CELF1 Promotes Matrix Metalloproteinases Gene Expression at Transcriptional Level in Lens Epithelial Cells

CELF1 Promotes Matrix Metalloproteinases Gene Expression at Transcriptional Level in Lens Epithelial Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CELF1 Promotes Matrix Metalloproteinases Gene Expression at Transcriptional Level in Lens Epithelial Cells Jun Xiao, Xi Tian, Siyan Jin, Yanhui He, Meijiao Song, He Zou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-785595/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Mar, 2022 Read the published version in BMC Ophthalmology → Version 1 posted 10 You are reading this latest preprint version Abstract Background : RNA binding proteins (RBPs)-mediated regulation plays important roles in many eye diseases, including the canonical RBP CELF1 in cataract. While the definite molecular regulatory mechanisms of CELF1 on cataract still remain elusive. Methods : In this study, we overexpressed CELF1 in lens epithelial SRA01/04 cells and applied whole transcriptome sequencing (RNA-seq) method to analyze the global differences mediated by CELF1. We then analyzed public RNA-seq and CELF1-RNA interactome data to decipher the underlying mechanisms. Results : The results showed that transcriptome profile was globally changed by CELF1 overexpression (CELF1-OE). Functional analysis revealed CELF1 specifically increased the expression of genes in extracellular matrix disassembly, extracellular matrix organization, and proteolysis, which could be classified into matrix metalloproteinases (MMPs) family. This finding was also validated by RT-qPCR and public mouse early embryonic lens data. Integrating analysis with public CELF1-RNA interactome data revealed that no obvious CELF1-binding peak was found on the transcripts of these genes, indicating an indirectly regulatory role of CELF1 in lens epithelial cells. Conclusions : Our study demonstrated that CELF1-OE promotes transcriptional level of MMP genes; and this regulation may be completed by other ways except for binding to RNA targets. These results suggest that CELF1-OE is implicated in the development of lens, which is associated with cataract and expands our understanding of CELF1 regulatory roles as an RNA binding protein. Ophthalmology CELF1 transcriptional regulation RNA-seq MMPs cataract Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Cataract, characterized by opacified lens, is the most common cause of reversible loss of vision which troubled nearly 24 million people worldwide [ 1 ]. White reflex of eye is an important symptom of cataract, named Leukocoria [ 2 ]. Lens is a transparent biconvex structure in eye that maintains the eye clarity and focus light onto the retina. It is composed of fibers, which are generated from the lens epithelium and then migrate from the periphery towards the center. Under normal circumstances, newly formed lens cells adhere externally to older cells. However, once epithelial cells are unable to shed so that differentiate into lens fibers, they will plie up centrally, with the oldest cells being in the center of the lens, ultimately develop into cataract with discoloration and opacities in the lens [ 1 ]. Drugs, chemical injury, mechanical trauma, ionising, infrared and ultraviolet radiate are common risk factors contributing to cataractogenesis [ 3 ]. RNA-binding proteins (RBPs) play an essential role in the process of post-transcriptional regulation. RNAs interact with RBPs through a series of canonical RNA-binding domains to form ribonucleoprotein complexes that involved in many post-transcriptional events including mRNA stability, polyadenylation, splicing, localization, and degradation [ 4 , 5 ]. The importance of RBPs in eye development and diseases has been received with concern in recent years [ 6 ]. RBPs are also important for lens development and cataract pathogenesis. For example, TDRD7 was firstly identified to be involved in cataract in human [ 7 ]. Caprin2 is another RBP that causes severe lens defects and features of Peter Anomaly in Caprin2-knockout mouse [ 8 ]. More recently, CELF1 was also identified as an important RBP in regulating post-transcription during lens development [ 9 ]. As a multifunctional RBP, CELF1 was known to preferentially bind to GU-rich elements (GREs) predominantly located in 3’ untranslated regions (UTRs) of target mRNAs [ 10 ]. Based on this structure, CELF1 has been implicated in various post-transcriptional processes, such as alternative splicing [ 11 ], localization [ 12 ], decay [ 13 ], and translation [ 14 ]. RNA binding sites of CELF1 was also identified by CLIP-seq, which promotes to characterize the genome-wide functions of CELF1 in human HeLa cells [ 15 ]. Regarding to the important roles of CELF1 in diverse biological processes, CELF1 dysregulation usually resulted in many diseases. Precocious myotube formation in mouse myoblasts is caused by knockdown of Celf1, which may play a negative role in terminal myocyte differentiation [ 16 ]; In type 1 diabetic mouse hearts, mutations of CELF1 binding sites impair alternative splicing regulation by CELF1, leading to abnormal gene expression [ 17 ]. Previous studies have also shown that CELF1 was associated with cataract by functioning as an RNA binding protein. Severe eye defects and cataract were presented in Celf1-knockout mice or Celf1-knockdown zebrafish and Xenopus[ 9 ]. To achieve lens transparency, Celf1 regulates key factors, such as p27 Kip1 and Dnase2b which are necessary for degradation of nuclear and fiber cell morphology by controlling the expression level of target mRNAs, providing new Celf1-regulated mechanisms involved in lens transparency. Using immunofluorescence assays, abnormally high expression of Pax6 protein was observed in CELF1 knockdown lenses compared to control lenes, suggesting that the eye transcription factor PAX6 is a key target regulated by CELF1 in lens development [ 18 ]. In addition, Aryal et al [ 19 ] found that Celf1 could post-transcriptionally control the spatiotemporal expression of the key homeodomain transcription factors Pax6 and Prox1 in the lens development in mice. RNA-immunoprecipitation assays showed that Celf1 negatively controls Pax6 and Prox1 translation through binding to their 3’ UTRs. These findings indicated that Celf1 plays a crucial role in lens development, and that it is a potential candidate for therapeutic treatment of cataract. However, the exact mechanism of gene expression level regulation mediated by CELF1 during lens development or cataractogenesis is largely unknown. To gain insight into the underlying molecular mechanisms, we obtained transcriptome profile after CELF1 overexpression in human lens epithelial SAR01/04 cells, and made a comprehensive analysis of the RNA-seq data with the public datasets. Our results revealed that a large number of genes in proteolysis were increased upon CELF1-overexpression; these genes were primarily MMPs, which were associated with cataract formation. Our findings expanded the understanding of critical CELF1 functions in lens development and provided a potential link with cataractogenesis. Methods Cell culture and transfection SRA01/04 cells, a lens epithelial cell line widely used in lens study, were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Science, Shanghai, China) and cultured in DMEM with 10% fetal bovine serum (FBS), penicillin (100 U/µl) and streptomycin (100 µg/ml). To increase the expression level of CELF1, a CELF1-overexpressing plasmid was transfected into SRA01/04 using Lipofectamine 2000 based on the manufacturer’s protocol. After 48 h, cells were pooled for RT-qPCR analyzing, SRA01/04 cells transfected with empty vector were used as control. Assessment of CELF1-overexpression To assess the efficiency of CELF1-overexpression, cDNA synthesis was conducted according to standard procedures followed by RT-qPCR, with the expression level of GAPDH as control. Transcript levels of CELF1 were measured by comparing with GADPH expression using 2 −ΔΔCT method [ 20 ]. Western blot analysis After transfection for 48 h, SRA01/04 were grouped and lysed in RIPA buffer, the samples were centrifuged at a speed of 12,000 rpm for 5min, supernatants were heated at 100 ℃ for 10 min. The samples were subjected to SDS-PAGE and subsequently transferred onto PVDF membranes. After blocking with 5% skim milk for one hour, the membranes were incubated with monoclonal Flag antibody (1:1,000 dilution; polyclonal antibody; cat. no.2368S; CST) at 4 ℃ overnight and GADPH as control (1:2000 dilution; polyclonal antibody; cat. no.A19056; ABClonal). Then the membranes were incubated with secondary antibody for an hour. Finally, the membranes were visualized by enhanced chemiluminescence (ECL). Complementary DNA (cDNA) library preparation and RNA-seq Total RNA was extracted by Trizol and further purified with two phenol-chloroform treatments, to acquire purified RNA, RQ1 DNase was added to remove DNA. Further, absorbance at 260 nm/280 nm was detected by Agilent Bioanalyzer 2100 to assess the quantity and quality of RNA. Before RNA-sequencing, 10 µg polyadenylated mRNA were prepared and concentrated with oligo (DT)-conjugated magnetic beads for each sample. Then, the mRNA samples were iron fragmented at 95 ℃, followed by end repair, A tailing, and reverse transcribed with RT primer harboring 3’ adaptor sequence and randomized hexamer. Finally, the cDNAs were amplified for sequencing. To insure high-quality reads, several criteria were set as following: Firstly, raw reads that were more than 2-N bases were abandoned; Secondly, filtered low quality bases and adaptors from raw reads; Thirdly, short reads that were less than 16nt were also removed; At last, clean reads were mapped to the GRch38 genome by TopHat2 [ 21 ]. The libraries were applied to NextSeq 500 system for 150 nt paired-end sequencing (ABLife Inc., Wuhan). Bioinformatics analysis Fragments per Kilobase per Million (FPKM) was used to evaluate gene expression level [ 22 ]. Meanwhile, R package edgeR [ 21 ] was applied to filter out differentially expressed genes (DEGs), fold change (FC ≥ 1.5) and false discovery rate (FDR < 0,05) were set as criteria of DEGs. In addition, Gene