RESEARCH ARTICLE Open Access

WRN inhibits oxidative stress-inducedapoptosis of human lensepithelial cellsthrough ATM/p53 signaling pathway andits expression is downregulated by DNAmethylationShengqun Jiang1,2 and Jiansu Chen1*

Abstract

Background: Apoptosis and oxidative stress are the main etiology of age related cataract (ARC). This article aims toinvestigate the role of WRN in lens epithelial cells (LECs).

Methods: We estimated the methylation level of WRN in anterior lens capsule tissues of ARC patients. SRA01/04 (LECs)cells were treated with H2O2 or combined with 5-aza-2-deoxycytidine (5-Aza-CdR) or chloroquine. CCK8 and flowcytometry were performed to explore proliferation and apoptosis. The content of ROS was detected by fluorescentprobe DCFH-DA. The gene and protein expression was assessed by quantitative real-time PCR or western blot.

Results: WRN was down-regulated and the methylation level of WRN was increased in the anterior lens capsuletissues. WRN overexpression and 5-Aza-CdR enhanced proliferation and repressed apoptosis and oxidative stress ofSRA01/04 cells. 5-Aza-CdR enhanced WRN expression. WRN knockdown inhibited proliferation and promoted apoptosisand oxidative stress of SRA01/04 cells, which was rescued by 5-Aza-CdR. WRN overexpression and 5-Aza-CdR repressedATM/p53 signaling pathway. Furthermore, chloroquine inhibited proliferation and promoted apoptosis and oxidativestress of SRA01/04 cells by activating ATM/p53 signaling pathway. The influence conferred by chloroquine wasabolished by WRN overexpression.

Conclusion: Our study reveals that DNA methylation mediated WRN inhibits apoptosis and oxidative stress of humanLECs through ATM/p53 signaling pathway.

Keywords: WRN, DNA methylation, Apoptosis, Oxidative stress, ATM/p53, Age related cataract

BackgroundAge related cataract (ARC), also known as senile cata-ract, is the world’s first blind eye disease (Zhang et al.2011). At present, surgery is still the only effective treat-ment for cataract. Therefore, it is urgent to study thepathogenesis of ARC and find the cause of ARC. Lens

epithelial cells (LECs) are the only cleavage-active cellsin the lens that can divide and differentiate into lensfiber cells and produce lens proteins. LECs play a crucialrole in maintaining the stability of the lens environmentand the osmotic pressure of the lens and resisting thedamage of external harmful factors (Su et al. 2011; Mat-tioli and Thomas 2010). The occurrence of ARC isclosely associated with DNA damage caused by oxidativestress in the lens (Yang et al. 2018). H2O2 induces oxida-tive stress in LECs by causing abnormal expression of

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* Correspondence: chenjsu30@163.com1Ophthalmology Department, The First Affiliated Hospital of Jinan UniversityGuangzhou, No.601 Huangpu Avenue West, Guangzhou, GuangdongProvince, ChinaFull list of author information is available at the end of the article

Molecular MedicineJiang and Chen Molecular Medicine (2020) 26:68 https://doi.org/10.1186/s10020-020-00187-x

some functional genes to cause ARC, including encodingDNA repair proteins, antioxidant defense enzymes, mo-lecular chaperones, protein biosynthesis enzymes, andtrafficking and degradation proteins (Sumanta Goswamiet al. 2003; Wang et al. 2018a). Oxidative stress producesexcessive reactive oxygen radicals that can damage DNAin cells. Reactive oxygen radicals convert to form hy-droxyl radicals and act on DNA to cause DNA breakage,thereby resulting in apoptosis and ARC formation (Roo-ban et al. 2012). Oxidative stress also causes apoptosis ofthe LECs by causing cell membrane permeabilitychanges, protein conformational changes, thereby result-ing in lens opacity (Goswami 2003). Apoptosis of LECsis the cytological basis for the formation of all types ofcataracts other than congenital cataracts (Li 1995). Onestudy reports that oxidative damage is increased in LECsand peripheral blood lymphocytes in patients with ARC(Zhang et al. 2014).The DNA damage repair gene, WRN, is one of the

