DCLRE1A Contributes to DNA Damage Repair and Apoptosis in Age-Related Cataracts by Regulating the lncRNA/miRNA/mRNA Axis

Abstract Purpose Age-related cataract (ARC) is associated with the deregulation of transcription and defects in DNA repair in lens epithelial cells (LECs). DCLRE1A acted in DNA interstrand cross-links pathway to improve DNA replication and transcription. The aim of this study was to examined the further regulatory effect on DCLRE1A in the lncRNA-miRNA-mRNA network using a cell model of DCLRE1A overexpression (OE-DCLRE1A) in LECs. Methods The expression level of DCLRE1A in ARC tissues and SRA01/04 cells after H2O2 treatment was measured as protein and mRNA by qRT-PCR and Western Blot(WB). CCK8, and TUNEL assays detected the change in cell viability and apoptosis, respectively. Furthermore, Immunofluorescence assays detect the expression of DNA damaged and repair marker proteins after OE-DCLRE1A. The global expression profiles of lncRNAs, miRNAs, and mRNAs were determined using high-throughput sequencing. KEGG and GO enrichment analysis disclose the possible function of differentially expressed (DE) lncRNA, miRNA, and mRNA. Results The protein and mRNA of DCLRE1A were decreased in the anterior capsule of ARC and SRA01/04 cells treated by H2O2. OE-DCLRE1A improved damaged-DNA repair and enhanced cell viability against apoptosis after H2O2 treatment. Furthermore, we demonstrated the DE-molecules between the OE-DCLRE1A and control groups including 595 DE-lncRNAs, 221 DE-miRNAs, and 4718 DE-mRNAs. Next, bioinformatics analysis not only found that the DE-mRNAs are mainly involved in DNA repair-related signaling pathways after OE-DCLRE1A, but also screened two lncRNA-miRNA-mRNA networks focusing on DNA damage activated by OE-DCLRE1A, which involved 2 lncRNAs, 2 miRNAs, and 53 mRNAs. Conclusion We revealed that DCLRE1A activated the lncRNA/miRNA/DNA-repair network to take part in DNA repair processes, which not only represents a new regulatory mechanism employed by DCLRE1A but also uncovers the screening lncRNA may hold potential therapeutic values in ARC formation. However, these conclusions will need to be confirmed by future studies in vitro and in vivo models.