Ontology (GO) terms and KEGG pathways were carried out to sort out functional categories of DEGs [ 23 ]. Hypergeometric test and Benjamini-Hochberg FDR controlling procedure were used to define the enrichment of each pathway. Validation of DEGs by qPCR To elucidate the validity of our RNA-seq data, quantitative real-time PCR was performed in several candidate genes. PCR amplifications were performed in triplicate for each sample with conditions consisting of denaturing at 95 ℃ for 10 min, 40 cycles of denaturing at 95 ℃ for 15 s, annealing and following by extension at 60 ℃ for 1 min. To explore the mRNA binding profile of CELF1, we obtained and analyzed the RNA ligands and binding sites of CELF1 in HeLa cells from two sets of published data SRP0935 and PRJEB12208 [ 11 , 15 ]. Furthermore, we also analyzed correlation between CELF1 and candidate DEGs in house mouse of different embryonic lens development from published data GSE119596 [ 24 ], in which the total number and expression level of cataract or lens defects-related genes, including CELF1 progressively increased as the lens develops from E10.5 through E16.5 stages. It implies that older stages might exhibit higher risks of cataract formation than that of younger stages. Statistical analysis For statistical method, all values were presented with mean ± standard deviation, SPSS statistical software (Chicago, IL) was used to analyze data. Only if the p -value was less than 0.05 (p < 0.05) then the difference was considered significant. Results Assessment of CELF1-overexpression in SRA01/04 cells To comprehensively investigate CELF1-mediated transcriptional regulation, SRA01/04 cells were transfected with a CELF1 -overexpressing (OE) plasmid using Lipofectamine 2000 followed by whole transcriptome sequencing (RNA-seq). We constructed six RNA-seq libraries and sequenced for CELF1-overexpression and control SRA01/04 cells, with three replicates for each group. The protein level of CELF1 was increased in CELF1-OE samples compared with control samples, as assessed by western blot in Fig. 1 A and Fig S1. Accordingly, the mRNA level of CELF1 was increased approximately 4 times compared with control by RT-qPCR (Fig. 1 B). Further, the efficiency of CELF1-overexpression was also confirmed by RNA-seq analysis (Fig. 1 C), indicating that overexpression of CELF1 in SRA01/04 cells was successful. FPKM values were applied for calculating the variation contribution of principal components between CELF1-overexpression and control cells, the distribution of principal component analysis (PCA) showed that the biological replicates were clustered together by the second component (Fig. 1 D). CELF1 overexpression regulates transcription in SRA014/01 cells After obtaining the raw RNA-seq reads, we discarded the adaptor sequences and low-quality reads, generating a total of 77.6 ± 5.4 million raw reads per sample and 74.3 ± 5.8 million clean reads per sample (Table 1 ), in which an average of 72.0 ± 5.2 million were paired-end reads. Based on the mapping results by TopHat2 [ 21 ], 95.59–96.72 % of those paired-end reads were mapped to the human GRCH38 genome, and about 85.74–96.95 % were uniquely aligned (Table 1 ). These results supported the validity of sequencing data in our experiments. To identify the gene expression profiles regulated by CELF1, FPKM values were applied to calculate the expression level of candidate genes. RNA-seq yielded 19,703 genes, in which 9,585 genes were detected at an expression level of FPKM > 1 in at least one sample. Table 1 Information of RNA-seq reads in experiment Sample CELF1_SRA_1st CELF1_SRA_2nd CELF1_SRA_3rd Ctrl_1st Ctrl_2nd Ctrl_3rd Mean Raw reads 79902626 84904120 81594846 70549626 75180916 73338412 77578424.33 ± 5446634.377 Clean reads 77004105 81761685 78505488 66213178 71926994 70345341 74292798.5 ± 5785186.607 Paired-end reads 74701846 79255492 76261510 64028722 69738738 68075308 72010269.33 ± 5198713.892 Total mapped(%) 71677060(95.95%) 76091134(96.01%) 73420345(96.27%) 61822687(96.55%) 67454303(96.72%) 65835489(96.71%) 69383503 ± 4830889.588 Uniquely mapped(%) 63361981(88.4%) 68826896(90.45%) 62950881(85.74%) 57030340(92.25%) 63156202(93.63%) 63830263(96.95%) 63192760.5 ± 3420862.379 Splice reads (%) 32010772(50.52%) 35210711(51.16%) 33379884(53.03%) 29918008(52.46%) 32659050(51.71%) 33157280(51.95%) 32722617.5 ± 1742879.187 To analyze genes responding to elevated CELF1 in SRA01/04 at the whole transcriptome level, edgeR [ 25 ] was used to perform differentially expressed genes (DEGs) analysis on our data. When we set the cut-off as fold change ≥ 2 or ≤ 0.5 and a 5% false discovery rate (FDR), 97 down-regulated and 225 up-regulated genes were identified, respectively (Fig. 2 A; Table S1). The results demonstrated that CELF1 significantly changed gene expression profile in SRA01/04 cells. Hierarchical clustering heat map analysis of DEGs expression pattern successfully and clearly separated CELF1-OE and control samples, revealing a high consistency among the triplicates data sets (Fig. 2 B). To further characterize the potential functions of these DEGs, all the identified DEGs were annotated by GO and KEGG functional enrichment analysis. The results revealed that upregulated DEGs were significantly enriched in GO biological process pathways, which were primarily associated with regulation of transcription, collagen catabolic process, extracellular matrix disassembly, proteolysis and extracellular matrix organization (Fig. 2 C). The top two GO BP pathways were regulation of transcription related pathways, suggesting that CELF1 could promote expression level of transcription regulators. While downregulated DEGs were only enriched in one GO terms: Small molecule metabolic process (Fig. 2 D). When we set p -value as < 0.05 for KEGG pathways, we analyzed and presented the top ten KEGG pathways for upregulated DEGs (Fig S2A), including cytokine-cytokine receptor interaction, chemokine signaling pathway and apoptosis. Whereas, down-regulated DEGs were enriched primarily in fat digestion and absorption and pathogenic Escherichia coli infection (Fig S2B). Elevated CELF1 alters expression of genes involved in proteolysis To further confirm the reliability of the RNA-seq results, we downloaded a set of RNA-seq data from early embryonic lens of mouse [ 24 ], in which the total number and expression level of contact or lens defects-related genes progressively increased as the lens developed from lens pit stage (embryonic day (E) 10.5) through secondary fiber differentiation stage (E16.5), implying that the embryonic lens of mouse in older stages might exhibit higher risks of cataract than that of younger stages. More important, a significant elevated Celf1 was observed when early embryonic lens developed from E10.5 to E14.5 (Fig. 3 A), indicating that elevated Celf1 might associated with lens formation and development in mouse. Based on the elevated expression level of Celf1, the transcriptome profile of embryonic lens both in E10.5 and E14.5 could be a substitute of Celf1 overexpression dataset and were selected for further analysis to make a comparison with our data. A total of 322 and 8186 protein coding genes were identified as DEGs in our dataset and downloaded dataset, respectively. However, among those genes, only 26 up-regulated DEGs were overlapped between two datasets. Accordingly, 7 down-regulated DEGs were overlapped (Fig. 3 B). Species differences may result in the low overlap rate of DEGs between human and mouse. In our present study, SRA01/04 cells which are monoplast originated from human were subjected to RNA-seq, whereas embryonic lens of mouse were preferred in downloaded dataset. GO molecular function enrichment analysis revealed that up-regulated genes in mouse embryonic lens were enriched in cell adhesion, lens development in camera-type eye, eye development and lens fiber cell development, indicating elevated CELF1 extensively regulated expression of genes implicated in lens development (Fig. 3 C). In addition, up-regulated genes were also enriched in proteolysis, which was also present in top 10 representative GO biological processes of up-regulated genes in SRA01/04 cells (Fig. 2 C). Thus, a heat map was generated to compare expression profile of genes enriched in proteolysis. Hierarchical clustering of normalized FPKM values of genes involved in proteolysis pathway showed separation between CELF1 overexpression SRA01/04 cells and control cells (Fig. 3 D), while a clear separation of proteolysis-related genes was observed between E10.5 and E14.5 (Fig. 3 E). These results indicated that elevated CELF1 significantly changed the expression level of genes associated with proteolysis both in SRA01/04 cells and mouse lens at embryonic stages, implying proteolysis may plays an essential role in lens development. Further validation of CELF1-regulated gene transcription in proteolysis In order to verify the DEGs results in RNA-seq, we performed RT-qPCR analysis to determine the gene expression levels. Five up-regulated DEGs from proteolysis were selected for RT-qPCR analysis, including MMP1, MMP7, MMP9, MMP13, and CTSS (Fig. 4 A). The primers of these genes were shown in Table 2 . A statistically significant increase of those genes was observed in RT-qPCR experiment (Fig. 4 B), which was in line with the FPKM values (Fig. 4 A). These results demonstrated the high correlation between RNA-seq and RT-qPCR results, confirming the discoveries in RNA-seq dataset. Furthermore, we also found that increased CELF1 expression level was coupled with elevated expression of MMP9 in embryonic lens of mouse (Fig. 4 C), when embryonic lens developed from E10.5 and E14.5. We also analyzed their expression level changes using the Celf1 knockout (Celf1-KO) microarray data GSE101393 [ 9 ], and found all of them showed no significant change between Celf1-KO and WT samples. Collectively, the results suggested that CELF1 may participate in lens development or cataract formation by regulating expression level of genes in proteolysis, particularly, matrix metalloproteinases. Table 2 Primers for detected genes Primer Name Sequence 5’ to 3’ MMP1-F CAGGGACAGAATGTGCTA MMP1-R TTTCCAGTGTTTTCCTCA MMP7-F