MicroRNA-29b-3p Promotes Human Retinal Microvascular Endothelial Cell Apoptosis via Blocking SIRT1 in Diabetic Retinopathy

Background Diabetic retinopathy (DR) is a main complication of diabetes mellitus (DM). Recent studies have implicated microRNAs in human retinal microvascular endothelial cell (HRMEC) dysfunction. In this study, we aim to investigate the apoptotic promotion of miR-29b-3p by blocking SIRT1 in HRMEC for DR situation. Method Blood samples were obtained from DR patients and controls. Dual-luciferase reporter assay using HEK-293T cells was performed to show the direct interaction of miR-29b-3p and the 3′UTR of SIRT1. HRMECs were exposed to 5.5 mmol/L of glucose (normal control), 5.5 mmol/L of glucose and 24.5 mmol/L of mannitol (osmotic pressure control), 30 mmol/L of glucose [hyperglycemia (HG)], 150 μmol/L of CoCl2 (hypoxia), and 30 mmol/L of glucose plus 150 μmol/L of CoCl2 (HG-CoCl2). To identify the regulating relationship between miR-29b-3p and SIRT1, HRMECs were transfected with miR-29b-3p mimics/inhibitors or their negative controls. SRT1720 was used as a SIRT1 agonist. Cell viability was assessed with the cell counting kit-8 (CCK-8) assay, and apoptotic cells were stained by one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit. Gene and protein expression were assayed by quantitative real-time reverse transcriptase-PCR (RT-qPCR) and western blotting separately. Result MiR-29b-3p was upregulated to 3.2-fold, and SIRT1 protein was downregulated to 65% in DR patients. Dual-luciferase reporter assay showed the direct interaction of miR-29b-3p and SIRT1. HRMECs were identified as >95% positive for CD31 and von Willebrand factor (vWF). MiR-29b-3p and Bax/Bcl-2 ratio was upregulated, whereas SIRT1 was downregulated in HRMECs in the HG-CoCl2 condition. Decreased cell viability and upregulated apoptosis were also found in HRMECs of the HG-CoCl2 condition. Upregulated miR-29b-3p decreased the expression of SIRT1 and increased the ratio of Bax/Bcl-2, whereas downregulated miR-29b-3p increased the expression of SIRT1 protein and downregulated the ratio of Bax/Bcl-2. SRT1720 rescued miR-29b-3p-induced HRMEC apoptosis via upregulating the expression of SIRT1 protein. Conclusion The dysregulation of miR-29b-3p/SIRT1 is a potential mechanism of HRMEC apoptosis in DR. MiR-29b-3p/SIRT1 may be a potential therapeutic target for DR.


INTRODUCTION
Diabetic retinopathy (DR) is a main complication of diabetes mellitus (DM), and it is a leading cause of blindness in working-aged adults worldwide (Chong et al., 2017). Vascular damage, which may be caused by cell apoptosis, inflammation, oxidative stress, and a series of metabolic disorders, is an initial characteristic and will exacerbate DR progression (Kannenkeril et al., 2018;Miloudi et al., 2019). Once the blood-retina barrier is broken down, dangerous factors from the circulating blood will leak into the retinal tissue and cause irreversible damage to the retinal neural cells (Trost et al., 2016;Xu and Chen, 2017). Although a series of studies have been carried out to investigate the pathogenesis of human retinal vascular endothelial cell apoptosis (Santiago et al., 2018;Whitehead et al., 2018), the mechanism is largely unknown.
Recent studies have revealed the decrease of SIRT1 in diabetes patients (Balestrieri et al., 2013) and the increase of miR-29b-3p in diabetes (Esteves et al., 2018). Furthermore, Su et al. (2019) have proved the direct regulation of miR-29b-3p to SIRT1 in insulin resistance. To our knowledge, there is no research to illuminate the regulatory relationship of miR-29b-3p/SIRT1/Bax/Bcl-2 pathway in human retinal microvascular endothelial cell (HRMEC). Here, we performed this study to verify the role of miR-29b-3p in DR.

