CircMAP3K4 protects human lens epithelial cells from H2O2-induced dysfunction by targeting miR-193a-3p/PLCD3 axis in age-related cataract

ABSTRACT Circular RNAs (circRNAs) have shown pivotal regulatory roles in multiple human ocular diseases, including age-related cataract (ARC). Here, we explored the role of circRNA mitogen-activated protein kinase kinase kinase 4 (circMAP3K4, hsa_circ_0078619) in ARC pathology and its associated mechanism. The expression of RNAs and proteins was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Cell viability, senescence, proliferation, and apoptosis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, senescence-associated-β-galactosidase (SA-β-Gal) staining, 5-ethynyl-20-deoxyuridine (EdU) assay, and flow cytometry. The oxidative stress status of SRA01/04 cells was analyzed using the commercial kits. The interaction between microRNA-193a-3p (miR-193a-3p) and circMAP3K4 or phospholipase C delta 3 (PLCD3) was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA-pull down assay. CircMAP3K4 was significantly down-regulated in ARC patients and H2O2-induced SRA01/04 cells. H2O2 treatment restrained the viability and proliferation and promoted the senescence, apoptosis, and oxidative stress of SRA01/04 cells, and circMAP3K4 overexpression protected SRA01/04 cells from H2O2-induced dysfunction. MiR-193a-3p was a direct target of circMAP3K4, and circMAP3K4 overexpression-mediated protective effects in H2O2-induced SRA01/04 cells were largely reversed by the accumulation of miR-193a-3p. MiR-193a-3p interacted with the 3’ untranslated region (3’UTR) of PLCD3, and PLCD3 knockdown largely overturned miR-193a-3p silencing-induced protective effects in H2O2-induced SRA01/04 cells. CircMAP3K4 up-regulated the expression of PLCD3 via sponging miR-193a-3p in SRA01/04 cells. In conclusion, circMAP3K4 protected SRA01/04 cells from H2O2-induced dysfunction in ARC through mediating miR-193a-3p/PLCD3 axis.


Introduction
Cataract is a vital cause of visual impairment and blindness [1]. The pathology of age-related cataract (ARC) is related to multiple factors, containing aging, diabetes, genetics, oxidative stress, and UV exposure [2]. Surgical intervention is the most effective therapeutic strategy for ARC. Nevertheless, surgical intervention may lead to severe postoperative complications [3]. Therefore, understanding the pathogenesis of ARC is important to prevent or delay the progression of this disease. Accumulating evidence have pointed out that the senescence, apoptosis, and oxidative injury of human lens epithelial cells (HLECs) are important pathological foundation of noncongenital cataracts [4][5][6]. Here, we explored the molecular mechanism behind ARC pathology using H 2 O 2 -induced HLECs.
Circular RNAs (circRNAs), a class of noncoding RNA with circular structure, have been confirmed to play important roles in human diseases [7,8]. Accumulating studies demonstrated that circRNAs can regulate cell biological behaviors via acting as sponges for microRNAs (miRNAs) [9,10]. Meanwhile, miRNAs can modulate gene expression by inducing messenger RNA (mRNA) degradation or suppressing mRNA translation [11]. Previous studies have associated the aberrant expression of circRNAs with the pathology of several ocular diseases [12][13][14]. Reduced expression of circRNA mitogen-activated protein kinase 4 (circMAP3K4, hsa_circ_0078619) has been reported in ARC lens samples in comparison with that in the transparent lens samples [15]. However, the role of circMAP3K4 in ARC pathology remains to be disclosed.
The purpose of this study is to explore the role and mechanism of circMAP3K4 in ARC progression. Through predicting the downstream miRNA/mRNA axis of circMAP3K4 by bioinformatics analysis, we found that circMAP3K4 might interact with miR-193a-3p, and miR-193a-3p could bind to phospholipase C delta 3 (PLCD3) 3'UTR. Therefore, we proposed the hypothesis of circMAP3K4/miR-193a-3p/PLCD3 axis and tested it. Our study hopes to provide a potential molecular target for the treatment of ARC.