AAGCCAAACTCAAGGAGA MMP7-R AGTCCATTTTGGGCTATT MMP9-F CCTTCTACGGCCACTACT MMP9-R ATCCTTGAACAAATACAGC MMP13-F TTTTGGGCTCTTAATGGT MMP13-R AGTCTTGCCTGTATCCTC CTSS-F CCAAGGCAGGCATATCAA CTSS-R TGGGTTCAAGGAATCTCG Functional analysis of genes bound by CELF1 We downloaded a set of CELF1 RIP-seq data [ 11 ] and a set of CELF1 CLIP-seq data for HeLa cells [ 15 ] to evaluate whether CELF1 directly bind to those genes and subsequently regulate their expression levels. After performing overlapping analysis, we detected 38 DEGs in our study overlapped with CELF1-targeted genes in RIP-seq experiment (Fig. 5 A) and only 18 DEGs overlapped in CLIP-seq experiment (Fig. 5 D). GO functional enrichment analysis was performed on overlapped DEGs between RIP-seq data and our data, 5 up-regulated DEGs were enriched in transcription, DNA-dependent (Fig. 5 B), which is consistent with the GO BP enrichment analysis of DEGs (Fig. 2 C). KEGG pathways (p-value < 0.05) were primarily enriched in Systemic lupus erythematosus, Phagosome, DNA replication, and Staphylococcus aureus infection (Fig. 5 C). By analyzing the genes validated by RT-qPCR in Fig. 4 , we did not find obvious CELF1 peaks that were enriched on the transcripts of MMP1, MMP9, MMP13, or CTSS. These results indicate that CELF1 might regulate expression of most DEGs by an indirect manner, while it could directly bind to TF related genes and regulate their expression levels in SRA01/04 cells. Discussion Cataract has been characterized as severe eye disease with opacified lens, and loss of epithelial polarity and cell multi-layering are primary signs of pathogenesis in cataractogenesis. Siddam teams applied a bioinformatics tool iSyTE to identify a novel RNA binding protein, CELF1, which is important for lens development and cataract formation [ 9 ]. Their results showed that Celf1 -knockout in mice, or knockdown in zebrafish or Xenopus morphants resulted in severe eye defects or cataract. Except for TDRD7 and Caprin2 [ 8 , 26 , 27 ], CELF1 was identified as another RBP that plays a crucial role during cataract formation. However, CELF1-overexpression meditated transcriptional or post-transcriptional regulations in lens development or cataract formation remains to be resolved. To have a deeper understanding of CELF1 functions on lens development and cataract, we performed RNA-seq experiments in CELF1-overexpressed SRA01/04 cells and control cells. Our results showed that elevated CELF1 globally changed expression profile in SRA01/04 cells. As an RNA binding protein, overexpression of CELF1 has been associated with many diseases, for example, CELF1-OE was correlated with lower levels of endogenous p27, at the same time, repressing p27 IRES activity in human breast cancer cell line MCF7 [ 14 ]. Moreover, elevated CELF1 expression mediated defects of myocytes with CUG-expansion, by increasing myocyte cycling [ 16 ]. In breast epithelial cells, CELF1-OE promoted the translation of epithelial to mesenchymal transition (EMT) and ultimately tumor progression [ 28 ]. All together, these findings indicated that CELF1-OE plays an essential role in pathological processes. In agreement with previous studies, we provided evidence that elevated CELF1 may also play a role in cataract regulation and lens development. Based on GO analysis results, the upregulated DEGs by CELF1-OE were highly enriched in transcriptional regulation, extracellular matrix disassembly and organization, and proteolysis, which were presented in the top 10 pathways. As we know, protein turnover in extracellular matrix is common and important as a fundamental feature of many normal and pathological processes [ 29 ]. Undoubtedly, alteration in extracellular matrix turnover is also associated with cataract [ 30 , 31 ]. Posterior capsule opacification (PCO), which is also known as secondary cataract, was characterized by cellular migration onto the posterior lens capsule, coupling with deposition of abnormal extracellular matrix and capsular wrinkling, all of which could lead to opacification of lens and ultimately cataract [ 32 ]. In addition, proteolysis is another typical characteristic in cataract formation [ 33 – 35 ]. Wang et al proposed that elevated proteolysis resulted from S129R mutation might induce pathology since significant decline of functional proteins changed the protein-protein interaction network [ 36 ]. Both in SRA01/04 cells of human and embryonic lens of mouse, DEGs were enriched in proteolysis pathway accompanying with elevated CELF1 indicating proteolysis might be a crucial pathway regulated by CELF1 in lens development. In the present study, all five genes enriched in proteolysis were up-regulated in CELF1-OE cells, and four of them were also belong to matrix metalloproteinase family, including MMP1, MMP7, MMP9, and MMP13. The result suggests the potential importance of MMPs in lens development and cataract regulation. In virtually, MMPs are widely distributed in every tissue of the eye under conditions of health and disease, they present a family of proteolytic enzymes that are involved in the breakdown of extracellular matrix in normal physiological process, ultimately influencing cell biological activities and morphogenesis [ 37 ]. MMP2 and MMP9 were most widely investigated for their important role in cataract. The level of MMP2 and MMP9 activities of patients with steroid induced posterior subcapsular cataract (PSC) in lens epithelial cells (LECs) and the serum was evaluated, MMP2 and MMP9 activities in both LECs and serum were significantly higher in cases with steroid induced PSC [ 38 ]. However, MMP9 plays a more important role in mediating TGF-β-induced anterior subcapsular cataract formation than MMP2 [ 39 ]. Besides, in human lens epithelial cells, LDL receptor related protein 5 like (LRP5L) may promote angiogenesis by increasing active MMP9. While its mutant, LRP5L-P36R, may inhibit angiogenesis by decreasing active MMP9 and laminin γ1, indicating that LRP5L-P36R may promote the formation of cataract via attenuating biological function of MMP9 [ 40 ]. These results suggest the indefinite roles of MMP9 in cataract formation. In this study, we propose that CELF1-OE might regulate lens development and cataract formation via up-regulating the expression of MMPs, especially MMP9. To further explore the mechanism that CELF1 regulated expression of MMPs, analysis of target genes to which CELF1 was bound. Previous study provided evidence that CELF1 associated to the 5’-UTR of human p27 mRNA and suppressed expression of p27 [ 14 ]; CELF1 was also inclined to binding with 3’UTR and intron regions of mRNA and further globally changed alternative splicing and translation of multiple genes [ 11 , 28 ]. Recently, an investigation revealed that Tristeraprolin (TTP) peaks were enriched in CELF1 binding motifs indicating that TTP might cooperate with other RBPs to participate in multiple post-transcriptional process [ 41 ]. While, in the current study, we did not find any obvious CELF1 peaks enriched on proteolysis-related genes. Genome-wide chromatin immunoprecipitation method revealed pervasive chromatin-RBP interactions, indicating the transcriptional regulatory ability of RBPs [ 42 ]. Based on these existing results, we speculate that CELF1 may regulate expression of proteolysis-related genes at transcriptional level or with an indirectly manner, such as protein-protein interactions. Furthermore, GO analysis showed that CELF1 targeted genes were primarily involved in DNA-dependent transcription regulation, including CDH9, SP4, ZNF420 and ZNF587 that are classical transcriptional factors. Through binding with DNA, these genes could regulate gene transcription [ 43 , 44 ]. Direct interacting signals between CELF1 and these transcripts were also found from RIP/CLIP sequencing data (Fig. 5 A). These CELF1-targeted genes implying that CELF1 might involve in lens development or cataract pathogenesis by changing expression levels of transcription factors. Besides, CELF1 may also regulates expression of MMPs by producing various transcripts, which need further investigation. Conclusions A genome-wide profile of CELF1-OE functions in regulating RNA-levels of genes in lens epithelial cells was offered in this study. The expression levels of hundreds of genes regulated by CELF1 were identified, and most of them were linked to multiple biological processes related to lens development. The main definite discovery is that CELF1 may indirectly regulate expression level of MMPs at transcriptional level, suggesting its potential important roles in lens development and cataract formation. Our study contributes to a precise understanding of potential CELF1-targeted therapies in cataract diagnosis and treatment. Abbreviations CTSS Cathepsin S; CELF1:CUG-BP, Elav-like family member 1; DEGs:differentially expressed genes; FPKM:fragments per kilobase of exon per million fragments mapped; GO:Gene Ontology; GREs:GU-rich elements; KEGG:Kyoto Encyclopedia of Genes and Genomes; MMPs:matrix metalloproteinase; PCO:Posterior capsule opacification; RBPs:RNA-binding proteins; UTRs:untranslated regions; Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The RNA-seq data has been deposited in NCBI Gene Expression Omnibus (GEO) under accession code GSE153022. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Natural Science Foundation of Jilin Province (20200201347JC). Authors' contributions JX and HZ design the experiment. 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Prog Retin Eye Res 2002, 21 (1):1–14. Alapure BV, Praveen MR, Gajjar DU, Vasavada AR, Parmar TJ, Arora AI: Matrix metalloproteinase-2 and – 9 activities in the human lens epithelial cells and serum of steroid induced posterior subcapsular cataracts . Mol Vis 2012, 18 :64–73. Korol A, Pino G, Dwivedi D, Robertson JV, Deschamps PA, West-Mays JA: Matrix metalloproteinase-9-null mice are resistant to TGF-beta-induced anterior subcapsular cataract formation . Am J Pathol 2014, 184 (7):2001–2012. Sun L, Song F, Du L, Wang J: LRP5L-P36R Attenuating Matrix Metalloproteinase 9 Biological Function May Contribute to the Development of Cataract . 