Patients and Tissues
This study was conducted in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of Aier Eye Hospital (Changsha, Hunan, China). This study was approved by the ethics committee of Aier Eye Hospital (AIER2018IRB21) and registered on the International Clinical Trials Registry Platform (ChiCTR1900025449). Consent was obtained from all the participants before collection. Blood samples were obtained from 21 DR patients aged 37-71 years. Negative control (NC) blood samples came from 11 pterygium patients without DM aged 53-69 years. All the samples were stored at −80 • C for further experiments. Human retinas in this study were obtained from organ donors without DM or retinal diseases.

Dual-Luciferase Reporter Assay
We predicted SIRT1 as a potential target of miR-29b-3p by using miRNA database (TargetScanHuman 7.2). Then the 3 -UTR of human SIRT1 containing the predicted binding sites [wild type (WT)] or mutated binding sites [mutant type (MUT)] was amplified and inserted into pmir-RB-Report TM vector. The reporter plasmids and miR-29b-3p mimics or NC were co-transfected into HEK-293T cells using Lipofectamine 2000 (Invitrogen) to determine if SIRT1 is a direct target of miR-29b-3p. Firefly and Renilla luciferase activities were measured 48 h after transfection using the Dual-Glo R Luciferase Assay System (Promega, Madison, WI, United States).

Cell Culture and Treatment
Primary HRMECs were isolated according to methods described previously (Fan et al., 2016); briefly, retinas were immersed in phosphate-buffered saline (PBS) with 5% penicillinstreptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, United States) for 5 min and then transferred into Dulbecco's modified Eagle's medium (DMEM) to remove vitreous. Retinas were then minced into small pieces and digested with type II collagenase (Sigma Aldrich, St. Louis, MO, United States) at 37 • C for 30 min. Cell suspension was filtered through a 70-µm mesh and cultured in endothelial cell medium (ECM; ScienCell, San Diego, CA, United States). Culture medium was replaced every 2-3 days.
To explore the HRMEC damage in DR, we constructed an in vitro model of hyperglycemia and hypoxia conditions. HRMECs were cultured in 5.5 mmol/L of glucose (normal control), 5.5 mmol/L of glucose and 24.5 mmol/L of mannitol (osmotic pressure control), 30 mmol/L of glucose [hyperglycemia (HG)], 150 µmol/L of CoCl 2 (hypoxia), 30 mmol/L of glucose, and 150 µmol/L of CoCl 2 (HG-CoCl 2 ). Culture medium was refreshed every 24 h. SRT 1720 Hydrochloride (MedChemExpress, Monmouth Junction, NJ, United States) was used as an activator to upregulate the expression of SIRT1.

Cell Transfection
Cells were seeded in 6-well and 96-well plates with a density of 2 × 10 5 /well and 4 × 10 3 /well. The miR-29b-3p mimics, inhibitors, and their NCs were purchased from RiboBio (Guangzhou, China) and transfected into cells using riboFECT TM CP Reagent (Guangzhou, China) according to the manufacturer's protocols. NC mimics labeled with Cy3 fluorescence (Guangzhou, China) were transfected to observe the transfect efficiency directly. After 30 h of transfection, the HRMECs were collected for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain, cell counting kit-8 (CCK-8), quantitative real-time reverse transcriptase-PCR (RT-qPCR), and Western blot (WB) assay.