major members of the human RecQ helicase family.Many studies have shown that mutations in WRN areassociated with the development of ARC. Our previousstudy has found that rs1346044 of WRN gene is associ-ated with the occurrence of ARC, and the C allele has aprotective effect on the occurrence of ARC. The C→Tof rs1346044 leads to the mutation of the cysteine atposition 1367 of the WRN gene to arginine, which in-creases the susceptibility of cortical ARC in ChineseHan population (Jiang et al. 2013a). The copy numbervariation of WRN may be related to the susceptibility ofARC in Chinese Han population, especially nuclear andposterior subcapsular ARC (Jiang et al. 2013b). Our pre-vious study has confirms that the mRNA and protein ex-pression levels of WRN in LECs of ARC patients aredown-regulated, and the CpG islands in the promoterregion of WRN gene are hypermethylated. And the pro-tein expression of WRN is increased in lens epithelialcells (HLEB-3) after treated with methylation transferaseinhibitor 5-aza-2-deoxycytidine (5-Aza-CdR) (Zhu et al.2015). It indicates that the WRN expression in ARC isregulated by DNA methylation.ATM protein kinase is a product encoded by the tel-

angiectasia ataxia mutated gene ATM, which sensesDNA damage, transmits DNA damage signals to down-stream target genes, initiates stress systems and pro-duces cycle arrest, cell repair and apoptosis (Berger et al.2017). In addition, the p53 pathway plays an importantrole in the body response to DNA damage (Speidel2015). The podophyllotoxin derivative XWL-1-48 trig-gers DNA damage by activating the ATM/p53/p21 sig-naling pathway (Wang et al. 2018b). Up-regulation ofp53 inhibits the antioxidant capacity and cell viability ofLECs, which is associated with the occurrence of ARC(Lu et al. 2018). Therefore, we hypothesized that WRN

down-regulation in ARC is regulated by DNA methyla-tion, and WRN may mediate apoptosis and oxidativedamage of LECs via ATM/p53 signaling pathway.

Materials and methodsCollection of the anterior lens capsule tissuesA continuous annular capsulorhexis is performed on theanterior lens capsule during cataract surgery. The anter-ior lens capsule with a diameter of 5–5.5 mm waswashed with PBS for 3 times and stored at − 80 °C. An-terior lens capsule tissues were obtained from the pa-tients with different types of ARC (cortical, nuclear andposterior subcapsular cataract). The healthy anterior lenscapsule tissues were obtained from age-matched personas control. All patients were informed and gave writtenconsent. All protocols were authorized by the EthicsCommittee of The First Affiliated Hospital of BengbuMedical College.

Cell culture and treatmentHuman lens epithelial cell line, SRA01/04, was pur-chased from ATCC (Manassas, VA, USA). SRA01/04cells were cultured in dulbecco’s modified eagle medium(DMEM) containing 10% fetal bovine serum and 1%penicillin/streptomycin in a humidified atmosphere at37 °C and 5% CO2. SRA01/04 cells at exponential stagewere seeded in six-well plate at a density of 1 × 106 cells/mL. These cells were treated with different concentra-tions (0, 25, 50 and 100 μM) of H2O2 (Sangon Biotech,Shanghai, China) for 24 h. In the subsequent experi-ments, H2O2 at a concentration of 100 μM was used totreat cells for 24 h. After that, H2O2-induced SRA01/04cells were treated with 10 μM 5-Aza-CdR (Sigma-Al-drich, St. Louis, MO, USA) for 72 h. 5-Aza-CdR was re-placed every 24 h. SRA01/04 cells were treated with50 μM chloroquine (ATM activator) for 24 h.

Methylation-specific PCR (MSP)The genome DNA of anterior lens capsule tissues wasextracted using Rapid Animal Genomic DNA IsolationKit (Sangon Biotech). The methylation of DNA sampleswas performed using Mag-DNA Modification Kit (San-gon Biotech). MSP was performed with the methylatedDNA as template. The primers set specific to methylatedDNA and un-methylated DNA were purchased fromSangon Biotech.

Cell transfectionPlasmid vector pcDNA3.1-WRN and its negative control(pcDNA3.1-NC) were constructed by RIBOBIO (Shang-hai, China) via standard molecular cloning approaches.Small interfering RNA (siRNA) specific targeting WRN(WRN siRNA) and the corresponding NC (Scramble)were purchased from RIBOBIO. These vectors were

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transfected into SRA01/04 cells separately. The celltransfection assay was performed using Lipofectamine2000 Transfection Reagent (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol.