Human lens epithelium tissues
Human lens epithelium tissues were collected from normal eyes (n = 15; free of ocular diseases) within 8 h after donor death and ARC patients (n = 15; free of other ocular diseases; grade 4 to 6). These lens epithelium tissues were obtained during cataract surgery at The Fifth Affiliated Hospital of Zhengzhou University by an experienced surgeon. All samples were immediately stored at −80°C. Clinical study was authorized by the Ethical Committee of The Fifth Affiliated Hospital of Zhengzhou University. All the participants or their families had signed the written informed consent before sample collection. The clinical features of ARC patients and normal controls are listed in Table 1.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNA samples were prepared with TRIzol reagent (Invitrogen, Waltham, MA, USA). M-MLV reverse transcriptase kit (Invitrogen) and miRNA cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA) were adopted to synthesize cDNA. Afterward, cDNA was amplified via the Power SYBR-Green PCR master mix (Applied Biosystems, Foster, CA, USA). The relative abundance was determined by the 2 −ΔΔCt formula=ΔCt value for target gene-ΔCt value for the average of internal gene (β-actin or U6). The primers are shown in Table 2.

RNase R treatment
RNA samples (2 µg) isolated from SRA01/04 cells were mixed with 3 U/µg RNase R (Epicenter Technologies, Madison, WI, USA) for 15 min, and then RNA samples were then subjected to RT-qPCR assay.

Actinomycin D treatment
SRA01/04 cells were incubated with 2 mg/mL Actinomycin D (Sigma) for the indicated time points (0, 4, 8, 12, and 24 h). Then, RNA samples were isolated with TRIzol reagent and were subjected to RT-qPCR analysis.

Oxidative stress analysis
The level of MDA and the activities of two oxidative stress-associated key enzymes (SOD and GSH-PX) were examined using the commercial kits (Jiancheng Biotech, Nanjing, China) according to the manufacturer's instructions.

Dual-luciferase reporter assay
The interacted miRNAs of circMAP3K4 and the interacted mRNAs of miR-193a-3p were sought by starbase database (http://starbase.sysu.edu.cn). The partial sequence of circMAP3K4 or PLCD3, including the WT or MUT base-pairing sequence with miR-193a-3p, was inserted into the pmirGLO vector (Promega, Shanghai, China). SRA01/04 cells at 24well plates were co-introduced with WT or MUT luciferase reporter and miR-193a-3p mimics/miR-NC for 48 h. The luciferase activities were determined using a commercial kit (Promega).

RNA immunoprecipitation (RIP) assay
SRA01/04 cells were lyzed with RIP buffer, and then cell extracts were co-immuno-precipitated with magnetic beads that pre-coated with anti-AGO2 or anti-IgG. The immuno-precipitated RNAs were isolated and subjected to RT-qPCR assay.

RNA pull-down assay
The biotinylated-circMAP3K4 probe was incubated with C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA) to obtain probe-covered beads. Subsequently, cell extracts were incubated with probecovered beads overnight at 4°C. The immunoprecipitated RNAs were subjected to RT-qPCR assay.

Statistical analysis
Figures were produced using GraphPad Prism 7.0 software and the results were expressed as mean ± standard deviation (SD). The differences were assessed by Student's t-test and analysis of variance (ANOVA). P < 0.05 was set as the threshold of significance.