Introduction
4][5] Among these mechanisms, oxidative stress triggered by reactive oxygen species (ROS) production is considered one of the main mechanisms of ARC. 6 Excess ROS leads to extensive oxidative DNA lesions in lens epithelial cells (LECs). 7To circumvent the deleterious effects of DNA damage cells timely recruitment of factors involved in the response to DNA damage (DDR) to repair damaged DNA. 8And if left unrepaired in time oxidative DNA can accumulate and eventually lead to apoptosis of LECs.For example, Cockayne syndrome type B (CSB) encoded by ERCC Excision Repair 6 (ERCC6) gene, play critical roles in nucleotide excision repair (NER) and transcription regulation, increases cellular vitality, and promotes LECs survival. 9Another study also found 8-Oxoguanine DNA Glycosylase (OGG1), through the base excision repair (BER) pathway, to protect LECs against oxidative DNA damage and apoptosis in ARC.Further research confirmed that the absence of OGG1 in zebrafish leads to cataract. 10Taken together, DNA repair genes (DRGs) play an important antioxidant role in the development of the ARC.DNA cross-link repair 1 A (DCLRE1A), also known as sensitive to nitrogen mustard 1 A, acted in DNA interstrand cross-links (ICLs) to improve DNA replication and transcription. 11In previous study, DCLRE1A was significantly decreased in LECs of ARC through microarray analysis. 12owever, the detailed mechanism, which was regulated by DCLRE1A to repair damaged DNA in ARC, has not been studied so far.
Long non-coding RNAs (lncRNAs), one of the non-coding RNAs, possess more than 200 nucleotides, which have limited ability to encode proteins in the cells. 13Recent studies have revealed that lncRNAs are widely involved in many different biological activities, such as functioning as miRNA sponges, binding to proteins as scaffolds, and serving as cofactors for transcription. 14,15With the combination of classical molecular biology technology and high-throughput sequencing technology, the competitive endogenous RNA network (ceRNA mechanism) based on interactions among lncRNA, miRNA, and mRNA has been beginning to be examined in ARC. 10,16,17For example, lncRNA KCNQ1OT1 not only directly binds miR-124-3p to protect cells from oxidative stress through BCL2L, 11,18 but also promotes the high expression of FBJ Murine Osteosarcoma Viral Oncogene Homolog via miR-29c-3p in ARC. 19In addition, lncRNA MIAT has a similar function in the progression of ARC via miR-181a/connective tissue growth factor signaling pathway. 20While many lncRNAs have emerged as critical regulators of transcriptional networks in ARC, 10,17,21 the upstream regulation of most lncRNAs is still lacking.Recently, Wang et al. have confirmed an interesting regulatory mechanism phenomenon, adenomatous polyposis coli (APC) could affect the expression of lncRNA, thus playing a critical role in the development of colorectal carcinoma. 22,23n light of this, a study found that in addition to ICLs mechanism DCLRE1A may regulate gene transcription to repair damaged DNA. 11Therefore, we speculate that DCLRE1A may also have a similar regulatory to integrated DDR influence on ARC.
In this study, we investigated DCLRE1A's fundamental role in the regulation of DNA damage repair and cell death in LECs.We next analyzed the expression of lncRNAs, miRNAs, and mRNAs in LECs by high-throughput sequencing to examine the influence of DCLRE1A on its activated potential lncRNA/miRNA to response on the DDR in LECs.Our study attempts to clarify a novel function for DCLRE1A in controlling DDR, which may activate a lncRNA/miRNA/mRNA axis involved in LECs' DNA repair.

Materials and methods
Clinical samples collection and establishment of DLRE1A-overexpressed cell model 22 ARC patients were randomly selected, based on the Lens Opacities Classification System (LOCS III).And 22 agematched patients with transparent lens, excluding vitreoretinal disease, were selected.Patients with systemic diseases such as hypertension, diabetes, high myopia, glaucoma, and uveitis were excluded.During cataract surgery, anterior continuous curvilinear capsulorhexis was used to collect LECs.The present study received its ethics approval from the Affiliated Hospital of Nantong University Ethics Committee, Nantong, China, as well as adhering to the principles of the Helsinki Declaration.We explained to all participants the objectives, procedures, as well as potential risks, and signed informed consents.
We purchased the SRA01/04 cell line in the Chinese Academy of Sciences Cell Ban.DMEM supplemented with 10% foetal bovine serum (FBS, Gibco, USA), 1% streptomycin, and 1% penicillin was used to culture the cells.The incubator, in which the cells are placed, is maintained at 37 C and given a humidified atmosphere containing 5% carbon dioxide.The pcDNA3.1-DCLRE1A and blank vector were transferred into SRA01/04 cells with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions.

Western blot assay (WB)
Human anterior capsule tissue samples and SRA01/04 cells were collected and lysed in RIPA containing phenylmethanesulphonyl fluoride (Beytime, Shanghai, China) for 30 min at 4 C. To measure protein concentrations, a BCA kit (thermo Fisher Scientific, Inc.) was used.And then, equal amount of protein samples was separated by SDS-PAGE and transferred to PVDF membrane (Thermos Fisher Scientific, Inc.), which was blocked with 5% skim milk after transferring.Rabbit anti-human-DCLRE1A and Rabbit anti-human-GAPDH were used to incubate the membrane overnight at 4 C. Three washes with TBST were followed by elution with 300 mM imidazole in TBST.Then, the membrane was again incubated with secondary antibodies at room temperature for 2 h.We visualized the protein band using the ECL Kit (Thermos Fisher Scientific, Inc.) and quantified immunoblots using Image J.