Quantitative real-time PCR (qRT-PCR)QRT-PCR was performed to measure the expression in-tensity of different genes. Total RNA was extracted fromanterior lens capsule tissues or SRA01/04 cells usingRNAprep Pure Tissue Kit (Tiangen, Beijing, China). Thepurity and concentration of RNA was detected using aNanoDrop 2000 spectrophotometer (Thermo Fisher Sci-entific, Waltham, MA, USA). The RNA was convertedto complementary DNA using PrimeScript™ RT ReagentKit (Takara, Tokyo, Japan). QRT-PCR was carried outusing SYBR Green PCR Mix Kit (Takara) according tothe instruction. The results were analyzed using theΔΔCT (cycle threshold) method for quantification.

Western blot (WB)The total protein was extracted from anterior lens cap-sule tissues or SRA01/04 cells using Tissue or Cell TotalProtein Extraction Kit (Sangon Biotech). Equivalent pro-tein from different samples were separated by proteinelectrophoresis and transferred on PVDF membranes.The membranes were incubated with antibodies againstthe test proteins after treated with sealed liquid. Afterthe membranes were washed with TBST for severaltimes, secondary antibodies labeled with horseradishperoxidase were incubated with the membranes.GAPDH or β-actin was used as a reference protein fornormalization. The gray level of the protein bands wasexamined by Image J software.

Cell viabilityThe CCK8 assay was performed to explore the prolifera-tion ability of SRA01/04 cells. The cells were treatedwith pancreatin after the cell density reached 80%. Thecells were then re-suspended in DMEM containing 10%fetal bovine serum and the cell density was adjusted to1 × 105/mL. The cell suspension (100 μL) and CCK8 re-agent (10 μL) were mixed and seeded into 96-well plates.Then, the cells were cultured in an incubator at 37 °Cfor 4 h. The optical density of samples was detected at450 nm wavelength using enzyme-labeled instrument(Thermo Fisher Scientific).

Cell apoptosisThe cells were collected by centrifuging for 5 min at thespeed of 500×g, 4 °C. The cells were washed with pre-cooling PBS for 2 times. Cells were then resuspended inthe Annexin V Binding buffer. The cell suspension wasdyed with Annexin V-FITC and PI and plunged intodarkness at room temperature for 15 min. Then, the cell

suspension was mixed with Annexin V Binding bufferand put on ice. The apoptosis rate of cells was deter-mined by flow cytometry in an hour. The assay was per-formed according to the instruction of Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen Bio-tech, Beijing, China).

Detection of intracellular reactive oxygen species (ROS)SRA01/04 cells were seeded into 24-well plate and cul-tured for 24 h. After centrifugation the supernatant wasdiscarded. The cells was resuspended in 1mL DCFH-DA(10 μM) and incubated in an incubator at 37 °C for 20min. The cells are mixed every 5min. The cells werewashed with serum-free DMEM for 3 times. The fluores-cence intensity of the cells was observed by confocal laserscanning microscope (LEICA, Wetzlar, Germany). Detec-tion conditions: excitation wavelength 488 nm, emissionwavelength 525 nm, and the grating width of the excita-tion and emission wavelengths is 5 nm. The assay was per-formed according to the instruction of Reactive OxygenSpecies Assay Kit (Solarbio, Beijing, China).

Statistical analysisAll experiments were independently repeated at least 3times. All values were exhibited as mean ± standard devi-ation and analyzed by SPSS 22.0 statistical software (IBM,Armonk, NY, USA). For comparison of two groups, atwo-tailed Student’s t test was used. Comparison of mul-tiple groups was made using a one- or two-way ANOVA.P < 0.05 was considered statistically significant.

ResultsThe expression of WRN is decreased and the methylationlevel of WRN is increased in the anterior lens capsuletissues of patients with ARCTo investigate the involvement of WRN in ARC, we ana-lyzed its expression and methylation level in the anteriorlens capsule tissues of patients with different types ofARC (cortical, nuclear and posterior subcapsular cata-ract). Compared with the healthy anterior lens capsuletissues of age-matched person, the gene and proteinexpression of WRN was significantly reduced in thethree types of diseased anterior lens capsule tissues(Fig. 1a and b). And the methylation level of WRNwas higher in the three types of diseased anterior lenscapsule tissues than that in the healthy anterior lenscapsule tissues (Fig. 1c).