CircMAP3K4 expression is down-regulated in ARC patients and H 2 O 2 -induced SRA01/04 cells
To explore the biological role of circMAP3K4 in ARC pathology, we first analyzed the expression pattern of circMAP3K4 in the lens epithelium tissues of ARC patients (n = 15) and normal controls (n = 15). We found that circMAP3K4 expression was down-regulated in ARC patients compared with normal controls ( Figure 1A). Then, we analyzed the optimal concentration and time of H 2 O 2 treatment in SRA01/04 cells. The results showed that the cell viability was significantly decreased at 24 h and 48 h after treatment with 50 µM H 2 O 2 for different times ( Figure 1B). After 24 h of treatment with different concentrations of H 2 O 2 , the cell viability was significantly decreased at 50 µM and 100 µM ( Figure 1C). Therefore, the cells treated with 50 µM of H 2 O 2 for 24 h had the best effect. The expression of circMAP3K4 was decreased in SRA01/04 cells treated with 50 µM H 2 O 2 for 24 h ( Figure 1D). We analyzed the stability of circMAP3K4 with exonuclease RNase R and transcriptional inhibitor Actinomycin D. The results showed that circMAP3K4 was resistant to RNase R ( Figure 1E), and the expression of circMAP3K4 was stable upon Actinomycin D treatment ( Figure 1F), suggesting that circMAP3K4 was a circular transcript. Taken together, circMAP3K4 expression might be associated with ARC pathology.

CircMAP3K4 overexpression protects SRA01/04 cells from H 2 O 2 -induced dysfunction
To investigate the molecular mechanism behind the abnormal down-regulation of circMAP3K4 in H 2 O 2 -induced SRA01/04 cells, we performed gain-offunction experiments. H 2 O 2 treatment reduced the expression of circMAP3K4, and the addition of circMAP3K4 overexpression plasmid largely rescued the level of circMAP3K4 in SRA01/04 cells (Figure 2(a)). MTT assay revealed that H 2 O 2 treatment reduced the viability of SRA01/04 cells, which was largely recovered by the addition of circMAP3K4 plasmid (Figure 2(b)). In addition, H 2 O 2 treatment enhanced SA-β-Gal positive cell rate, while this effect could be abolished by circMAP3K4 overexpression (Figure 2(c)). EdU assay showed that H 2 O 2 treatment suppressed the percentage of EdUpositive cells, and the introduction of circMAP3K4 plasmid largely recovered the proliferation ability of SRA01/04 cells (Figure 2(d)). H 2 O 2 treatmentinduced cell apoptosis, and circMAP3K4 overexpression attenuated the apoptosis of SRA01/04 cells (Figure 2(e)). We also determined the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax. Western blot assay showed that H 2 O 2 stimulation-mediated effects on the expression of Bax and Bcl-2 were largely overturned by the addition of circMAP3K4 plasmid (Figure 2(f)), further demonstrating that circMAP3K4 overexpression alleviated H 2 O 2 -induced apoptosis in SRA01/04 cells. H 2 O 2 stimulation promoted the production of MDA and the suppressed the activities of SOD and GSH-PX, and these effects were largely reversed by the overexpression of circMAP3K4 (Figure 2(g-i)). Overall, circMAP3K4 overexpression protected SRA01/04 cells from H 2 O 2 -induced dysfunction.