Immunohistochemistry (IHC)
In ARC patients, the anterior capsule was fixed by 4% paraformaldehyde and incubated for 1 h.The tissue was immersed in 3% and 10% sucrose solution for 1 h and then transferred to 30% sucrose over 24 h at 4 C to dehydrate the tissue.They were cut into 8 lm sections and subsequently rinsed with PBS.The sections were blocked with 1% Triton x-100 and 3%BSA for 2 h.Overnight at 4 C, sections were incubated with primary antibody for 4 nights, and then washed with PBS and incubated with secondary antibody for 1 h at room temperature.DAB was finally added to make the image visible.

TUNEL assay
A TUNEL assay kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) was used to detect apoptosis of the SRA01/04 cells.According to the manufacturer's protocol, SRA01/04 cells were fixed in 4% formaldehyde at room temperature for 10 min.Finally, SRA01/04 cells were incubated with a TUNEL mixture at room temperature for 1 h, counterstained with DAPI, and photographed by fluorescence microscopy.

Cell viability
Cell viability was assessed using CCK-8 (Dojindo Laboratory, Kumamoto, Japan).We seeded the cells into 96well plates at a density of 5 Â 10 4 cells/well.After 24 h, we added CCK8 solution into each well and incubated for 2 h.After that, the absorbance at 450 nm was calculated for cell viability.

Immunofluorescence (IF) assay
The LECs, SRA01/04, were fixed with 4% paraformaldehyde for 20 min at room temperature, and then Bovine serum albumin was used to block the nonspecific binding sites for 2 h after permeabilizing with 0.5% Triton X-100 for 15 min on ice.Primary antibody was applied to incubate the cells at 4 C overnight and then the cells were incubated with a secondary antibody for 2 h.The nuclear was stained by DAPI.IF was obtained by a fluorescence microscope (Leica, Germany).

RNA sequencing
RNA sequencing was divided into control and overexpression DCLRE1A(OE-DCLRE1A), the control group was transfected with the control plasmid HA, and the OE-DCLRE1A group was transfected with the plasmid of DCLRE1A in SRA01/04 cells and repeated 3 times per group.The expression profile of lncRNA, miRNA, and mRNA was tested by RNA sequencing.RNA sequencing was conducted and data was analyzed by RiboBio Co., Ltd.(Guangzhou, China).Trimmomatic tools were used to deal with raw fastq sequences.TRAILING:20, MINLEN:235, and CROP:235 are the options.To obtain a consistent sequence length, trailing sequences, less than a phred quality score of 20, were removed.p < 0.05 and fold-change !2.0 were considered to be the threshold of differentially expressed lncRNAs, miRNAs, and mRNAs.

Gene Ontology and pathway enrichment analysis
Enrichment analysis of DE lncRNAs, miRNAs, and mRNAs was performed by Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) database, respectively using Metascape online tools (http://metascape.org/gp/index.html#/main/step1)and hypergeometric distribution through R 'phyper' function.

Statistical analysis
All statistical data were presented as mean ± standard deviation. 24In all measurements, three independent trials were conducted.GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) was used to evaluate the data differences.Student's t-test was used to compare the differences between the two groups.A two-tailed p < 0.05 was identified as statistically significant.

Results
The expression of DCLRE1A was elevated in both ARC patients' specimens and H 2 O 2 -treated SRA01/04 cells As a first step, we measured the expression levels of DCLRE1A in capsule tissues from ARC and control patients.In a comparison with the control group, the protein of DCLRE1A was noticed to have a significantly decreased in the anterior capsule of ARC (Figure 1(A)).Meanwhile, the mRNA level change of DCLRE1A was also consistent with the protein level change of DCLRE1A (Figure 1(B)).IHC experiments showed that the expression of DCLRE1A was lower in anterior capsule tissues of ARC patients than in control patients (Figure 1(C)).These data suggested that DCLRE1A level might be strongly associated with ARC formation.To explore the expression of DCLRE1A in an oxidative damage environment, we treated SRA01/04 cells with four different concentrations of H 2 O 2 (0, 100, 200, 400 lmol/L).As shown in Figure 2(A,B), compared with the control group, we also detected that the protein and mRNA expression of DCLRE1A in SRA01/04 cells was significantly decreased as the concentration gradient increased, with the sharpest decline at 400 lmol/L H 2 O 2 .Based on this, we next detected cells with 400 lmol/L H 2 O 2 to explore the effect of DCLRE1A on SRA01/04 cell functions.