Cell Apoptotic and Viability Assay
For apoptosis and viability assay, 4 × 10 3 cells/well were seeded into 96-well plates and cultured at 37 • C with 5% CO 2 in a humidified environment. The One Step TUNEL Apoptosis Assay Kit (Beyotime) was used for detecting apoptotic cells. Nuclei were stained with DAPI (blue). Fluorescent images were acquired by a fluorescence microscope (ECLIPSE Ts2R, Nikon). The quantification of TUNEL-positive cells was obtained by ImageJ software and calculated by GraphPad Prism version 5.0. Cell viability was determined by a CCK-8 assay (MedChemExpress, Monmouth Junction, NJ, United States). Seven replicates per group and a group without cells served as the blank. After being treated with different conditions, 100 µl of fresh culture medium with 10% CCK-8 solution was added to each well and incubated at 37 • C for 1.5 h. The absorbance at 450 nm was observed by Synergy TM HTX Multi-Mode Microplate Reader (Bio-Tek Technologies, Winooski, VT, United States). The relative viability of cells was calculated according to the manufacturer's protocol.

Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reactions
MicroRNA was isolated with a microRNA kit (Omega Bio-Tek, Norcross, GA, United States) and reversed to cDNA with a reverse transcription kit (Roche, Basel, Switzerland); the stem-loop method was especially used for microRNA reverse transcription as described previously (Chen et al., 2005). The RT product was subjected to 45 cycles of qPCR reactions with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Jiangsu, China) in a Roche LightCycler R 96 System (Roche, Basel, Switzerland). U6 was used to normalize the expression FIGURE 1 | The clinical expression pattern and dual-luciferase reporter assay of miR-29b-3p and SIRT1. (A) In DR patients' blood, miR-29b-3p was upregulated to 3.2-fold compared with that of control group. (B) DR patients' blood SIRT1 protein was downregulated to 65% compared with that of control group. (C) SIRT1 is a direct target of miR-29b-3p predicted by the miRNA online database (TargetScanHuman7.2 and miRBase). (D) Dual-luciferase reporter assay using HEK-293T cells showed the direct interaction of miR-29b-3p and the 3 UTR of SIRT1. After 48-h cotransfection, overexpression of miR-29b-3p reduced the luciferase activity of WT reporter but had no inhibition on the MUT reporter. Data are shown as mean ± SD, *p < 0.05, **p < 0.01. NC, negative control; DR, diabetic retinopathy; WT, wild type; MUT, mutant type.
of microRNA. The relative expression level of miRNA was calculated by the 2 − CT method. The specific primers for miR-29b-3p and U6 are listed in Table 1.

Western Blots
After being treated with different conditions, cells were washed twice with ice-cold PBS and lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Lysates were then centrifuged at 12,000 rpm for 20 min at 4 • C to collect the supernatant. Protein quantification was performed using BCA Protein Assay Kit (Solarbio) according to the company's protocol. The supernatant proteins were concentrated with the method described previously (Zaiss et al., 2013). Briefly, supernatant, methanol, and chloroform were mixed thoroughly. The mixture was centrifuged at 10,000 rpm for 10 min at 4 • C, and the supernatant was discarded carefully. Then another volume of methanol was added to the pellet and vortexed to mix thoroughly. The mixture was again centrifuged at 12,000 rpm for 10 min at 4 • C, and the supernatant was discarded. After being air-dried for 5 min, the proteins were dissolved with the lysis buffer from a Caspase 3 Activity Assay Kit (Beyotime), and the quantification was performed using a Bradford Protein Assay Kit (Solarbio).
Protein was denatured using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Solarbio) by heating the samples at 98 • C for 6 min. Electrophoresis was performed using 10% SDS-PAGE gel and transferred onto nitrocellulose membranes (Pall) and blocked with 5% non-fat milk containing Tween-20 for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4 • C. IRDye R 800CW goat anti-rabbit/mouse secondary antibody (LI-COR) was used to detect primary antibody binding. The immunoblots were analyzed and quantified using ImageJ software. Antibodies to SIRT1(19A7AB4), Bax (E63), and Bcl-2 (E17) were obtained from Abcam. Caspase-3 and β-actin (8H10D10) antibodies were obtained from Cell Signaling Technology. Total protein stain was performed by using a REVERT Total Protein Stain kit (LI-COR). Relative quantification of cleaved caspase-3 in supernatant was achieved by normalizing each target to the value of total proteins.

