miR-19a promotes proliferation and migration of lens epithelial cells by regulating AKT/pAKT signaling

miR-19a promotes proliferation and migration of lens epithelial cells by regulating AKT/pAKT signaling | 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 miR-19a promotes proliferation and migration of lens epithelial cells by regulating AKT/pAKT signaling Yuanbin Li, Yanfang Wang, Yadi Pu, Shujun Liu, Fenglan Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.13691/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Posterior capsule opacification (PCO) occurs frequently following surgical treatment of cataracts, representing a common adverse outcome. PCO develops as a result of lens epithelial cell (LEC) transdifferentiation, migration, and proliferation. In the present report we sought to explore the role of microRNA (miR)-19a in this process and to establish the underlying molecular mechanisms. Methods: miR-19a was transduced into HLE-B3 LECs, with microscopy and RT-qPCR used to confirm transfection efficiency. We then employed MTT, wound healing, and transwell assay approaches to monitor the proliferation and migration of these LECs. We further assessed levels of the proteins PTEN, AKT, and phosphorylated AKT (pAKT) via western blotting in WT and miRNA-transfected cells. Results: HLE‑B3 proliferation was markedly enhanced by miR-19a transduction, as well the migration activity of these cells (both P<0.01). Furthermore, overexpressing miR‑19a failed to reduce PTEN expression whereas it did enhance pAKT levels within these LECs (P<0.05). Conclusions: This suggests that miR-19a can enhance LEC proliferation and migratory activity through a mechanism that may be linked with regulating AKT activation and signaling, thus highlighting a potential avenue for therapeutic treatment of PCO patients. Ophthalmology Epigenetics & Genomics miR-19a Lens epithelial cells Proliferation Migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Posterior capsule opacification (PCO) is a significant adverse event that occurs following surgical treatment of cataracts, impairing the vision of as many as 28% of individuals within 5 years of intraocular lens (IOL) implantation [1]. PCO develops as a result of tissue fibrosis that occurs as a result of some combination of inflammation induced by the surgical operation and an immunological response to the IOL. During PCO development and progression, lens epithelial cells (LECs), which are normally present in a single layer on the inner side of the anterior lens capsule, transdifferentiate, proliferate, and migrate in a pathogenic manner [2]. MicroRNAs (miRNAs) are short RNAs that lack coding potential but which play broad roles in the post-transcriptional regulation of a wide array of target genes, influencing processes including but not limited to proliferation, apoptotic cell death, and differentiation [3]. The miR–17–92 cluster encodes for 6 distinct oncogenic miRNAs with significant roles in regulating genes linked to cellular proliferation (miR–17, miR–18a, miR–19a, miR–19b, miR–20a, and miR–92) [4]. Of these, miR–19a overexpression has been detected in the context of colorectal cancer [5], and this miRNA is known to be induced in colorectal cancer cells by PRL3 whereupon it can target TG2 and thereby drive their metastasis and proliferation [6, 7]. miRNAs have been shown to play tissue-specific roles within the eye [8], with unique roles for multiple miRNAs including miR–19 in this setting [9, 10]. However, at present the specific understanding of the role of individual miRNAs in the eye is limited, and further investigation is thus warranted. In this study, we assessed the role of miR–19 in regulating the ability of LECs to proliferate and migrate and we explored the underlying molecular mechanisms in an effort to highlight new potential targets for treating PCO. Methods Cell Culture The human HLE-B3 LC line was from Shanghai Genechem (Shanghai, China). Cells were grown in DMEM (Gibco, MA, USA) containing 15% FBS (Zhejiang Tianhang Biotechnology, Hangzhou, China), along with penicillin and streptomycin in a 37°C 5% CO 2 incubator. Cell Transduction HLE-B3 cells were transduced with control or miR–19-encoding lentiviral particles (Shanghai Genechem) in a 96-well plate, with 5x10 4 cells/mL in 100 uL per well. Lentiviral particles were added to cells at a range of multiplicities of infection (MOIs), and then after 72 h the GFP expression in transduces cells was assessed via fluorescent microscope (Leica DMI6000B). RT-qPCR The RNAiso for Small RNA kit (TaKaRa, Dalian, China) was used to extract total RNA from WT or lentivirally-transduced cells, with a BioSpectrometer (Eppendorf, Germany) used to gauge RNA quality. A total of 0.25–8 μg RNA in a 3.75 μl volume was then used with the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa) for reverse transcription based on provided directions, and the Step One Plus Real-time PCR System (Applied Biosystems) was used to measure relative miR–19a expression. TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa) was used for all reactions, which consisted of a total of 25 µl made up of 12.5 µl TB Green Advanced premix, 0.5 µl ROX, 0.5 µl of the appropriate miR–19a primer, 0.5 µl of the mRQ 3′ primer, 2 µl cDNA, and 9 µl dd H 2 O. Thermocycler settings were: 95°C for 30 sec; 40 cycles of 95°C for 5 sec, 60°C for 30 sec. PCR products were confirmed via melting curve analyses. Relative miR–19a levels were normalized to levels of the U6 RNA, with the 2 - △△ Ct method used for quantification. miR–19a primer sequences were as follows: Forward - 5′-GGAACGATACAGAGAAGATTAGC–3′; Reverse - 5′-TGGAACGCTTCACGAATTTGCG –3′ (TaKaRa). Cell proliferation assay The MTT assay approach was used to quantify the proliferation of HLE-B3 cells, which were plated into 96-well plates (2×10 4 cells/ml, 100uL/well). After 24 h post-plating, cells were lentivirally transfected. After an additional 24, 48, 72, or 96 h, 20 μl MTT (5 mg/ml; Biofroxx, Germany) was added per well, and cells were incubated at 37°C for 4 h. Media was then exchanged for 150 µl DMSO (Beijing Solarbio, Beijing, China). A microplate reader (SepectraMax M3, Molecular Devices, Shanghai, China) was then used to assess absorbance values at 490 nm. Wound healing assay At 72 h post-lentiviral transduction, HLE-B3 cells were replated into 12-well plates at 3.5×10 5 cells/well. After an additional 24 h, a wound was generated in the monolayer within each well using a sterile 20µl pipette tip, and PBS was used to wash away non-adherent cells. After washing, fresh media was added and cells were imaged for 24 h, with the size of the wound being imaged at 0 and 24 h via an inverted microscope (Leica DMI6000B), with Image J used to quantify wound size changes over time. Cell Migration Assay At 72 h post-lentiviral transduction, HLE-B3 cells (5x10 4 ) were resuspended into 200 µl of serum-free DMEM, and were then added to the upper portion of a Transwell insert (Corning, High Wycombe, UK). This insert was then inserted into a well, with media supplemented with 20% FBS added to the lower chamber. Cells were incubated for 24 h, after which 4% paraformaldehyde was used to fix membranes prior to crystal violet (Beyotime) staining and counting of cells via Axio vert A 1 inverted fluorescence microscope. Western blotting After a 30 minute lysis step on ice, HLE-B3 cell lysates were spun at 12,000 rpm for 10 minutes at 4°C, with supernatants being collected and protein levels therein being quantified via a BCA assay. After boiling for 5 min in sample buffer, proteins from each sample were separated via SDS-PAGE, transferred to PVDF membranes, blocked for 4 h using 5% non-fat dried milk at room temperature, and probed overnight with primary antibodies specific for AKT, pAKT, and PTEN (1:1000, Wanlei Biotechnology, China) at 4°C. Secondary HRP-conjugated antibodies were then used for antigen detection for 1 h at room temperature. Blots were then washed using TBST, and an ECL detection kit (Beyotime) was used to quantify protein levels. Actin served as a loading control Statistical analysis Data are means ± SD. Values were compared via one-way ANOVAs with Bonferroni post-hoc testing for multiplecomparisons using SPSS. P < 0.05 was the significance threshold. Results Overexpression of miR–19a in HLE-B3 cells We observed significantly enhanced GFP expression in HLE-B3 cells lentivirally transduced with viral particles encoding