Expression profiles of mRNAs in DCLRE1Aoverexpressing (OE-DCLRE1A) cells
To investigate the specific functions of DCLRE1A in oxidative stress conditions, we intended to construct an OE-DCLRE1A cell model by transfection pcDNA3.1-DCLRE1Ainto SRA01/04 cells.Then, through western blot assay, we confirmed that the protein expression of DCLRE1A was increased in this cell model (Figure 3(A)).Next, using high throughout mRNA sequencing, we found that compared with the control group, there were 4781 differentially expressed mRNAs (2617 upregulated and 2164 downregulated) in the OE-DCLRE1A group (Figure 3(B)).Subsequently, KEGG enrichment analysis was conducted to study the functions of up-regulated and down-regulated mRNAs, respectively.Spliceosome, RNA transport, and DNA replication are the most enriched pathways of up-regulated genes (Figure 3(C)).Likewise, DNA repair pathways, including, BER, NER, and mismatch repair were closely related to the up-regulated genes.In addition, the most enriched pathways of downregulated genes were the FoxO signaling pathway, apoptosis, and p53 signaling pathway (Figure 3(D)), which are the main pathways of ARC caused by oxidative stress. 25O analysis showed that the main terms of up-regulated genes (Figure 3(E)) were rich in cell cycle, chromosome organization, and DNA metabolic process in biological processes (BP).In cellular components (CC) and molecular functions (MF), chromosome and RNA binding are respectively the most enriched terms.Additionally, the main terms of down-regulated genes (Figure 3(F)) were rich in intracellular signal transduction, cell death, and programmed cell death in BP.In CC and MF categories, intracellular vesicle and enzyme binding respectively are the most enriched terms.
Oxidative damage is considered to be the most important cause of ARC. 26 Among them, excessive ROS is the primary factor that causes oxidative damage, which usually leads to decreased cell viability, 27 DNA damage, 10 apoptosis, 28 and other results.Furthermore, A mass of DE genes are involved in cell functions, including DNA damage, DNA repair, and apoptosis (Fig. The topA).The top 25 genes, related to cell death, were listed in the hot map (Figure S1B).

Overexpression of DCLRE1A improved damaged-DNA repair and enhanced cell viability against apoptosis after H 2 O 2 treatment
The above results show that the increase of DCLRE1A enhances overall DNA damage response in LECs.As shown in Figure 4A, the hot map analysis revealed the significant changing expression of DRGs after OE-DCLRE1A.To probe the connection between DCLRE1A and DDR, 15A3 was employed to identify DNA/RNA Damage sensor proteins and cH2A was utilized to recognize double-stranded DNA damage sensor proteproteins.IF assay analysis demonstrated that the fluorescence intensity of 15A3 and cH2A was elevated in H 2 O 2 -treated cells, but DCLRE1A inhibited the increase of 15A3 and cH2A (Figure 4(B,C)).Simultaneously, western blot assay revealed that the change of cH2A protein was consistent with those described previously (Figure 4(E)).Furthermore, compared with the control group, the cell viability was significantly dampened after H 2 O 2 treatment, while the SRA01/04 cell viability was heightened after OE-DCLRE1A (Figure 4

(D)).
To further validate the function of DCLRE1A in the apoptosis of SRA01/04 cells, we examined the most classical pro-apoptotic and anti-apoptotic genes of the Bcl-2 family, Bax, and Bcl-2.29 WB assay showed that the ratio of Bcl-2/Bax decreased in H 2 O 2 -treated cells.However, this ratio was significantly elevated by DCLRE1A (Figure 5(A)).In the meantime, the TUNEL assay disclosed that apoptotic cells in the OE-DCLRE1A group were significantly less than the HA group (Figure 5(B)).Taken together, DCLRE1A contributed to the increasement of cells viability and DNA repair, while it blunted apoptosis.