2019. Tu Y, Wu X, Yu F, Dang J, Wei Y, Yu H, Liao W, Zhang Y, Wang J: Tristetraprolin-RNA interaction map reveals a novel TTP-RelB regulatory network for innate immunity gene expression . Mol Immunol 2020, 121 :59–71. Xiao R, Chen JY, Liang Z, Luo D, Chen G, Lu ZJ, Chen Y, Zhou B, Li H, Du X et al : Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription . Cell 2019, 178 (1):107–121 e118. Wang C, Pan YH, Wang Y, Blatt G, Yuan XB: Segregated expressions of autism risk genes Cdh11 and Cdh9 in autism-relevant regions of developing cerebellum . Mol Brain 2019, 12 (1):40. Sheehan K, Lee J, Chong J, Zavala K, Sharma M, Philipsen S, Maruyama T, Xu Z, Guan Z, Eilers H et al : Transcription factor Sp4 is required for hyperalgesic state persistence . PLoS One 2019, 14 (2):e0211349. Additional Declarations No competing interests reported. Supplementary Files FigS1.tif FigS2.tif SupplementaryInformation.docx TableS1ListofDEGsidentifiedinthisstudy.xlsx Cite Share Download PDF Status: Published Journal Publication published 14 Mar, 2022 Read the published version in BMC Ophthalmology → Version 1 posted Editorial decision: Major revision 29 Nov, 2021 Reviews received at journal 25 Nov, 2021 Reviewers agreed at journal 19 Nov, 2021 Reviews received at journal 26 Oct, 2021 Reviewers agreed at journal 26 Oct, 2021 Reviewers invited by journal 25 Oct, 2021 Editor assigned by journal 09 Oct, 2021 Editor invited by journal 11 Aug, 2021 Submission checks completed at journal 11 Aug, 2021 First submitted to journal 05 Aug, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-785595","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":45038893,"identity":"2ab52718-f27f-48f2-83d1-a824d2747603","order_by":0,"name":"Jun Xiao","email":"","orcid":"","institution":"The Second Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Xiao","suffix":""},{"id":45038894,"identity":"df642a2c-190e-4a14-9980-50c0328cf67c","order_by":1,"name":"Xi Tian","email":"","orcid":"","institution":"The Second Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Tian","suffix":""},{"id":45038895,"identity":"723efc46-8857-4228-ad35-0af73ab35b6f","order_by":2,"name":"Siyan Jin","email":"","orcid":"","institution":"The Second Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siyan","middleName":"","lastName":"Jin","suffix":""},{"id":45038896,"identity":"5b1c9dea-47cb-4497-a875-709e923ded44","order_by":3,"name":"Yanhui He","email":"","orcid":"","institution":"The Second Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanhui","middleName":"","lastName":"He","suffix":""},{"id":45038897,"identity":"f18f9f4e-10ff-44c7-9181-f347e13fc9b1","order_by":4,"name":"Meijiao Song","email":"","orcid":"","institution":"The Second Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meijiao","middleName":"","lastName":"Song","suffix":""},{"id":45038898,"identity":"99a9bdab-6e39-4e24-931f-0ffa49e4e241","order_by":5,"name":"He Zou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie3PvYrCQBSG4SMDA8Jo2iOR5BZGhFR7MTMErLJgOYWwgsuk8KeOd2G5ZSQwNqO2FlskzbbrdlbiBbgk2W6LeerzwncAHOcfouG5Kq/qHtDwuyqFmjUnfWRklNl87EFCeGlNcxIgo35X53I7T+igeicthvnr3Af72dnlR6PknIKXLkV9MjyJ8VR9Eb7fTC7yYwhoj7v6BASPM0soLyC6SEuB42tzUvQ0YdxANJWatEgwGS16usDBikXQLmEm7mR2wj2kMQprWOMvYbo43K7q5U0j2f/c1Czw0nV98oT97dxxHMf51QPrrE1mclk+IgAAAABJRU5ErkJggg==","orcid":"","institution":"The Second Hospital of Jilin University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Zou","suffix":""}],"badges":[],"createdAt":"2021-08-06 01:14:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-785595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-785595/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12886-022-02344-8","type":"published","date":"2022-03-14T20:04:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":12368842,"identity":"9d95cd2c-035e-4ccc-b6f1-59255eaec700","added_by":"auto","created_at":"2021-08-12 13:45:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191878,"visible":true,"origin":"","legend":"Assessment of CELF1-overxpression in SRA01/04 cells. (A) Validation of CELF1-overexpression by Western Blot. (B) Validation of CELF1-overexpression by qPCR. (C) The FPKM values of CELF1 were calculated in RNA-seq. (D) Principal component analysis of all expressed genes between the control and CELF1 overexpression samples. Data are represented as the mean ± standard deviation. Student’s test was performed to compare CELF1-overexpression SRA01/04 cells and control with significance set at a p value of less than 0.05. *P \u003c 0.05, ***P \u003c 0.001.","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/74a9c1b7d9382a367171703e.png"},{"id":12368839,"identity":"1c4e67e8-79d5-4a14-b20a-cf78b331311e","added_by":"auto","created_at":"2021-08-12 13:45:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":327943,"visible":true,"origin":"","legend":"RNA-seq analysis of CELF1 regulated transcriptome in SRA01/04 cells. (A) Identification of CELF1 regulated genes. Red dots indicated up-regulated genes, whereas blue dots indicated down-regulated genes. (B) Heatmap of all DEGs in control and CELF1 overexpression samples. The most representative GO biological processes of up-regulated genes (C) and down-regulated genes (D). ","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/efed4bdddc1f21c872e2e17e.png"},{"id":12368840,"identity":"9bce37b7-faa0-4ba4-8912-e98973cf29f2","added_by":"auto","created_at":"2021-08-12 13:45:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":317036,"visible":true,"origin":"","legend":"Elevated CELF1 extensively regulates expression of genes involved in proteolysis. (A) Relative expression level of CELF1 determined by RNA-seq between E10.5 and E14.5 in mouse lens. (B) Venn diagram analysis exhibited the overlapping differentially upregulated or downregulated genes between our data and download data. (C) The top 20 representative GO biological processes of differentially expressed genes between E10.5 and E14.5 in mouse lens. (D) Heatmap of proteolysis-related genes in control and CELF1-overexpression SRA01/04 cells. (E) Heatmap of proteolysis-related genes in E10.5 and E14.5 in mouse lens. ","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/5603128ca18f8b32d4794159.png"},{"id":12369034,"identity":"658d1d75-6683-414e-a7af-0d975b48cd50","added_by":"auto","created_at":"2021-08-12 13:48:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":588192,"visible":true,"origin":"","legend":"Validation of CELF1-regulated genes. Validation of DEGs in SRA01/04 cells measured by RNA-seq (A) and qPCR (B). (C) Relative expression levels of MMP9 in mouse lens measured by RNA-seq. For qPCR, GAPDH was used as the reference gene. ","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/9b8e1851d0632bc699f2ae47.png"},{"id":12368843,"identity":"8be448c6-0721-48b3-bd82-9215a4fb1003","added_by":"auto","created_at":"2021-08-12 13:45:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":220606,"visible":true,"origin":"","legend":"Function analysis of CELF1-bound genes. (A) CELF1-regulated DEGs overlapped with the CELF1-bound genes from SRR503. (B) The most representative GO biological processes of overlapped genes between DEGs and CELF1-bound genes from SRR503. (C) The top representative KEGG pathways of overlapped genes between DEGs and CELF1-bound genes from SRR503. (D) CELF1-regulated DEGs overlapped with the CELF1-bound genes from PRJEB12208. (E) The top representative KEGG pathways of overlapped genes between DEGs and CELF1-bound genes from PRJEB12208.","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/926c8d6ffc9c66e45d165772.png"},{"id":19703137,"identity":"ce258ced-f616-4884-95d8-ec3dde524752","added_by":"auto","created_at":"2022-03-28 20:06:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2564333,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/133187c1-6e32-4672-947b-5392fe93ce1b.pdf"},{"id":12368846,"identity":"bfadfcbb-c8c0-4b64-81ba-ce2789af9666","added_by":"auto","created_at":"2021-08-12 13:45:26","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2207084,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/8654a26bf9e38478bb40552d.tif"},{"id":12368847,"identity":"b91fe65d-6cec-4d6b-be07-63df14de7094","added_by":"auto","created_at":"2021-08-12 13:45:26","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1047372,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/f8a1c3f77f8baebbc3a185db.tif"},{"id":12369035,"identity":"5804c87f-a253-4cd8-8bd1-7fe30682f0e1","added_by":"auto","created_at":"2021-08-12 13:48:26","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":538367,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/db0674492c1792cbe6d4d43b.docx"},{"id":12369036,"identity":"183a01fc-200e-4aaf-8a63-d1edbd448609","added_by":"auto","created_at":"2021-08-12 13:48:26","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":59032,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1ListofDEGsidentifiedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-785595/v1/389ac1ded02a2adcf4858cd4.