miR–19a, consistent with elevated miR–19a expression relative to control cells (Figure 1A), and this was confirmed via RT-qPCR (Figure 1B). We found an optimal MOI of infection to be 60, and as such this MOI was used for subsequent experiments. Figure 1.miR–19a in lentivirally transduced HLEB3 cells. (A) GFP was used to assess miR–19a expression in HLE-B3 cells following lentiviral transduction. (B) miR–19a expression was assessed by RT-qPCR following lentiviral transduction. * P <0.05 vs. NC. miR, microRNA; NC, negative control miR–19a regulates HLEB3 cell proliferation We next used an MTT assay approach to assess how miR–19a impacted HLE-B3 cell proliferation. We observed a significant increase in the proliferation of these cells upon mIR–19a overexpression, with a significant increase in proliferation at 96 h post-transduction relative to NC cells (Figure 2). Figure 2. miR–19a impacts the proliferation of HLEB3 cells. * P <0.05 vs. NC. miR, microRNA; NC, negative control miR–19a regulates HLEB3 cell migration We further explored the impact of miR–19a overexpression on HLEB3 cell migration using wound healing and Transwell assay approaches. We observed significantly enhanced rates of migration following miR–19a overexpression relative to NC cells in the wound healing assay (Figure 3A, 3B), and consistent with this the Transwell assay similarly confirmed enhanced migration of cells overexpressing miR–19a (Figure 3C, 3D). Figure 3. miR–19a regulates HLEB3 cell migration. (A) Images and (B) quantification of relative cell migration in a wound healing assay. (C) Images and (D) quantification of cell migration and invasion in a Transwell assay. ** P <0.01. miR, microRNA; NC, negative control. miR–19a influences AKT signaling in HLE-B3 cells We next measured the levels of proteins in HLE-B3 cells following lentiviral transduction via western blotting, revealing that miR–19a overexpression did not significantly impact PTEN levels, whereas it did significantly increase pAKT levels relative to NC cells ( P <0.05; Figure 4). Figure 4. miR–19a influences AKT signaling in HLE-B3 cells. (A) AKT, pAKT, and PTEN levels in miR–19a-transfected HLE-B3 and NC cells were assessed via western blotting; (B) AKT, pAKT, and PTEN levels were quantified. * P <0.05. miR, microRNA; NC, negative control. Discussion PCO frequently arises following surgical treatment of cataracts through a fibrotic process wherein LECs undergo proliferation, migration, and transdifferentiation within the lens capsule. While surgeons generally attempt to remove as many of these cells as possible during surgery, it is inevitable that some remain within the capsular bag. At 3–4 days post-surgery, these cells reach their maximal rate of proliferation in a manner that is age-dependent, such that younger individuals are at a higher risk of PCO. LECs are also able to contribute to PCO via migrating over the posterior lens capsule and towards the posterior visual axis through the use of a range of adhesion molecules, including integrins and intracellular adhesion molecule 1 (ICAM1), which allow them to adhere to other cells and to the extracellular matrix [11]. The specific mechanisms that initiate this LEC proliferation and migration, however, remain uncertain. miR–19a is a member of the miR–17–92 family, which consists of a polycistronic locus encoding miRNAs that play key roles in regulating the survival, proliferation, and differentiation of many different cell types [12]. Multiple recent studies have detected both physiological and pathological roles for miRNAs within the eye [13]. In this report, we provided direct evidence of the role of miR–19a in LECs, suggesting it may contribute to their proliferation and migration after surgical treatment of cataracts. LEC expression of miR–19a at baseline is very low, and as such to examine its function in this study we transduced HLE-B3 LECs with a lentivirus encoding this miRNA. We found that miR–19a overexpression significantly enhanced the migration and proliferation of these LECs, although the specific underlying mechanism still requires further clarification. AKT proteins are kinases that play essential roles in central signaling events necessary to mediate the growth and