miR-122 levels is associated with DR (Pastukh et al., 2019). MiR-1273g-3p is involved in DR development (Ye et al., 2017). Recently, it has been reported that miR-124-3p participates in the regulation of various diseases, such as traumatic brain injury (Schindler et al., 2020) and neuropathic pain . Importantly, miR-124-3p was reported to be associated with DR (You et al., 2018).
Subsequently, experimental data revealed that miR-124-3p overexpression restrained the HG-induced cell injury of HRMECs by suppressing tube formation ability, reducing pro-angiogenic factors and increasing tight junction proteins. Consistent with this study, previous research has shown that miR-124-3p restrains cell injury in some other diseases, for example, miR-124-3p alleviates the neuronal injury of SH-SY5Y cells induced by MPP + (Geng et al., 2017). MiR-124-3p inhibits cell injury in traumatic brain injury (Vuokila et al., 2018;Schindler et al., 2020). MiR-124-3p alleviates neuronal apoptosis induced by mechanical injury .
Additionally, in this study, G3BP2 was identified as a downstream target of miR-124-3p in HRMECs. G3BP2 is found to be involved in diabetic nephropathy (Carney, 2016;Zhao et al., 2016). We identified that G3BP2 is upregulated in HG-stimulated HRMECs, which indicated the potential involvement of G3BP2 in DR. G3BP2 overexpression rescued the suppressive effects of miR-124-3p on HG-induced injury of HRMECs by promoting tube formation ability, increasing pro-angiogenic factors and decreasing tight junction proteins. Moreover, the p38MAPK signaling pathway is frequently reported to be activated during DR (Zhang et al., 2013;Dong et al., 2020;Liu et al., 2020). A previous research has reported that G3BP2 is involved in the regulation of the p38MAPK signaling pathway (Zhao et al., 2016). In this study, we observed that G3BP2 promoted the HG-induced injury of HRMECs by the p38MAPK pathway. MiR-124-3p suppressed the ratio of p-p38/p38 and p-p53/p53, and the trend was rescued by G3BP2, indicating that miR-124-3p suppressed the p38MAPK signaling pathway by G3BP2.

CONCLUSION
The current study provided an evidence that miR-124-3p suppressed the dysfunction of HG-stimulated HRMECs by targeting G3BP2 and suppressing MAPK signaling pathway. Our finding pointed out the potential of miR-124-3p in the development of novel therapeutic methods for DR.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
HZ and YH wrote first draft of the manuscript and commented on previous versions of the manuscript. Both authors read and approved the final manuscript, contributed to the study conception and design, material preparation, and data collection and analysis.