WRN overexpression suppresses apoptosis and oxidativestress of SRA01/04 cells in vitroIn order to define the role of WRN in the anterior lenscapsule tissues, we detected the expression and methyla-tion level of WRN in SRA01/04 cells after treated withdifferent concentrations of H2O2. The gene and protein

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expression level of WRN was gradually decreased withthe increase of H2O2 concentration (Fig. 2a and b). Themethylation level of WRN was gradually increased withthe increase of H2O2 concentration (Fig. 2c). And H2O2

at a concentration of 100 μM had the greatest influenceon the expression and methylation level of WRN inSRA01/04 cells. Therefore, in the subsequent experi-ments, H2O2 at a concentration of 100 μM was used totreat the SRA01/04 cells. In addition, WRN was up-regulated in SRA01/04 cells, and then the modifiedSRA01/04 cells were treated with 100 μM H2O2. CCK8assay and flow cytometry were assessed cell proliferationand apoptosis of SRA01/04 cells. H2O2 treatment led toa decrease in cell proliferation of SRA01/04 cells, whichwas effectively rescued by WRN overexpression (Fig.2d). H2O2 treatment markedly facilitated apoptosis ofSRA01/04 cells, and its promoting effect on apoptosiswas dramatically reduced by WRN overexpression (Fig.2e). In addition, the level of ROS and malondialdehyde

(MDA) was significantly increased in SRA01/04 cellsafter H2O2 treatment, and significantly decreased inSRA01/04 cells after transfected with pcDNA3.1-WRN(Fig. 2f and g). On the contrary, H2O2-treated SRA01/04cells exhibited a decrease in the expression of superoxidedismutase (SOD) and catalase (CAT), and WRN overex-pression promoted the expression of SOD and CAT inSRA01/04 cells (Fig. 2g). These results taken together re-veal that WRN overexpression suppressed apoptosis andoxidative stress and promoted cell proliferation ofSRA01/04 cells in vitro.

5-Aza-CdR treatment suppresses apoptosis and oxidativestress of SRA01/04 cells in vitroTo explore the effect of 5-Aza-CdR on apoptosis andoxidative stress of SRA01/04 cells, H2O2-inducedSRA01/04 cells were treated with 5-Aza-CdR. We foundthat the gene and protein expression of WRN were obvi-ously reduced in H2O2-induced SRA01/04 cells, which

Fig. 1 The expression of WRN is decreased and the methylation level of WRN is increased in the anterior lens capsule tissues of patients withARC. Anterior lens capsule tissues were obtained from the patients with different types of ARC (cortical, nuclear and posterior subcapsularcataract). The healthy anterior lens capsule tissues from age-matched person were as control. a QRT-PCR and b WB were performed to detect thegene and protein expression of WRN in the anterior lens capsule tissues. c MSP was performed to detect the methylation level of WRN in theanterior lens capsule tissues. M: methylation; U: unmethylation. (**P < 0.01 compared with the Control group)

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was effectively abolished by 5-Aza-CdR treatment(Fig. 3a and b). Furthermore, we performed CCK8assay and flow cytometry to examine cell proliferationand apoptosis of SRA01/04 cells. The cell prolifera-tion was lower in SRA01/04 cells after H2O2 treat-ment than that in normal SRA01/04 cells. 5-Aza-CdR-treated SRA01/04 cells exhibited an increase incell proliferation (Fig. 3c). The apoptosis was mark-edly promoted by H2O2 treatment and repressedby 5-Aza-CdR treatment in SRA01/04 cells (Fig. 3d).

In addition, the level of ROS, SOD, CAT and MDAin the SRA01/04 cells were detected by fluorescentprobe DCFH-DA and WB. As shown in Fig. 3e and f,the level of ROS and MDA were significantly in-creased in SRA01/04 cells after H2O2 treatment, andsignificantly decreased in SRA01/04 cells after treatedwith 5-Aza-CdR and H2O2. On the contrary, H2O2

treatment inhibited the expression of SOD and CATin SRA01/04 cells. And the influence conferred byH2O2 treatment was abolished by 5-Aza-CdR (Fig. 3f).