Statistical Analysis
Statistical Package for Social Science (SPSS) software version 19.0 and GraphPad Prism version 5.0 were used for descriptive analysis. The data were shown as mean ± standard deviation (SD). The results presented in the paper were representative of at least three different repetitions. Student's t test was performed to assess differences between two means. A chisquare test for qualitative data was applied. One-way or two-way ANOVA followed by Bonferroni's post hoc test was performed in multiple means comparison. Statistical significance was defined as p < 0.05.

SIRT1 Is a Direct Target of MiR-29b-3p in Diabetic Retinopathy
The baseline data of clinical samples are shown in Table 2.
To explore the expression pattern of miR-29b-3p and SIRT1 in DR patients, RT-qPCR and WB were performed. MiR-29b-3p RNA was upregulated to 3.2-fold (Figure 1A), and SIRT1 protein was downregulated to 65% ( Figure 1B) in DR patients' blood samples. With the miRNA online database (TargetScanHuman7.2 and miRBase), we found that SIRT1 is a direct target of miR-29b-3p ( Figure 1C). Dual-luciferase reporter assay using HEK-293T cells showed the direct interaction of miR-29b-3p and the 3 UTR of SIRT1. After 48-h cotransfection, overexpressed miR-29b-3p reduced the luciferase activity of WT reporter but had no inhibition on the MUT reporter ( Figure 1D). The results of this study showed that miR-29b-3p could inhibit the expression of SIRT1 by binding with the 3 -UTR of SIRT1, and SIRT1 might be the downstream target gene of miR-29b-3p.

Verification of Human Retinal Microvascular Endothelial Cell
Human retinal microvascular endothelial cell clusters began to form on the third day after plating. After 10-day culture, the cells showed an oval morphology and a contact-inhibited monolayer (Figures 2A,B). Immunofluorescence was performed to detect CD31 and vWF, which were well-known typical vascular endothelial cell markers. As a result, both CD31 ( Figure 2C) and vWF ( Figure 2D) were positive on the same cells ( Figure 2E). These results verified the cell type and purification.