Expression profiles of OE-DCLRE1A-related lncRNAs and miRNAs
To identify the DE ncRNAs, the expression profile of lncRNAs and miRNAs in SRA01/04 cells after OE-DCLRE1A, was tested by high-throughput sequencing.The hot map and scatter plot of DE lncRNAs are shown in Figures 6(A,B) respectively.A total of 595 DE lncRNAs (172 upregulated and 423 downregulated) were identified in the OE-DCLRE1A groups as compared to the control group.GO analysis of down-regulated DE-lncRNAs (Figure S2A) disclosed that single-organism, receptor binding, and extracellular region are respectively enriched in BP, CC, and MF.GO analysis of up-regulated DE lncRNAs (Figure S2B) displayed that DNA replication initiation, MCM complex, and nucleoside-triphosphataseactivity are respectively enriched in BP, CC, and MF.KEGG analysis revealed that down-regulated DE-lncRNAs are tightly associated with the mTOR signaling pathway (Figure S2C).Albeit, DNA replication, which is closely connected to ARC, was the principal function, related to up-regulated DE lncRNAs, through KEGG (Figure S2D).According to the genomic location of DE lncRNAs, most of them were distributed in intergenic (35.1%), exon antisense (25.2%), and bidirectional (20.7%) regions (Figure S2E).In addition, 221 DE miRNAs (123 upregulated and 98 downregulated) were also found in OE-DCLRE1A groups by miRNA sequencing and the top 20 miRNAs with significantly different expressions were listed in the hot map (Figure 6(C,D)).GO analysis was performed to explain the functions of DE miRNAs (Figure 6(E)).Cell cycle, cellular response to stress, RNA metabolic process, and symbiont process were involved in the BP.RNA binding, enzyme binding, and nucleotide binding were involved in the MF.DNA replication, base excision repair, and apoptosis were the largely enriched terms in KEGG pathway analysis (Figure 6(F)).The potential DNA repair-related lncRNA-miRNA-mRNA regulatory network Firstly, to further assess the critical role of DCLRE1A in repairing DNA damage, we predicted potential mRNAs related to DNA repair of the DE-miRNAs according to their interaction sites through starBase (Figure 7(A)).
Subsequently, to investigate DCLRE1A-related ceRNA network, lncRNA-seq and starBase are applied to predict the putative target miRNAs, which were differentially expressed in SRA01/04 cells overexpressing DCLRE1A.As Figure 7(B) showed that we screened two downregulated miRNAs (miRNA let-7a-5p and miRNA let-7c-5p) and identified putative upstream lncRNA (lnc XIST and lnc NUTM2A-AS1) and miRNA-DRGs interactions using miRanda software and RNA-seqencing.To confirm the lncRNA-seq and miRNA-seq results, qRT-PCR verified the hypothesis that lncRNA XIST, lncRNA NUTM2A-AS1, miRNA let-7a-5p and miRNA let-7c-5p at the RNA level by H 2 O 2 treatment, raise after overexpressing DCLRE1A (Figure 7(C-F)).The results indicated that the expression of the two lncRNAs were upregulated, while the expression of two miRNAs were downregulated by overexpressing DCLRE1A in highly oxidative environment.