CircMAP3K4 acts as a molecular sponge for miR-193a-3p in SRA01/04 cells
It is well established that circRNAs can act as miRNA sponges to regulate cell biological behaviors [9,16]. To unveil the working mechanism of circMAP3K4, we predicted the downstream miRNAs of circMAP3K4 using bioinformatics database starbase. MiR-193a-3p was one of the predicted target of circMAP3K4, and the putative binding sites between circMAP3K4 and miR-193a-3p are shown in Figure 3(a). RT-qPCR confirmed the overexpression and knockdown efficiencies of miR-193a-3p mimics and in-miR-193a-3p in SRA01/04 cells (Figure 3(b)). Then, we conducted dual-luciferase reporter assay, RIP assay, and RNApull down assay to verify the target relationship between circMAP3K4 and miR-193a-3p. As shown in Figure 3(c), the overexpression of miR-193a-3p markedly reduced the luciferase activity of wild-type vector (circMAP3K4 WT) but not that of mutant vector (circMAP3K4 MUT), suggesting that circMAP3K4 interacted with miR-193a-3p via the predicted sites. RIP assay displayed that circMAP3K4 and miR-193a-3p expression were enriched in AGO2 antibody-associated RISC (Figure 3(d)), suggesting the spatial interaction between circMAP3K4 and miR-193a-3p. RNA-pull down assay showed that miR-193a-3p was pulled down when using biotin-labeled circMAP3K4WT probe (Figure 3(e)), further demonstrating the interaction between circMAP3K4 and miR-193a-3p. The expression of miR-193a-3p was notably up-regulated in ARC patients and H 2 O 2 -induced SRA01/04 cells (Figure 3(f,g)). CircMAP3K4 overexpression reduced the expression of miR-193a-3p, and the addition of miR-193a-3p mimics rescued the expression of miR-193a-3p level in SRA01/04 cells (Figure 3(h)). Taken together, miR-193a-3p was a direct target of circMAP3K4 in SRA01/04 cells.

MiR-193a-3p interacts with the 3'UTR of PLCD3 in SRA01/04 cells
Previous articles have demonstrated that miRNAs can interact with the 3"UTR of mRNAs, thereby leading to their degradation or translational repression [11,17]. To explore the working mechanism by which miR-193a-3p regulated the biological behaviors of H 2 O 2induced SRA01/04 cells, we predicted the possible mRNA targets of miR-193a-3p using starbase database. PLCD3 was predicted as one of the candidate target of miR-193a-3p, and their putative binding sequences were shown in Figure 5(a). The luciferase activity of wild-type plasmid (PLCD3 3"UTR WT) was dramatically reduced by the overexpression of miR-193a-3p ( Figure 5(b)), indicating the direct interaction between miR-193a-3p and the 3'UTR of PLCD3. The mRNA and protein expression of PLCD3 was decreased in ARC patients compared with normal controls (Figure 5(c,d)). H 2 O 2 stimulation down-regulated the protein level of PLCD3 in SRA01/ 04 cells ( Figure 5(e)). MiR-193a-3p overexpression reduced the protein level of PLCD3, and this effect could be abolished by PLCD3 overexpression plasmid ( Figure 5(f)). CircMAP3K4 overexpression upregulated the protein level of PLCD3, and the addition of miR-193a-3p reduced the protein expression of PLCD3 in SRA01/04 cells ( Figure 5(g)). These results demonstrated that PLCD3 was a direct target of miR-193a-3p, and its expression was positively regulated by circMAP3K4 in SRA01/04 cells.

SRA01/04 cells, we performed rescue experiments.
The silence of miR-193a-3p up-regulated PLCD3 protein level, which was reduced by the addition of si-PLCD3 in SRA01/04 cells (Figure 6(a)). The knockdown of miR-193a-3p enhanced cell viability, suppressed SA-β-Gal positive cell rate, and promoted the proliferation of H 2 O 2 -induced SRA01/04 cells, while these effects were eliminated by PLCD3 knockdown (Figure 6(b-d)). The introduction of si-PLCD3 also reversed the suppressive effects of miR-193a-3p inhibitor on cell apoptosis rate, Bax protein level, MDA level, as well as the increasing effect on Bcl-2 protein level, SOD activity and GSH-PX level in H 2 O 2 -induced SRA01/04 cells ( Figure 6(e-i)). Overall, these findings suggested that miR-193a-3p silencing protected SRA01/04 cells from H 2 O 2 -induced dysfunction largely by up-regulating PLCD3.