DISCUSSION
Diabetic retinopathy is a serious complication of DM, which may cause irreversible vision loss or even blindness. Microvascular damage is a typical worsening characteristic as DM progresses (Stehouwer, 2018). However, the mechanism of microvascular damage in DR is complicated and should be further studied. In this study, we illuminated the dysregulation of miR-29b-3p/SIRT1 in DR patient blood samples. Additionally, using cultured HRMEC apoptotic model induced by HG-CoCl 2 , we proved that miR-29b-3p promoted HRMEC apoptosis. These findings indicate that miR-29b-3p may be an important regulator in vascular damage in DR progression.
Previous studies revealed that dysregulation of miR-29 family promoted cell apoptosis in many human diseases including cerebral ischemia/reperfusion injury, pulmonary arterial hypertension, and myocarditis (Chen et al., 2018;Huang et al., 2018;Zhang et al., 2018). Yuan et al. (2018) found that miR-29b would activate NF-κB, thus aggravating endothelial cell inflammatory damage. Overexpression of miR-29b increased the expression of cleaved caspase-3, which is a typical apoptotic factor (Yuan et al., 2018). Saravanan et al. (2019) reported that miR-29b-3p was selectively released in exosomes from inflammation and hypoxia induced islets before apoptosis and cell death, which coincided with activation of endoplasmic reticulum (ER) stress response markers IRE-1α, XBP1, HIF-1α, and CHOP. They also detected the selective release of miR-29b-3p in plasma exosomes after these cells were transplanted into streptozotocin (STZ) diabetic nude mice. Therefore, miR-29b-3p might be an early indicator of human islet cell apoptosis during prediabetic conditions (Saravanan et al., 2019). MiR-29b-3p was obviously upregulated in high glucose-induced endothelial cells (Silambarasan et al., 2016), and overexpression of miR-29b-3p could cause insulin resistance in mice (Su et al., 2019). These findings indicate that miR-29b-3p may be involved in vascular damage and DM progression. Our findings in clinical samples and HG-CoCl 2 -induced HRMEC are consistent with these studies.
SIRT1 was proved to alleviate inflammation and apoptosis via deacetylating inflammatory transcription factors; therefore, SIRT1 was gradually coming to be interpreted as a DR protector (Mishra et al., 2018), whereas in DM patients and especially in patients with poor glycemic control, SIRT1 was significantly downregulated (Balestrieri et al., 2013). We also verified this and found that SIRT1 protein was downregulated in DR blood samples. Furthermore, we revealed the tendency that SIRT1 was decreased in DR patients' plasma using the ELISA method (Supplementary Figure 1). Mariani et al. (2015Mariani et al. ( , 2016 and Khalyfa et al. (2019) reported that SIRT1 was downregulated in DM-associated metabolic diseases plasma and the decreased exosome SIRT1 might be correlated with endothelial dysfunction. However, the regulatory mechanism of diabetes to SIRT1 is not clear. Recent studies revealed that the microRNA might be an indispensable regulator (Yamakuchi and Hashiguchi, 2018). Thounaojam et al. (2019) found that overexpression of miR-34a could decrease the expression of SIRT1 directly and induce mitochondrial dysfunction in high glucose-induced retinal endothelial cells. Thus, miR-155-5p, miR-106b, etc. were successively identified to target SIRT1 directly in diabetes conditions (Chen and Yang, 2017;Jiao et al., 2018;Wang et al., 2018). We predicted that SIRT1 was a direct target of miR-29b-3p by the miRNA online database (TargetScanHuman7.2 and miRBase). We also found the different expression patterns of miR-29b-3p/SIRT1 in DR patient and control blood samples. However, the regulatory mechanism of miR-29b-3p to SIRT1 in DR is still unknown. Our in vitro research displayed that overexpression of miR-29b-3p in HRMEC downregulated SIRT1 protein expression and promoted cell apoptosis and that the apoptotic promotion of upregulated miR-29b-3p could be rescued by a SIRT1 specific agonist SRT1720. Furthermore, we verified that both miR-29b-3p inhibitor and SRT1720 could increase SIRT1 protein expression and alleviate cell apoptosis in HG-CoCl 2induced HRMEC. Therefore, we suggest that SIRT1 is a direct target of miR-29b-3p in DR patients' retinal microvascular endothelial cells.
In summary, in this study, we investigated the different expression of miR-29b-3p/SIRT1 in blood samples from DR patients and controls. We found that miR-29b-3p was upregulated and SIRT1 was downregulated in DR blood samples. We further explored the interaction mechanism of miR-29b-3p and SIRT1 in cultured HRMEC apoptotic model induced by HG-CoCl 2 . To the best of our knowledge, the relevant reports on the mechanism of miR-29b-3p/SIRT1 in diabetic HRMEC have not yet found. Our study illuminated this mechanism preliminarily. Our data provided the proof that miR-29b-3p/SIRT1 may be a potential therapeutic target for DR. However, DR is a very complicated disease that may be affected by aging, diabetes duration, smoking, etc. (Varma et al., 2014;Chen et al., 2019). We need more clinical samples to verify our findings especially the vitreous or retinal tissues from DR patient surgeries. We need more in vitro research on the apoptotic regulation mechanism of miR-29b-3p/SIRT1 via deacetylation method, as epigenetics plays an important role in DM and its complications (Menzies et al., 2016).

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of Aier eye Hospital. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
ST and JC conceived the project. YZ carried out most of the experiments and wrote the manuscript. JL assisted in the cell culture and Western blotting. ZC helped in data analysis. All authors approved the final version of the manuscript.

FUNDING
This work was supported by grants from the National Natural Science Foundation of China (No. 81570876).

ACKNOWLEDGMENTS
Thanks to the help of Dr. Zhongping Chen, Dr. Qichang Wang, Dr. Juan Chen, and the residents. Without their help, it would not have been possible to collect the clinical samples.