Fig. 2 WRN overexpression suppresses apoptosis and oxidative stress of SRA01/04 cells in vitro. SRA01/04 cells were treated with differentconcentrations (0, 25, 50 and 100 μM) of H2O2. a QRT-PCR and (b) WB were performed to estimate the gene and protein expression of WRN in theSRA01/04 cells. c MSP was performed to detect the methylation level of WRN in the SRA01/04 cells. SRA01/04 cells were transfected with pcDNA3.1-WRN or pcDNA3.1-NC, and then the modified SRA01/04 cells were treated with 100 μM H2O2. d CCK8 was performed to explore the cell proliferationof the modified SRA01/04 cells. e The apoptosis of the modified SRA01/04 cells was determined by flow cytometry. f The content of ROS in themodified SRA01/04 cells was detected by fluorescent probe DCFH-DA. g WB was performed to detect the expression of SOD, CAT and MDA in themodified SRA01/04 cells. M: methylation; U: unmethylation. (*P < 0.05 compared with the 0 μM H2O2 or Control group,

**P < 0.01 compared with the0 μM H2O2 or Control group,

#P < 0.05 compared with the H2O2 + Vector group, ##P < 0.01 compared with the H2O2 + Vector group)

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These data suggest that 5-Aza-CdR treatment inhibitsapoptosis and oxidative stress and promotes cell pro-liferation of SRA01/04 cells in vitro.

5-Aza-CdR treatment suppresses apoptosis and oxidativestress of SRA01/04 cells by promoting WRN expressionin vitroTo further investigate the molecular mechanism of 5-Aza-CdR in regulating apoptosis and oxidative stress ofSRA01/04 cells, we silenced WRN in SRA01/04 cells.

Then, we performed qRT-PCR and WB to estimate thegene and protein expression of WRN in the SRA01/04cells. We found that WRN silencing led to a decrease inthe gene and protein expression of WRN in the SRA01/04 cells (Fig. 4a and b). Furthermore, the normal andmodified SRA01/04 cells were consecutively treated withH2O2 and 5-Aza-CdR. The gene and protein expressionof WRN in the SRA01/04 cells was notably enhanced by5-Aza-CdR. WRN-silenced SRA01/04 cells exhibited adecrease in the gene and protein expression of WRN,

Fig. 3 5-Aza-CdR treatment suppresses apoptosis and oxidative stress of SRA01/04 cells in vitro. SRA01/04 cells were consecutively treated with 100 μMH2O2 and 5-Aza-CdR. Normal SRA01/04 cells served as control. a QRT-PCR and (b) WB were performed to detect the gene and protein expression of WRNin the SRA01/04 cells. c CCK8 was performed to explore the cell proliferation of the SRA01/04 cells. d The apoptosis of the SRA01/04 cells was determinedby flow cytometry. e The content of ROS in the SRA01/04 cells was detected by fluorescent probe DCFH-DA. f WB was performed to explore theexpression of SOD, CAT and MDA in the SRA01/04 cells. (**P< 0.01 compared with the Control group, ##P< 0.01 compared with the H2O2 group)

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Fig. 4 (See legend on next page.)

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which was effectively recused by 5-Aza-CdR (Fig. 4c and d).Moreover, the cell proliferation and apoptosis of SRA01/04cells were estimated by CCK8 assay and flow cytometry. 5-Aza-CdR significantly promoted cell proliferation ofSRA01/04 cells, whereas WRN knockdown severely re-pressed apoptosis of SRA01/04 cells. The inhibiting effectof WRN down-regulation on cell proliferation of SRA01/04cells was rescued by 5-Aza-CdR (Fig. 4e). The apoptosis ofSRA01/04 cells was repressed by 5-Aza-CdR and enhancedby WRN silencing. WRN-silenced SRA01/04 cells displayedan increase in apoptosis after 5-Aza-CdR treatment (Fig.4f). In addition, the content of ROS and the expression ofSOD, CAT and MDA in the SRA01/04 cells were detectedby fluorescent probe DCFH-DA and WB. Figure 4g and hshow that the level of ROS and MDA in the SRA01/04 cellswas suppressed by 5-Aza-CdR and enhanced by WRN si-lencing. The influence conferred by WRN silencing wasabolished by 5-Aza-CdR treatment. 5-Aza-CdR caused aboost in the levels of SOD and CAT in the SRA01/04 cells.And WRN-silenced SRA01/04 cells exhibited a decrease inthe level of SOD and CAT, which was effectively rescuedby 5-Aza-CdR treatment (Fig. 4h). Therefore, these findingsconfirm that 5-Aza-CdR treatment suppresses apoptosisand oxidative stress of SRA01/04 cells by promoting WRNexpression in vitro.