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCELF1 Promotes Matrix Metalloproteinases Gene Expression at Transcriptional Level in Lens Epithelial Cells\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eCataract, characterized by opacified lens, is the most common cause of reversible loss of vision which troubled nearly 24\u0026nbsp;million people worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. White reflex of eye is an important symptom of cataract, named Leukocoria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Lens is a transparent biconvex structure in eye that maintains the eye clarity and focus light onto the retina. It is composed of fibers, which are generated from the lens epithelium and then migrate from the periphery towards the center. Under normal circumstances, newly formed lens cells adhere externally to older cells. However, once epithelial cells are unable to shed so that differentiate into lens fibers, they will plie up centrally, with the oldest cells being in the center of the lens, ultimately develop into cataract with discoloration and opacities in the lens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Drugs, chemical injury, mechanical trauma, ionising, infrared and ultraviolet radiate are common risk factors contributing to cataractogenesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRNA-binding proteins (RBPs) play an essential role in the process of post-transcriptional regulation. RNAs interact with RBPs through a series of canonical RNA-binding domains to form ribonucleoprotein complexes that involved in many post-transcriptional events including mRNA stability, polyadenylation, splicing, localization, and degradation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The importance of RBPs in eye development and diseases has been received with concern in recent years [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. RBPs are also important for lens development and cataract pathogenesis. For example, TDRD7 was firstly identified to be involved in cataract in human [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Caprin2 is another RBP that causes severe lens defects and features of Peter Anomaly in Caprin2-knockout mouse [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. More recently, CELF1 was also identified as an important RBP in regulating post-transcription during lens development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a multifunctional RBP, CELF1 was known to preferentially bind to GU-rich elements (GREs) predominantly located in 3\u0026rsquo; untranslated regions (UTRs) of target mRNAs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on this structure, CELF1 has been implicated in various post-transcriptional processes, such as alternative splicing [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], localization [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], decay [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and translation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. RNA binding sites of CELF1 was also identified by CLIP-seq, which promotes to characterize the genome-wide functions of CELF1 in human HeLa cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Regarding to the important roles of CELF1 in diverse biological processes, CELF1 dysregulation usually resulted in many diseases. Precocious myotube formation in mouse myoblasts is caused by knockdown of Celf1, which may play a negative role in terminal myocyte differentiation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; In type 1 diabetic mouse hearts, mutations of CELF1 binding sites impair alternative splicing regulation by CELF1, leading to abnormal gene expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies have also shown that CELF1 was associated with cataract by functioning as an RNA binding protein. Severe eye defects and cataract were presented in Celf1-knockout mice or Celf1-knockdown zebrafish and Xenopus[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To achieve lens transparency, Celf1 regulates key factors, such as p27\u003csup\u003eKip1\u003c/sup\u003e and Dnase2b which are necessary for degradation of nuclear and fiber cell morphology by controlling the expression level of target mRNAs, providing new Celf1-regulated mechanisms involved in lens transparency. Using immunofluorescence assays, abnormally high expression of Pax6 protein was observed in CELF1 knockdown lenses compared to control lenes, suggesting that the eye transcription factor PAX6 is a key target regulated by CELF1 in lens development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, Aryal et al [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] found that Celf1 could post-transcriptionally control the spatiotemporal expression of the key homeodomain transcription factors Pax6 and Prox1 in the lens development in mice. RNA-immunoprecipitation assays showed that Celf1 negatively controls Pax6 and Prox1 translation through binding to their 3\u0026rsquo; UTRs. These findings indicated that Celf1 plays a crucial role in lens development, and that it is a potential candidate for therapeutic treatment of cataract.\u003c/p\u003e \u003cp\u003eHowever, the exact mechanism of gene expression level regulation mediated by CELF1 during lens development or cataractogenesis is largely unknown. To gain insight into the underlying molecular mechanisms, we obtained transcriptome profile after CELF1 overexpression in human lens epithelial SAR01/04 cells, and made a comprehensive analysis of the RNA-seq data with the public datasets. Our results revealed that a large number of genes in proteolysis were increased upon CELF1-overexpression; these genes were primarily MMPs, which were associated with cataract formation. Our findings expanded the understanding of critical CELF1 functions in lens development and provided a potential link with cataractogenesis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eSRA01/04 cells, a lens epithelial cell line widely used in lens study, were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Science, Shanghai, China) and cultured in DMEM with 10% fetal bovine serum (FBS), penicillin (100 U/\u0026micro;l) and streptomycin (100 \u0026micro;g/ml). To increase the expression level of CELF1, a CELF1-overexpressing plasmid was transfected into SRA01/04 using Lipofectamine 2000 based on the manufacturer\u0026rsquo;s protocol. After 48 h, cells were pooled for RT-qPCR analyzing, SRA01/04 cells transfected with empty vector were used as control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of CELF1-overexpression\u003c/h2\u003e \u003cp\u003eTo assess the efficiency of CELF1-overexpression, cDNA synthesis was conducted according to standard procedures followed by RT-qPCR, with the expression level of GAPDH as control. Transcript levels of CELF1 were measured by comparing with GADPH expression using 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eAfter transfection for 48 h, SRA01/04 were grouped and lysed in RIPA buffer, the samples were centrifuged at a speed of 12,000 rpm for 5min, supernatants were heated at 100 ℃ for 10 min. The samples were subjected to SDS-PAGE and subsequently transferred onto PVDF membranes. After blocking with 5% skim milk for one hour, the membranes were incubated with monoclonal Flag antibody (1:1,000 dilution; polyclonal antibody; cat. no.2368S; CST) at 4 ℃ overnight and GADPH as control (1:2000 dilution; polyclonal antibody; cat. no.A19056; ABClonal). Then the membranes were incubated with secondary antibody for an hour. Finally, the membranes were visualized by enhanced chemiluminescence (ECL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eComplementary DNA (cDNA) library preparation and RNA-seq\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted by Trizol and further purified with two phenol-chloroform treatments, to acquire purified RNA, RQ1 DNase was added to remove DNA. Further, absorbance at 260 nm/280 nm was detected by Agilent Bioanalyzer 2100 to assess the quantity and quality of RNA.\u003c/p\u003e \u003cp\u003eBefore RNA-sequencing, 10 \u0026micro;g polyadenylated mRNA were prepared and concentrated with oligo (DT)-conjugated magnetic beads for each sample. Then, the mRNA samples were iron fragmented at 95 ℃, followed by end repair, A tailing, and reverse transcribed with RT primer harboring 3\u0026rsquo; adaptor sequence and randomized hexamer. Finally, the cDNAs were amplified for sequencing.\u003c/p\u003e \u003cp\u003eTo insure high-quality reads, several criteria were set as following: Firstly, raw reads that were more than 2-N bases were abandoned; Secondly, filtered low quality bases and adaptors from raw reads; Thirdly, short reads that were less than 16nt were also removed; At last, clean reads were mapped to the GRch38 genome by TopHat2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The libraries were applied to NextSeq 500 system for 150 nt paired-end sequencing (ABLife Inc., Wuhan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eFragments per Kilobase per Million (FPKM) was used to evaluate gene expression level [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Meanwhile, R package edgeR [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] was applied to filter out differentially expressed genes (DEGs), fold change (FC\u0026thinsp;\u0026ge;\u0026thinsp;1.5) and false discovery rate (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0,05) were set as criteria of DEGs.\u003c/p\u003e \u003cp\u003eIn addition, Gene Ontology (GO) terms and KEGG pathways were carried out to sort out functional categories of DEGs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Hypergeometric test and Benjamini-Hochberg FDR controlling procedure were used to define the enrichment of each pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eValidation of DEGs by qPCR\u003c/h2\u003e \u003cp\u003eTo elucidate the validity of our RNA-seq data, quantitative real-time PCR was performed in several candidate genes. PCR amplifications were performed in triplicate for each sample with conditions consisting of denaturing at 95 ℃ for 10 min, 40 cycles of denaturing at 95 ℃ for 15 s, annealing and following by extension at 60 ℃ for 1 min.