Discussion
A growing body of research shows that supports a critical role of oxidatively induced DNA damage in the etiology of ARC. 9,30-32Accumulated oxidative DNA damage will activate the DNA repair pathways and LECs undergo apoptosis if DNA repair fails. 33It is well known that DNA damage repair pathways, mainly including BER, NER, mismatch repair (MMR), double strand break repair (DSBR) and DNA inter-strand crosslink repair (ICLR), are involved in the repairment of DNA damage and their normal functions mainly depend on DRGs expression and function. 34,35s shown in our previous study, MSH3, a key member of MMR pathway, plays a fundamental role of DNA repair in ARC.Reduced MSH3 leads to inefficient or slowed DNA repair, which triggers LECs apoptosis. 36Another study also demonstrated that KU80 was differentially decreased in ARC tissues and UVB-exposed cells. 28Further study, KU80 ubiquitination exerted indispensable role in DSBR pathway for the removal of Ku heterodimers at sites of DNA damage. 28,37In addition, NER proteins ERCC6 and BER repair proteins (OGG1, and NEIL2) plays an important role in both LECs apoptosis and DNA repair pathways. 9,10,38In this study, we mainly explored the expression and function of DCLRE1A in ARC, which is an important role of ICLR pathway.DCLRE1A serves DNA repair roles by binds to multiple repair factors. 39,40Previous studies have found that DCLRE1A was recruited to the double-strand break site by ATM and played a role as a phosphorylation substrate for ATM in the promotion of G1 cell cycle checkpoint. 41In addition, researchers have also found that DCLRE1A binds directly to CSB and co-localizes to DNA ICL in response to DNA damage. 42Both our previous study and the present study find that there is significant decrease in DCLRE1A expression in ARC patients and H 2 O 2 -treated cells. 12oreover, we also demonstrated that DCLRE1A have repair capacity for damaged DNA in LECs.Simultaneously, overexpressed DCLRE1A enhanced cell viability and weakened cell apoptosis caused by H 2 O 2 -treated.The data suggested that DCLRE1A might play a protective role in DNA repair and apoptosis to prevent ARC formation.
To confirm our findings, we also assayed the mRNA expression profiling and focused on DRGs expression change.After overexpression DCLRE1A in SRA01/04 cells, gene expression profiling revealed changes in the expression of DRGs, for example FEN1, MSH2, LIG1, and BLM.Among them, we had been detecting a significant downregulation of expression of LIG1 and MSH2 in ARC, as previously described microarray results. 12In addition, it has been identified that BLM can regulate cell vitality and apoptosis involving in ARC formation. 43During DNA recombination, replication, and repair processes, BLM, as a key enzyme of the DSBR pathway, plays crucial roles as a RecQ helicase. 44n ARC, BLM can influence cell vitality and apoptosis to regulate ARC progression. 43Moreover, we also observed a significant enrichment of DNA repair related pathways in genes upregulated during overexpression DCLRE1A, for example DNA replication, MMR, NER, BER, and RNA degradation through KEGG analysis.At the same time, downregulated genes after overexpression DCLRE1A were mainly enriched in apoptosis pathways, suggesting that DCLRE1A protects against ARC by inhibiting LECs apoptosis.In summary, the following phenomenon may suggest a role for DCLRE1A in enhanced DNA repair and inhibited apoptosis by regulating upstream of other DDR genes transcription.
As our previous studies, lncRNAs regulate DRGs expression programs through ceRNA mechanisms. 10,16,17For example, PLCD3 and TDG, belongs to oxidative damage repair gene, plays a protective role in LECs and that PLCD3-OT1 and lncRNA H19 regulates PLCD3 and TDG expression by sponging miR-224-5p and miR29a in ARC, respectively. 16,17In addition, OGG1, as a BER pathway gene, regulated by NONHSAT143692.2/miR-4728-5p/OGG1 axis involving in damaged DNA repairing in the development of ARC. 10 As described above, lncRNA are exquisitely regulated and control ODGs expression in ARC.On the contrary, a study found that lncRNAs activated by the APC gene mediate the role of APC in the pathogenesis of colorectal carcinoma. 22Moreover, another study reported that NER pathway gene ERCC6 can regulate the expression of ncRNAs to involving in various aspects of biological processes of ARC, for example DNA repair, DNA damaged, and cell death. 