Discussion
CircRNAs have attracted a lot of attention due to their important modulatory activities in many physiological and pathological processes, including ocular diseases [18,19]. ARC is featured by the accumulation of insoluble crystalline proteins and the abnormal death and oxidative damage of HLECs [20]. Previous articles have demonstrated the functions of several circRNAs in ARC pathology. Cui et al. demonstrated that circHIPK3 exhibited a protective role in HLECs by absorbing miR-221-3p in ARC pathology [21]. Xu et al. reported that circZNF292 overexpression alleviated H 2 O 2 -induced damage in HLECs through mediating the miR-222-3p/ E2F3 axis [22]. A previous article reported that circMAP3K4 abundance was decreased in ARC lens samples compared with the transparent lens samples [15]. However, its role and molecular mechanism in regulating ARC pathology remain to be clarified. Consistent with former study [15], we observed that circMAP3K4 abundance was markedly reduced in the lens epithelium samples of ARC cases than that in normal controls.
Lens epithelial cell monolayer is responsible for lens proliferation, differentiation and damage repair, which exerts vital protective effects in the transparency and the homeostasis in the lens [23]. It has also to be remind that senescence is a leading cause of ARC that plays an important role in the pathological process of ARC [24,25] CircRNAs have shown pivotal regulatory roles in gene expression by acting as miRNA sponges [16]. We identified miR-193a-3p as a direct target of circMAP3K4 for the first time. Liu et al. demonstrated that circHIPK3 exerts a protective role in HLECs by enhancing CRYAA expression via absorbing miR-193a in ARC [15], indicating that miR-193a facilitates the pathological progress of ARC. We observed that miR-193a-3p abundance was elevated in the lens epithelium tissues of ARC patients and H 2 O 2induced SRA01/04 cells. CircMAP3K4 negatively regulated miR-193a-3p expression in SRA01/04 cells. The silence of miR-193a-3p protected SRA01/04 cells from H 2 O 2 -induced dysfunction, suggesting that miR-193a-3p contributed to ARC pathology. Furthermore, we found that circMAP3K4 overexpression-mediated protective effects in H 2 O 2 -induced SRA01/04 cells were largely reversed by miR-193a-3p overexpression, indicating that circMAP3K4 protected SRA01/04 cells from H 2 O 2 -induced dysfunction largely by down-regulating miR-193a-3p. miRNAs can interact with the 3'UTR of mRNAs to induce their degradation or translational repression [11]. Here, PLCD3 was identified as a downstream target of miR-193a-3p for the first time. PLCD3 abundance was decreased in the lens epithelium samples of ARC cases and H 2 O 2 -exposed SRA01/04 cells. PLCD3 is a member of the PLC family, a vital enzyme in the phosphoinositide signaling. PLC isozymes are involved in the pathology of multiple human diseases, including schizophrenia [26], diabetes [27], obesity [28], and tumorigenesis [29,30]. Liu et al. demonstrated that PLCD3 plays a pro-tumor activity in nasopharyngeal carcinoma by facilitating cell proliferation and motility [31]. Xiang et al. demonstrated that upregulation of PLCD3 expression restrained the progression of ARC [32]. We found that PLCD3 was reversely modulated by miR-193a-3p in SRA01/04 cells. In addition, circMAP3K4 can positively regulate the expression of PLCD3 by serving as miR-193a-3p sponge. The results of compensation assays demonstrated that miR-193a-3p silencing-mediated protective effects in H 2 O 2 -induced SRA01/04 cells were largely counteracted by PLCD3 interference, indicating that miR-193a-3p silencing protected SRA01/04 cells from H 2 O 2 -induced dysfunction largely by upregulating PLCD3.
In conclusion, circMAP3K4 was down-regulated in ARC patients and H 2 O 2 -induced SRA01/04 cells. CircMAP3K4 overexpression protected SRA01/04 cells from H 2 O 2 -induced dysfunction. Furthermore, we found that circMAP3K4 overexpression-mediated protective role was largely based on miR-193a-3p/ PLCD3 axis (Figure 7). This study provided several novel potential targets for the prevention and therapy of ARC.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by Henan Province Medical Science and Technology Tackling Program (Jointly Co-construction) Project (no, LHGJ20190423), (no. LHGJ20190424)