\u003c/p\u003e \u003cp\u003eTo explore the mRNA binding profile of CELF1, we obtained and analyzed the RNA ligands and binding sites of CELF1 in HeLa cells from two sets of published data SRP0935 and PRJEB12208 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, we also analyzed correlation between CELF1 and candidate DEGs in house mouse of different embryonic lens development from published data GSE119596 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], in which the total number and expression level of cataract or lens defects-related genes, including CELF1 progressively increased as the lens develops from E10.5 through E16.5 stages. It implies that older stages might exhibit higher risks of cataract formation than that of younger stages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor statistical method, all values were presented with mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, SPSS statistical software (Chicago, IL) was used to analyze data. Only if the \u003cem\u003ep\u003c/em\u003e-value was less than 0.05 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) then the difference was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of CELF1-overexpression in SRA01/04 cells\u003c/h2\u003e \u003cp\u003eTo comprehensively investigate CELF1-mediated transcriptional regulation, SRA01/04 cells were transfected with a \u003cem\u003eCELF1\u003c/em\u003e-overexpressing (OE) plasmid using Lipofectamine 2000 followed by whole transcriptome sequencing (RNA-seq). We constructed six RNA-seq libraries and sequenced for CELF1-overexpression and control SRA01/04 cells, with three replicates for each group. The protein level of CELF1 was increased in CELF1-OE samples compared with control samples, as assessed by western blot in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig S1. Accordingly, the mRNA level of CELF1 was increased approximately 4 times compared with control by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Further, the efficiency of CELF1-overexpression was also confirmed by RNA-seq analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating that overexpression of CELF1 in SRA01/04 cells was successful. FPKM values were applied for calculating the variation contribution of principal components between CELF1-overexpression and control cells, the distribution of principal component analysis (PCA) showed that the biological replicates were clustered together by the second component (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCELF1 overexpression regulates transcription in SRA014/01 cells\u003c/h2\u003e \u003cp\u003eAfter obtaining the raw RNA-seq reads, we discarded the adaptor sequences and low-quality reads, generating a total of 77.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u0026nbsp;million raw reads per sample and 74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u0026nbsp;million clean reads per sample (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), in which an average of 72.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u0026nbsp;million were paired-end reads. Based on the mapping results by TopHat2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], 95.59\u0026ndash;96.72 % of those paired-end reads were mapped to the human GRCH38 genome, and about 85.74\u0026ndash;96.95 % were uniquely aligned (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results supported the validity of sequencing data in our experiments. To identify the gene expression profiles regulated by CELF1, FPKM values were applied to calculate the expression level of candidate genes. RNA-seq yielded 19,703 genes, in which 9,585 genes were detected at an expression level of FPKM\u0026thinsp;\u0026gt;\u0026thinsp;1 in at least one sample.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInformation of RNA-seq reads in experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCELF1_SRA_1st\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCELF1_SRA_2nd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCELF1_SRA_3rd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCtrl_1st\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCtrl_2nd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCtrl_3rd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRaw reads\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79902626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84904120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81594846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70549626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75180916\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e73338412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e77578424.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5446634.377\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClean reads\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77004105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81761685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78505488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66213178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71926994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e70345341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e74292798.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5785186.607\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePaired-end reads\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74701846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79255492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76261510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64028722\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69738738\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68075308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e72010269.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5198713.892\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal mapped(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71677060(95.95%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76091134(96.01%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73420345(96.27%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e61822687(96.55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e67454303(96.72%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65835489(96.71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e69383503\u0026thinsp;\u0026plusmn;\u0026thinsp;4830889.588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUniquely mapped(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63361981(88.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68826896(90.45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62950881(85.74%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57030340(92.25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63156202(93.63%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63830263(96.95%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e63192760.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3420862.379\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSplice reads (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32010772(50.52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35210711(51.16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33379884(53.03%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29918008(52.46%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32659050(51.71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33157280(51.95%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e32722617.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1742879.187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo analyze genes responding to elevated CELF1 in SRA01/04 at the whole transcriptome level, edgeR [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] was used to perform differentially expressed genes (DEGs) analysis on our data. When we set the cut-off as fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 or \u0026le;\u0026thinsp;0.5 and a 5% false discovery rate (FDR), 97 down-regulated and 225 up-regulated genes were identified, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; Table S1). The results demonstrated that CELF1 significantly changed gene expression profile in SRA01/04 cells. Hierarchical clustering heat map analysis of DEGs expression pattern successfully and clearly separated CELF1-OE and control samples, revealing a high consistency among the triplicates data sets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the potential functions of these DEGs, all the identified DEGs were annotated by GO and KEGG functional enrichment analysis. The results revealed that upregulated DEGs were significantly enriched in GO biological process pathways, which were primarily associated with regulation of transcription, collagen catabolic process, extracellular matrix disassembly, proteolysis and extracellular matrix organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The top two GO BP pathways were regulation of transcription related pathways, suggesting that CELF1 could promote expression level of transcription regulators. While downregulated DEGs were only enriched in one GO terms: Small molecule metabolic process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). When we set \u003cem\u003ep\u003c/em\u003e-value as \u0026lt;\u0026thinsp;0.05 for KEGG pathways, we analyzed and presented the top ten KEGG pathways for upregulated DEGs (Fig S2A), including cytokine-cytokine receptor interaction, chemokine signaling pathway and apoptosis. Whereas, down-regulated DEGs were enriched primarily in fat digestion and absorption and pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e infection (Fig S2B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eElevated CELF1 alters expression of genes involved in proteolysis\u003c/h2\u003e \u003cp\u003eTo further confirm the reliability of the RNA-seq results, we downloaded a set of RNA-seq data from early embryonic lens of mouse [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], in which the total number and expression level of contact or lens defects-related genes progressively increased as the lens developed from lens pit stage (embryonic day (E) 10.5) through secondary fiber differentiation stage (E16.5), implying that the embryonic lens of mouse in older stages might exhibit higher risks of cataract than that of younger stages. More important, a significant elevated Celf1 was observed when early embryonic lens developed from E10.5 to E14.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that elevated Celf1 might associated with lens formation and development in mouse. Based on the elevated expression level of Celf1, the transcriptome profile of embryonic lens both in E10.5 and E14.5 could be a substitute of Celf1 overexpression dataset and were selected for further analysis to make a comparison with our data. A total of 322 and 8186 protein coding genes were identified as DEGs in our dataset and downloaded dataset, respectively. However, among those genes, only 26 up-regulated DEGs were overlapped between two datasets. Accordingly, 7 down-regulated DEGs were overlapped (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Species differences may result in the low overlap rate of DEGs between human and mouse. In our present study, SRA01/04 cells which are monoplast originated from human were subjected to RNA-seq, whereas embryonic lens of mouse were preferred in downloaded dataset.