WRN overexpression inhibits ATM/p53 signaling pathwayTo investigate whether WRN overexpression has aninhibiting effect on ATM/p53 signal pathway, we de-tected the effect of WRN overexpression and 5-Aza-CdRtreatment on the expression of ATM/p53 signalingpathway-related proteins in SRA01/04 cells by WB. Asshown in Fig. 5a and b, H2O2 treatment had no effect onATM expression in SRA01/04 cells. And H2O2 treat-ment significantly promoted the expression of p-ATM,p53, p-p53, p21, Bax, PUMA and NOXA in SRA01/04cells. WRN overexpression and 5-Aza-CdR treatmenthad on effect on the expression of ATM in SRA01/04 cells. However, WRN overexpression and 5-Aza-CdR treatment obviously suppressed the expressionof p-ATM, p53, p-p53, p21, Bax, PUMA and NOXAin SRA01/04 cells (Fig. 5a and b). These data showthat WRN overexpression inhibits ATM/p53 signal-ing pathway.

WRN overexpression inhibits apoptosis and oxidativestress of SRA01/04 cells via ATM/p53 signaling pathwayTo explore the molecular mechanism of WRN in ARC,SRA01/04 cells were treated with chloroquine to activateATM/p53 signaling pathway. As shown in Fig. 6a, WRNoverexpression and chloroquine treatment had no effecton ATM expression in SRA01/04 cells. WRN overex-pression apparently inhibited the expression of p-ATM,p53, p-p53, p21, Bax, PUMA and NOXA, and chloro-quine treatment distinctly promoted the expression of p-ATM, p53, p-p53, p21, Bax, PUMA and NOXA inSRA01/04 cells. And the promoting effect of chloroquinetreatment on the expression of p-ATM, p53, p-p53, p21,Bax, PUMA and NOXA in SRA01/04 cells was rescuedby WRN overexpression (Fig. 6a). WRN overexpressioncaused an increase in cell proliferation, and chloroquinetreatment led to a decrease in cell proliferation inSRA01/04 cells. The influence conferred by chloroquinetreatment was abolished by WRN overexpression (Fig.6b). Apoptosis was lower in SRA01/04 cells after trans-fected with pcDNA3.1-WRN than that in the noramlSRA01/04 cells. Chloroquine-treated SRA01/04 cells dis-played a boost in apoptosis, which was effectively abol-ished by WRN up-regulation (Fig. 6c). As shown in Fig.6d and e, the levels of ROS and MDA were significantlydecreased in SRA01/04 cells after transfected withpcDNA3.1-WRN, and distinctly increased in SRA01/04cells after chloroquine treatment. WRN up-regulationmarkedly inhibited the promoting effect of chloroquinetreatment on the levels of ROS and MDA in SRA01/04cells. On the contrary, WRN up-regulation promotedthe expression of SOD and CAT, and chloroquine treat-ment inhibited the expression of SOD and CAT inSRA01/04 cells. And the inhibiting effect of chloroquinetreatment on the expression of SOD and CAT inSRA01/04 cells was rescued by WRN overexpression(Fig. 6e). Therefore, these data indicate that WRN over-expression inhibits apoptosis and oxidative stress ofSRA01/04 cells via ATM/p53 signaling pathway.

DiscussionARC is a common disease in the elderly and is one ofthe phenomena of human aging. As the age increases,the intraocular lens will slowly harden, turbid, and affect

(See figure on previous page.)Fig. 4 5-Aza-CdR treatment suppresses apoptosis and oxidative stress of SRA01/04 cells by promoting WRN expression in vitro. SRA01/04 cellswere transfected with WRN siRNA or Scramble. Normal SRA01/04 cells served as control. a QRT-PCR and (b) WB were performed to detect thegene and protein expression of WRN in the modified SRA01/04 cells. The normal and modified SRA01/04 cells were consecutively treated with100 μM H2O2 and 5-Aza-CdR. c QRT-PCR and d WB were performed to detect the gene and protein expression of WRN in the modified SRA01/04cells. e CCK8 was performed to explore the cell proliferation of the modified SRA01/04 cells. f The apoptosis of the modified SRA01/04 cells wasdetermined by flow cytometry. g The content of ROS in the modified SRA01/04 cells was detected by fluorescent probe DCFH-DA. h WB wasperformed to detect the expression of SOD, CAT and MDA in the modified SRA01/04 cells. (**P < 0.01 compared with the Scramble group,##P < 0.01 compared with the Control or Control+Scramble group, $$P < 0.01 compared with the Control+WRN siRNA group)