45In these present studies, we have also found that over-expression of DCLRE1A could significantly change the expression of lncRNAs.RNA sequencing revealed a total of 595 differentially expressed lncRNAs in the overexpressed DCLRE1A group compared with the control group, of which 172 lncRNAs were up-regulated and 423 lncRNAs were down-regulated.][48][49] The present study was primarily aimed at elucidating the ceRNA mechanism of DCLRE1A activated lncRNAs.For this purpose, we also measure the miRNA expression profiles after DCLRE1A overexpression.Among differentially expressed miRNAs, we found 123 upregulated and 98 downregulated miRNAs.Among the top 20 differentially expressed miRNAs, we found that miR-486-3p, 50 miR-486-5p, 51,52 miR-126-3p, 53,54 miR-1246,55 miR-4488,56 miR-451a, 57,58 and miR-122-5p [59][60][61] have confirmed the correlation with apoptosis, aging, and the development of the eye disease after reference and citation searches.For example, miR-451a serves as a prosenescence factor that is differentially expressed, and has an essential role in regulating ROS production and mitophagy activation in senescent HDFs. 57Moreover, one study also identified that miR-451a targeting IL-6R activates JAK2/STAT3 pathway, thus regulates apoptosis in multiple myeloma cells. 58This was further substantiated using KEGG to analysis the DE miRNAs.In addition to this, a significant enrichment for DNA repairrelated pathways (DNA replication, BER, MMR, and P53 signaling pathway) was observed in DE miRNAs.In addition, our results showed that the association between the significance of the miRNA-DRGs results and the number of miRNA target DRGs, suggesting that indirect coregulation of the miRNA and the DRGs contributes to our results.Subsequently, our intial screening identified miRNAs as a regulator of DNA repair, after predicting lncRNAs based on ceRNA mechanisms which was integrated with lncRNA-Sequencing and starbase software.Hence, our findings reveal novel lncRNA/miRNA regulatory networks in the DNA repair,which including DCLRE1A activated lncRNA (XIST and NUTM2A-AS1,) and these targeted miRNA (let-7a-5p, let-7c-5p).LncRNA NUTM2A antisense RNA 1(NUTM2A-AS1) is related to various cell phenotype, including apoptosis 62 and cell viability. 63The previous study has demonstrated that NUTM2A-AS1 could affect cell apoptosis by changing the expression of high-mobility Group Box 1 (HMGB1). 62In most eukaryotic cells, HMGB1 is highly conserved non-histone nuclear protein and binds to the DNA in the nucleus to plays an important role in DNA repair. 64hus, DCLRE1A activated NUTM2A-AS1 may also play a positive role in the regulation of DNA repair pathway.6][67] Furthermore, XIST can regulate MMR gene MSH6 expression through directly targetting miR-29c via SP1 and MGMT. 68XIST may be a potential therapeutic target through regulate LECs DNA repair pathway after overexpression DCLRE1A.Furthermore, we validated the siginificant increasing two lncRNAs after overexpression DCLRE1A.In addition, miRNA let-7c-5p and let-7a-5p were significantly increased in ARC. 9,69The screening miRNAs may be involved in the pathogenesis of ARC, in particular, related to DNA repair and autophagy. 9,69As our previous study identified that let-7c-5p can downregulated the expression of ERCC6 might prevent the NER pathway. 9Herein, our analysis found that two predicted interactions miRNAs, as let-7c-5p and let-7a-5p, were associated with DCLRE1A-activated lncRNAs XIST and NUTM2A-AS1.
Nevertheless, the current study also had some shortcomings.Firstly, the molecular mechanisms leading to lncRNA activation after overexpression DCLRE1A remain to be explored.Secondly, the DCLRE1A biological functions need to be explore further.Lastly, the relationship among DCLRE1A activated lncRNAs, screening miRNAs, and DNA repair genes remains to be further validated and explored.
In conclusion, this study proved that DCLRE1A was decrease in ARC tissues and H 2 O 2 treated cells.Functionally, elevated DCLRE1A expression can enhance LECs viability, promote damaged-DNA repairing, and ultimately inhibits apoptosis.Next, we demonstrate that DCLRE1A may regulated lncRNAs in LECs and DCLRE1Arelated lncRNAs could affect DNA damage repair processes by ceRNA networks.Our study lays the foundation for further exploration of the pathogenesis and therapeutic targets of ARC.