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGO molecular function enrichment analysis revealed that up-regulated genes in mouse embryonic lens were enriched in cell adhesion, lens development in camera-type eye, eye development and lens fiber cell development, indicating elevated CELF1 extensively regulated expression of genes implicated in lens development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, up-regulated genes were also enriched in proteolysis, which was also present in top 10 representative GO biological processes of up-regulated genes in SRA01/04 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Thus, a heat map was generated to compare expression profile of genes enriched in proteolysis. Hierarchical clustering of normalized FPKM values of genes involved in proteolysis pathway showed separation between CELF1 overexpression SRA01/04 cells and control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), while a clear separation of proteolysis-related genes was observed between E10.5 and E14.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results indicated that elevated CELF1 significantly changed the expression level of genes associated with proteolysis both in SRA01/04 cells and mouse lens at embryonic stages, implying proteolysis may plays an essential role in lens development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFurther validation of CELF1-regulated gene transcription in proteolysis\u003c/h2\u003e \u003cp\u003eIn order to verify the DEGs results in RNA-seq, we performed RT-qPCR analysis to determine the gene expression levels. Five up-regulated DEGs from proteolysis were selected for RT-qPCR analysis, including MMP1, MMP7, MMP9, MMP13, and CTSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The primers of these genes were shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A statistically significant increase of those genes was observed in RT-qPCR experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which was in line with the FPKM values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These results demonstrated the high correlation between RNA-seq and RT-qPCR results, confirming the discoveries in RNA-seq dataset. Furthermore, we also found that increased CELF1 expression level was coupled with elevated expression of MMP9 in embryonic lens of mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), when embryonic lens developed from E10.5 and E14.5. We also analyzed their expression level changes using the Celf1 knockout (Celf1-KO) microarray data GSE101393 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and found all of them showed no significant change between Celf1-KO and WT samples. Collectively, the results suggested that CELF1 may participate in lens development or cataract formation by regulating expression level of genes in proteolysis, particularly, matrix metalloproteinases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers for detected genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence 5\u0026rsquo; to 3\u0026rsquo;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP1-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAGGGACAGAATGTGCTA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP1-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTTCCAGTGTTTTCCTCA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP7-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAGCCAAACTCAAGGAGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP7-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGTCCATTTTGGGCTATT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP9-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTTCTACGGCCACTACT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP9-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCCTTGAACAAATACAGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP13-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTTTGGGCTCTTAATGGT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP13-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGTCTTGCCTGTATCCTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTSS-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCAAGGCAGGCATATCAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTSS-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGGGTTCAAGGAATCTCG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFunctional analysis of genes bound by CELF1\u003c/h2\u003e \u003cp\u003eWe downloaded a set of CELF1 RIP-seq data [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and a set of CELF1 CLIP-seq data for HeLa cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] to evaluate whether CELF1 directly bind to those genes and subsequently regulate their expression levels. After performing overlapping analysis, we detected 38 DEGs in our study overlapped with CELF1-targeted genes in RIP-seq experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and only 18 DEGs overlapped in CLIP-seq experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). GO functional enrichment analysis was performed on overlapped DEGs between RIP-seq data and our data, 5 up-regulated DEGs were enriched in transcription, DNA-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), which is consistent with the GO BP enrichment analysis of DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). KEGG pathways (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were primarily enriched in Systemic lupus erythematosus, Phagosome, DNA replication, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). By analyzing the genes validated by RT-qPCR in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, we did not find obvious CELF1 peaks that were enriched on the transcripts of MMP1, MMP9, MMP13, or CTSS. These results indicate that CELF1 might regulate expression of most DEGs by an indirect manner, while it could directly bind to TF related genes and regulate their expression levels in SRA01/04 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCataract has been characterized as severe eye disease with opacified lens, and loss of epithelial polarity and cell multi-layering are primary signs of pathogenesis in cataractogenesis. Siddam teams applied a bioinformatics tool iSyTE to identify a novel RNA binding protein, CELF1, which is important for lens development and cataract formation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Their results showed that \u003cem\u003eCelf1\u003c/em\u003e-knockout in mice, or knockdown in zebrafish or Xenopus morphants resulted in severe eye defects or cataract. Except for TDRD7 and Caprin2 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], CELF1 was identified as another RBP that plays a crucial role during cataract formation. However, CELF1-overexpression meditated transcriptional or post-transcriptional regulations in lens development or cataract formation remains to be resolved.\u003c/p\u003e \u003cp\u003eTo have a deeper understanding of CELF1 functions on lens development and cataract, we performed RNA-seq experiments in CELF1-overexpressed SRA01/04 cells and control cells. Our results showed that elevated CELF1 globally changed expression profile in SRA01/04 cells. As an RNA binding protein, overexpression of CELF1 has been associated with many diseases, for example, CELF1-OE was correlated with lower levels of endogenous p27, at the same time, repressing p27 IRES activity in human breast cancer cell line MCF7 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, elevated CELF1 expression mediated defects of myocytes with CUG-expansion, by increasing myocyte cycling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In breast epithelial cells, CELF1-OE promoted the translation of epithelial to mesenchymal transition (EMT) and ultimately tumor progression [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. All together, these findings indicated that CELF1-OE plays an essential role in pathological processes. In agreement with previous studies, we provided evidence that elevated CELF1 may also play a role in cataract regulation and lens development.\u003c/p\u003e \u003cp\u003eBased on GO analysis results, the upregulated DEGs by CELF1-OE were highly enriched in transcriptional regulation, extracellular matrix disassembly and organization, and proteolysis, which were presented in the top 10 pathways. As we know, protein turnover in extracellular matrix is common and important as a fundamental feature of many normal and pathological processes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Undoubtedly, alteration in extracellular matrix turnover is also associated with cataract [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Posterior capsule opacification (PCO), which is also known as secondary cataract, was characterized by cellular migration onto the posterior lens capsule, coupling with deposition of abnormal extracellular matrix and capsular wrinkling, all of which could lead to opacification of lens and ultimately cataract [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, proteolysis is another typical characteristic in cataract formation [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Wang et al proposed that elevated proteolysis resulted from S129R mutation might induce pathology since significant decline of functional proteins changed the protein-protein interaction network [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Both in SRA01/04 cells of human and embryonic lens of mouse, DEGs were enriched in proteolysis pathway accompanying with elevated CELF1 indicating proteolysis might be a crucial pathway regulated by CELF1 in lens development.