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vision. The LECs play a vital role in the growth and de-velopment of lens and in maintaining the transparencyand stability of the lens (Mccarty 2001). Pathologicalchanges in LECs will inevitably lead to lens lesions, suchas the occurrence of cataracts and the development ofposterior cataracts (Zhang et al. 2002). A series of de-generative changes occurs in LECs during the formationof cataracts, in which the apoptosis of LECs participatesin this process. In addition, oxidative stress is a commonpathway for the occurrence of ARC caused by variousexternal factors (Goswami 2003). With the increase ofage, the content of antioxidants such as glutathione inthe human lens gradually decreases, and the ability toscavenge oxygen free radicals gradually decreases. Thedenaturation and aggregation of lens proteins caused byoxidative damage leads to the increase of lens scattering,thereby resulting in opacity of the lens and the occur-rence of ARC. But the molecular mechanisms of apop-tosis and oxidative stress in human lens epithelial cellsremain unknown. Our previous study has found that

WRN gene is closely associated with the occurrence ofARC (Jiang et al. 2013a). In our study, we further ex-plored the effect of WRN expression on apoptosis andoxidative stress in LECs of ARC. Our data showed thatthe expression of WRN is remarkably decreased and themethylation level of WRN is significantly increased inthe anterior lens capsule tissues of patients with ARC.Studies have found that hypermethylation of the WRN

promoter region results in low expression of WRN pro-tein in tumor tissues and increases chromosome in-stability in a variety of tumors. WRN shows thecharacteristics of inhibiting tumors (Agrelo et al. 2006).On the other hand, low expression of the WRN proteinpromotes the sensitivity of tumor cells to chemothera-peutic drugs (topological enzyme inhibition). The detec-tion of methylation in the WRN promoter region intumor tissues provides a good basis for screening pa-tients who are suitable for chemotherapy drugs (Masudaet al. 2012; Wang and Wang 2013). Many studies alsohave shown that WRN is closely associated with

Fig. 5 WRN overexpression inhibits ATM/p53 signaling pathway. SRA01/04 cells were transfected with pcDNA3.1-WRN or pcDNA3.1-NC, and thenthe modified SRA01/04 cells were treated with 100 μM H2O2. a WB was performed to detect the expression of p-ATM, ATM, p53, p-p53, p21, Bax,PUMA and NOXA in the modified SRA01/04 cells. SRA01/04 cells were consecutively treated with 100 μM H2O2 and 5-Aza-CdR. b WB wasperformed to estimate the expression of p-ATM, ATM, p53, p-p53, p21, Bax, PUMA and NOXA in the modified SRA01/04 cells.(**P < 0.01 comparedwith the Control group, ##P < 0.01 compared with the H2O2 + Vector group, $P < 0.05 compared with the H2O2 group,

$$P < 0.01 compared withthe H2O2 group)

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apoptosis. In microsatellite instability models, WRNknock-out induces double-stranded DNA breaks andpromotes apoptosis and cell cycle arrest selectively(Chan et al. 2019). The Werner syndrome ATP-dependent helicase encoded by WRN gene is closely re-lated to cell proliferation and DNA repair. The cell cyclearrest and apoptosis is obviously induced in human T-cell leukemia virus type 1-transformed leukemia cellsafter treated by WRN inhibitor (NSC 19630) (Moleset al. 2016). In our study, H2O2 treatment inhibited theexpression of WRN and promoted the methylation levelof WRN in SRA01/04 cells. H2O2 treatment led to a

decrease of cell proliferation and caused an increase ofapoptosis in SRA01/04 cells. H2O2-treated SRA01/04cells exhibited a boost in the levels of ROS and MDA,and a decrease in the levels of SOD and CAT. SOD2 isone of the endogenous antioxidant enzymes that protectagainst reactive oxygen species. Previous study has con-firmed that H2O2 induces the up-regulation of miR-146a, which interacts with SOD2 and inhibits the ex-pression of SOD2 (Ji et al. 2013). H2O2 treatment pro-motes the generation of excess ROS, which then causesMDA formation, thereby resulting in cell death (Xiaet al. 2018). Thus, these findings indicate that H2O2

Fig. 6 WRN overexpression inhibits apoptosis and oxidative stress of SRA01/04 cells via ATM/p53 signaling pathway. SRA01/04 cells weretransfected with pcDNA3.1-WRN or pcDNA3.1-NC, and then the modified SRA01/04 cells were treated with 50 μM chloroquine. a WB wasperformed to detect the expression of p-ATM, ATM, p53, p-p53, p21, Bax, PUMA and NOXA in the modified SRA01/04 cells. b CCK8 wasperformed to explore the cell proliferation of the modified SRA01/04 cells. c The apoptosis of the modified SRA01/04 cells was determined byflow cytometry. d The content of ROS in the modified SRA01/04 cells was detected by fluorescent probe DCFH-DA. e WB was performed toexplore the expression of SOD, CAT and MDA in the modified SRA01/04 cells. (*P < 0.05 compared with the Vector group, **P < 0.01 comparedwith the Vector group, #P < 0.05 compared with the Vector+Chl group, ##P < 0.01 compared with the Vector+Chl group)