Disclosure statement
No potential conflict of interest was reported by the author(s).
Finally, we performed IF assays to visualize the localization and indirectly detected the expression of DCLRE1A during H 2 O 2 treatment.Results showed that DCLRE1A was significantly reduced in H 2 O 2 -treated SRA01/04 cells and was distributed in both the nucleus and cytoplasm (Figure 2(C)).

Figure 1 .
Figure 1.DCLRE1A was decreased in ARC patients' specimens.(A) WB assay was performed to detect DCLRE1A protein expression level between in control patients and ARC patients.(B) DCLRE1A mRNA expression was detected by qRT-PCR.C. IHC assay found that DCLRE1A was decreased in the anterior capsular tissue in ARC pateints.Scale bar: 50 lm." Ã " represents difference compared to the control group.ÃÃÃÃ p < 0.0001.

Figure 3 .
Figure 3. Enrichment analysis of DE DRGs in OE-DCLRE1A cells.(A) SRA 01/04 cells were transfected with vector, pcDNA-DCLRE1A or untreated.DCLRE1A protein expression was elevated in the OE-DCLRE1A group.ÃÃÃÃ p < 0.0001.(B) Scatter plot was utilized to evaluate the expression of relative mRNA in OE-DCLRE1A cells.Intensity increases from blue (low) to red (high).(C-D) Analysis of mRNA changes between the OE-DCLRE1A and control group using KEGG enrichment.Blue and red represent downregulated and upregulated genes, respectively.(E-F) Comparison of OE-DCLRE1A and the control group's mRNA changes using GO analysis.The order is arranged by enrichment score (-log10(P value)).

Figure 4 .
Figure 4. DCLRE1A decreased DNA damage and enhanced SRA01/04 cell survival.(A) heat map was employed to evaluate the relative mRNA expression in OE-DCLRE1A cells.from low-intensity blue to high-intensity red (high).(B-C) IF was applied to detect DNA damage markers inside the cells.15A3, cH2A, and DAPI were each labeled in red, green, and blue, respectively.(D) Cell viability was assessed by using CCK8 assay.(E) WB analysis was performed to detected the expression of cH2A." Ã " represents difference compared to the control group." & " represents difference compared to the HA þ H 2 O 2 group.ÃÃÃÃ p < 0.0001.&&&& p < 0.0001.

Figure 5 .
Figure 5.Effect of DCLRE1A on cell apoptosis.(A) WB identified BAX and Bcl-2.The right column listed the Bcl-2/BAX relative ratio.(B) The TUNEL assay demonstrated cell apoptosis.Blue stands for DAPI and red for TUNEL stain." Ã " represents difference compared to the control group." & " represents difference compared to the HA þ H 2 O 2 group.ÃÃÃÃ p < 0.0001.&&&& p < 0.0001.

Figure 6 .
Figure 6.Profiles and enrichment analysis of DE lncRNA and miRNA in the OE-DCLRE1A and control group.(A-D) Heat maps (A and D) and scatter plots (B and C) were used to analysis the expression of DE lncRNAs and miRNAs between OE-DCLRE1A group and control group.The expression level is represented by a color scale, increasing from low (blue) to high (red).(E-F) GO and KEGG pathway analyses were used to evaluate the target DE-mRNAs of miRNAs.The order is based on enrichment score (-log10(p value)).

Figure 7 .
Figure 7. Building and identifying lncRNA-miRNA-DRG regulatory networks associated to DCLRE1A.(A) The top 20 miRNAs associated with DNA damage were ranked by the quantity of target mRNAs.(B) The lncRNA-miRNA-mRNA network for the DCLRE1A gene was created using XIST and NUTM2A-AS1.(C, D) The lncRNA XIST NUTM2A-AS1 alteration was verified by qRT-PCR.(E, F) The relative quantitative expression of miR let-7a-5p and let-7c-5p." Ã " represents difference compared to the control group." & " represents difference compared to the HA þ H 2 O 2 group.Data were shown as the mean ± SD. n ¼ 3. ÃÃÃ p < 0.001.