\u003c/p\u003e \u003cp\u003eIn the present study, all five genes enriched in proteolysis were up-regulated in CELF1-OE cells, and four of them were also belong to matrix metalloproteinase family, including MMP1, MMP7, MMP9, and MMP13. The result suggests the potential importance of MMPs in lens development and cataract regulation. In virtually, MMPs are widely distributed in every tissue of the eye under conditions of health and disease, they present a family of proteolytic enzymes that are involved in the breakdown of extracellular matrix in normal physiological process, ultimately influencing cell biological activities and morphogenesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. MMP2 and MMP9 were most widely investigated for their important role in cataract. The level of MMP2 and MMP9 activities of patients with steroid induced posterior subcapsular cataract (PSC) in lens epithelial cells (LECs) and the serum was evaluated, MMP2 and MMP9 activities in both LECs and serum were significantly higher in cases with steroid induced PSC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, MMP9 plays a more important role in mediating TGF-β-induced anterior subcapsular cataract formation than MMP2 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Besides, in human lens epithelial cells, LDL receptor related protein 5 like (LRP5L) may promote angiogenesis by increasing active MMP9. While its mutant, LRP5L-P36R, may inhibit angiogenesis by decreasing active MMP9 and laminin γ1, indicating that LRP5L-P36R may promote the formation of cataract via attenuating biological function of MMP9 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These results suggest the indefinite roles of MMP9 in cataract formation. In this study, we propose that CELF1-OE might regulate lens development and cataract formation via up-regulating the expression of MMPs, especially MMP9. To further explore the mechanism that CELF1 regulated expression of MMPs, analysis of target genes to which CELF1 was bound.\u003c/p\u003e \u003cp\u003ePrevious study provided evidence that CELF1 associated to the 5\u0026rsquo;-UTR of human p27 mRNA and suppressed expression of p27 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; CELF1 was also inclined to binding with 3\u0026rsquo;UTR and intron regions of mRNA and further globally changed alternative splicing and translation of multiple genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Recently, an investigation revealed that Tristeraprolin (TTP) peaks were enriched in CELF1 binding motifs indicating that TTP might cooperate with other RBPs to participate in multiple post-transcriptional process [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. While, in the current study, we did not find any obvious CELF1 peaks enriched on proteolysis-related genes. Genome-wide chromatin immunoprecipitation method revealed pervasive chromatin-RBP interactions, indicating the transcriptional regulatory ability of RBPs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Based on these existing results, we speculate that CELF1 may regulate expression of proteolysis-related genes at transcriptional level or with an indirectly manner, such as protein-protein interactions. Furthermore, GO analysis showed that CELF1 targeted genes were primarily involved in DNA-dependent transcription regulation, including CDH9, SP4, ZNF420 and ZNF587 that are classical transcriptional factors. Through binding with DNA, these genes could regulate gene transcription [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Direct interacting signals between CELF1 and these transcripts were also found from RIP/CLIP sequencing data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These CELF1-targeted genes implying that CELF1 might involve in lens development or cataract pathogenesis by changing expression levels of transcription factors. Besides, CELF1 may also regulates expression of MMPs by producing various transcripts, which need further investigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA genome-wide profile of CELF1-OE functions in regulating RNA-levels of genes in lens epithelial cells was offered in this study. The expression levels of hundreds of genes regulated by CELF1 were identified, and most of them were linked to multiple biological processes related to lens development. The main definite discovery is that CELF1 may indirectly regulate expression level of MMPs at transcriptional level, suggesting its potential important roles in lens development and cataract formation. Our study contributes to a precise understanding of potential CELF1-targeted therapies in cataract diagnosis and treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCathepsin S; CELF1:CUG-BP, Elav-like family member 1; DEGs:differentially expressed genes; FPKM:fragments per kilobase of exon per million fragments mapped; GO:Gene Ontology; GREs:GU-rich elements; KEGG:Kyoto Encyclopedia of Genes and Genomes; MMPs:matrix metalloproteinase; PCO:Posterior capsule opacification; RBPs:RNA-binding proteins; UTRs:untranslated regions;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data has been deposited in NCBI Gene Expression Omnibus (GEO) under accession code GSE153022.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Jilin Province (20200201347JC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJX\u003c/strong\u003e and \u003cstrong\u003eHZ\u0026nbsp;\u003c/strong\u003edesign the experiment.\u0026nbsp;\u003cstrong\u003eXT,\u003c/strong\u003e \u003cstrong\u003eSYJ\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;MJS\u0026nbsp;\u003c/strong\u003econtributed to experiments conduction and/or data analysis. \u003cstrong\u003eJX, ZGC\u003c/strong\u003e and \u003cstrong\u003eHZ\u003c/strong\u003e prepared the manuscript. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr. Yi Zhang\u0026rsquo;s team members for their help in discussion and critical reading of the manuscript. We thank International Science Editing for editing this manuscript (\u003ca href=\"http://www.internationalscienceediting.com\"\u003ehttp://www.internationalscienceediting.com\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThompson J, Lakhani N: \u003cb\u003eCataracts\u003c/b\u003e. \u003cem\u003ePrim Care\u003c/em\u003e 2015, \u003cb\u003e42\u003c/b\u003e(3):409\u0026ndash;423.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhokhar S, Pillay G, Agarwal E: \u003cb\u003ePediatric Cataract - Importance of Early Detection and Management\u003c/b\u003e. \u003cem\u003eIndian journal of pediatrics\u003c/em\u003e 2018, \u003cb\u003e85\u003c/b\u003e(3):209\u0026ndash;216.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBobrow, Breadsley, Jick: \u003cb\u003eLens and cataract\u003c/b\u003e. \u003cem\u003ePrimary 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\u003cb\u003e14\u003c/b\u003e(2):e0211349.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CELF1, transcriptional regulation, RNA-seq, MMPs, cataract","lastPublishedDoi":"10.21203/rs.3.rs-785595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-785595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: RNA binding proteins (RBPs)-mediated regulation plays important roles in many eye diseases, including the canonical RBP CELF1 in cataract. While the definite molecular regulatory mechanisms of CELF1 on cataract still remain elusive. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study, we overexpressed CELF1 in lens epithelial SRA01/04 cells and applied whole transcriptome sequencing (RNA-seq) method to analyze the global differences mediated by CELF1. We then analyzed public RNA-seq and CELF1-RNA interactome data to decipher the underlying mechanisms.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The results showed that transcriptome profile was globally changed by CELF1 overexpression (CELF1-OE). Functional analysis revealed CELF1 specifically increased the expression of genes in extracellular matrix disassembly, extracellular matrix organization, and proteolysis, which could be classified into matrix metalloproteinases (MMPs) family. This finding was also validated by RT-qPCR and public mouse early embryonic lens data. Integrating analysis with public CELF1-RNA interactome data revealed that no obvious CELF1-binding peak was found on the transcripts of these genes, indicating an indirectly regulatory role of CELF1 in lens epithelial cells. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our study demonstrated that CELF1-OE promotes transcriptional level of MMP genes; and this regulation may be completed by other ways except for binding to RNA targets. These results suggest that CELF1-OE is implicated in the development of lens, which is associated with cataract and expands our understanding of CELF1 regulatory roles as an RNA binding protein.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"CELF1 Promotes Matrix Metalloproteinases Gene Expression at Transcriptional Level in Lens Epithelial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-12 13:45:23","doi":"10.21203/rs.3.rs-785595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-11-29T07:14:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-11-25T17:50:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23eea1e1-63e9-4d59-8ced-346a9b6951f5","date":"2021-11-19T06:42:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-10-26T17:58:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3d23cf27-52a5-488f-a80a-6d36a6e236e1","date":"2021-10-26T15:45:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-10-25T14:25:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-10-09T15:31:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-08-11T07:31:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-08-11T07:30:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ophthalmology","date":"2021-08-06T01:13:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac9a606c-4f85-4c95-abd6-9d777523e072","owner":[],"postedDate":"August 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6402724,"name":"Ophthalmology"}],"tags":[],"updatedAt":"2022-03-28T20:04:29+00:00","versionOfRecord":{"articleIdentity":"rs-785595","link":"https://doi.org/10.1186/s12886-022-02344-8","journal":{"identity":"bmc-ophthalmology","isVorOnly":false,"title":"BMC Ophthalmology"},"publishedOn":"2022-03-14 20:04:29","publishedOnDateReadable":"March 14th, 2022"},"versionCreatedAt":"2021-08-12 13:45:23","video":"","vorDoi":"10.1186/s12886-022-02344-8","vorDoiUrl":"https://doi.org/10.1186/s12886-022-02344-8","workflowStages":[]},"version":"v1","identity":"rs-785595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-785595","identity":"rs-785595","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}