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treatment induces apoptosis and oxidative stress, and re-presses cell proliferation of SRA01/04 cells. In addition,the influence conferred by H2O2 treatment was rescuedby WRN overexpression. Furthermore, 5-Aza-CdR treat-ment significantly promoted the expression of WRN.And 5-Aza-CdR treatment enhanced cell proliferation,suppressed apoptosis and oxidative stress in SRA01/04cells. WRN silencing caused a decrease in cell prolifera-tion, and led to an increase in apoptosis and oxidativestress in SRA01/04 cells, which was effectively rescuedby 5-Aza-CdR treatment. Therefore, these data taken to-gether reveal that 5-Aza-CdR treatment inhibits apop-tosis and oxidative stress and promotes cell proliferationof SRA01/04 cells by up-regulating WRN expression.One study shows that the ATM/p53/p21 signaling

pathway induces G2/M arrest and mediates DNA dam-age (Li et al. 2018). Under DNA damage conditions, acti-vated ATM kinase phosphorylates p53 and drives cellsenescence or apoptosis. Phosphorylation of p53 is thefirst step in initiating oxidative stress response. Activa-tion of p53 protein activates cell cycle checkpoint p21expression, thereby inhibiting cell cycle and repairingdamaged DNA. Apoptosis occurs when DNA repair fails.Our research found that WRN overexpression and 5-Aza-CdR treatment inhibited the expression of p-ATM,p53, p-p53 and p21 in the SRA01/04. And the expres-sion of PUMA, NOXA and Bax was notably repressedby WRN overexpression and 5-Aza-CdR treatment.PUMA and NOXA were the target gene of pro-apoptotic p53. It indicates that WRN overexpression and5-Aza-CdR treatment inhibits apoptosis via ATM/p53signaling pathway. Moreover, chloroquine was used toinduce ATM activation. Chromatin and chromosomestructures can be altered in the absence of DNA breaksby exposure to chloroquine. Then, the changes in chro-matin structure lead to ATM activation (Bakkenist andMB K. 2003). We found that chloroquine treatmentinhibited cell proliferation, and enhanced apoptosis andoxidative stress by activating ATM/p53 signaling path-way, which was effectively abolished by WRN up-regulation.

ConclusionsIn conclusion, our study demonstrates that DNA methy-lation mediated WRN inhibits apoptosis and oxidativestress of human LECs via ATM/p53 singling pathway.The results imply that WRN might play certain roles inthe pathogenesis of ARC and supply the new targetpoint and strategy for the treatment of ARC.

Abbreviations5-Aza-CdR: 5-aza-2-deoxycytidine; ARC: Age related cataract; CAT: Catalase;DMEM: Dulbecco’s modified eagle medium; LECs: Lens epithelial cells;MDA: Malondialdehyde; MSP: Methylation-Specific PCR; qRT-PCR: Quantitativereal-time PCR; SOD: Superoxide dismutase; WB: Western blot

AcknowledgementsNot applicable.

Authors’ contributionsJiansu Chen designed the project, analyzed the data and reviewed themanuscript; Shengqun Jiang performed the experiments, interpreted thedata and drafted the manuscript. The author(s) read and approved the finalmanuscript.

FundingThis study was supported by the fund from the Department of EducationAnhui Province (KJ2019A0349).

Availability of data and materialsThe datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request.

Ethics approval and consent to participateAll patients were informed and gave written consent. All protocols wereauthorized by the Ethics Committee of The First Affiliated Hospital of BengbuMedical College.

Consent for publicationNot applicable.

Competing interestsNot applicable.

Author details1Ophthalmology Department, The First Affiliated Hospital of Jinan UniversityGuangzhou, No.601 Huangpu Avenue West, Guangzhou, GuangdongProvince, China. 2Ophthalmology Department, The First Affiliated Hospital ofBengbu Medical College, Bengbu, Anhui Province, China.

Received: 27 November 2019 Accepted: 16 June 2020

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