differentiation of cells, such that the PI3K/AKT signaling pathway is central to cellular survival, proliferation, and migration [14]. Cancer cells with enhanced proliferative and migratory activity typically also exhibit increased AKT activation, such that suppressing AKT activity is a promising approach to suppressing the aberrant proliferation and invasion of tumor cells in a variety of cancers [15]. PTEN serves to negatively regulate PI3K/AKT signaling in humans [16], and it is known to be regulated by specific miRNAs. For example, miR–17–5p was recently found to be expressed at elevated levels in ovarian cancer cells, and to therein target and suppress PTEN signaling, thereby promoting the epithelial-mesenchymal transition process within these tumor cells [17]. Similarly, in lung cancer cells it has been shown that miR–10a overexpression can promote both proliferation and metastasis through activation of PTEN/AKT/ERK signaling in vitro [ 18]. In this report, we determined that miR–19a overexpression is linked to enhanced LEC proliferation and migration through a mechanism that correlates with enhanced AKT phosphorylation but without any corresponding change in PTEN levels, suggesting miR–19a does not target PTEN directly. Conclusions Together these findings clearly demonstrate that overexpressing miR–19a can drive enhanced proliferation and migration of LECs through a mechanism potentially linked with regulation of AKT signaling. This thus suggests that targeting miR–19a may be a viable therapeutic strategy for treating PCO. Abbreviations PCO: Posterior capsule opacification; miR: microRNA; LECs: lens epithelial cells; PTEN: phosphatase and tensin homolog. Declarations Funding This study was supported by grants from the Yantai Technology Development Plan (No. 2017ws109) and Shandong Provincial Natural Science Foundation (No. ZR2017MH068, ZR2017LH043). The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on request. Authors’ contributions YBL, SJL and FLZ conceived and designed the study, YFW and FLZ drafted the manuscript. YFW, YBL, FLZ, SJL and YDP performed the experiments. YFW and FLZ analyzed the data. All authors read and approved the final manuscript. Acknowledgements Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors report no conflicts of interest. References Lu C, Yu S, Song H, Zhao Y, Xie S, et al. Posterior capsular opacification comparison between morphology and objective visual function. BMC Ophthalmol. 2019; 19: 40. Nibourg LM, Gelens E, Kuijer R, Hooymans JM, van Kooten TG, et al. Prevention of posterior capsular opacification. Exp Eye Res. 2015; 136: 100–15. Ambros V. The functions of animal microRNAs. Nature. 2004; 431: 350–5. Khan AA, Penny LA, Yuzefpolskiy Y, Sarkar S, Kalia V. MicroRNA–17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections. Blood. 2013; 121: 4473–83. Liu Y, Liu R, Yang F, Cheng R, Chen X, et al. miR–19a promotes colorectal cancer proliferation and migration by targeting TIA1. Mol Cancer. 2017; 16: 53. Zhang J, Xiao Z, Lai D, Sun J, He C, et al. miR–21, miR–17 and miR–19a induced by phosphatase of regenerating liver–3 promote the proliferation and metastasis of colon cancer. Br J Cancer. 2012; 107: 352–9. Cellura D, Pickard K, Quaratino S, Parker H, Strefford JC, et al. miR–19-Mediated Inhibition of Transglutaminase–2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer. Mol Cancer Res. 2015; 13: 1095–105. Karali M, Peluso I, Marigo V, Banfi S. Identification and characterization of microRNAs expressed in the mouse eye. Invest Ophthalmol Vis Sci. 2007; 48: 509–15. Hughes AE, Bradley DT, Campbell M, Lechner J, Dash DP, et al. Mutation altering the miR–184 seed region causes familial keratoconus with cataract. Am J Hum Genet. 2011; 89: 628–33. Lee SK, Teng Y, Wong HK, Ng TK, Huang L, et al. MicroRNA–145 regulates human corneal epithelial differentiation. PLoS One. 2011; 6: e21249. Nibourg LM, Gelens E, Kuijer R, Hooymans JM, van Kooten TG, et al. Prevention of posterior capsular opacification. Exp Eye Res. 2015; 136: 100–15. Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, et al. A polycistronic microRNA cluster, miR–17–92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res. 2005; 65: 9628–32. Raghunath A, Perumal E. Micro-RNAs and their roles in eye disorders. Ophthalmic Res. 2015; 53: 169–86. Fumarola C, Bonelli MA, Petronini PG, Alfieri RR. Targeting PI3K/AKT/mTOR pathway in non small cell lung cancer. Biochem Pharmacol. 2014; 90: 197–207. Revathidevi S, Munirajan AK. Akt in cancer: Mediator and more. Semin Cancer Biol. 2019; pii: S1044–579X: 30062–2. Zhang X, Chen Y, Zhao P, Zang L, Zhang Z, et al. MicroRNA–19a functions as an oncogene by regulating PTEN/AKT/pAKT pathway in myeloma. Leuk Lymphoma. 2017; 58: 932–40. Fang Y, Xu C, Fu Y. MicroRNA–17–5p induces drug resistance and invasion of ovarian carcinoma cells by targeting PTEN signaling. J Biol Res (Thessalon). 2015; 22: 12. Yu T, Liu L, Li J, Yan M, Lin H, et al. MiRNA–10a is upregulated in NSCLC and may promote cancer by targeting PTEN. Oncotarget. 2015; 6: 30239–50. Cite Share Download PDF Status: Posted Version 1 posted 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-4397","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":142016,"identity":"5b4b8ea8-8a86-4b64-b298-b0365a05b2a7","order_by":1,"name":"Yuanbin Li","email":"","orcid":"","institution":"Qindao University Medical College Affiliated Yantai Yuhuangding Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanbin","middleName":"","lastName":"Li","suffix":""},{"id":142017,"identity":"8cec9cb6-f56a-4105-9607-db13944af0b9","order_by":2,"name":"Yanfang Wang","email":"","orcid":"","institution":"Yantai University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanfang","middleName":"","lastName":"Wang","suffix":""},{"id":142018,"identity":"ae41fed6-d4b0-463b-a617-1ed116d8c0d9","order_by":3,"name":"Yadi Pu","email":"","orcid":"","institution":"Qindao University Medical College Affiliated Yantai Yuhuangding Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yadi","middleName":"","lastName":"Pu","suffix":""},{"id":142019,"identity":"93b660e4-091f-47f6-8eae-f039a78b1c1b","order_by":4,"name":"Shujun Liu","email":"","orcid":"","institution":"Qindao University Medical College Affiliated Yantai Yuhuangding Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shujun","middleName":"","lastName":"Liu","suffix":""},{"id":142020,"identity":"44dfff3d-cb9b-4bed-9aa2-cf259f570c93","order_by":5,"name":"Fenglan Zhang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-4426-0907","institution":"Qindao University Medical College Affiliated Yantai Yuhuangding Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Fenglan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2019-08-25 14:01:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.13691/v1","doiUrl":"https://doi.org/10.21203/rs.2.13691/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":613212,"identity":"f8f36f37-2447-4209-a836-5659d6a55fcd","added_by":"auto","created_at":"2020-03-09 14:26:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97590,"visible":true,"origin":"","legend":"Figure 1.miR-19a in lentivirally transduced HLE B3 cells. (A) GFP was used to assess miR-19a expression in HLE-B3 cells following lentiviral transduction. (B) miR-19a expression was assessed by RT-qPCR following lentiviral transduction. *P\u003c0.05 vs. NC. miR, microRNA; NC, negative control","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397/v1/Fig 1.jpg"},{"id":613213,"identity":"a2321fb8-1cf4-464e-be0f-e9fc3d74378c","added_by":"auto","created_at":"2020-03-09 14:26:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34622,"visible":true,"origin":"","legend":"Figure 2. miR-19a impacts the proliferation of HLE B3 cells. * P\u003c0.05 vs. NC.\nmiR, microRNA; NC, negative control","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397/v1/Fig 2.jpg"},{"id":613214,"identity":"50ffb5f9-db78-4965-ae02-bb2df73796fe","added_by":"auto","created_at":"2020-03-09 14:26:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126127,"visible":true,"origin":"","legend":"Figure 3. miR-19a regulates HLE B3 cell migration. (A) Images and (B) quantification of relative cell migration in a wound healing assay. (C) Images and (D) quantification of cell migration and invasion in a Transwell assay. **P\u003c0.01. miR, microRNA; NC, negative control.","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397/v1/Fig 3.jpg"},{"id":613215,"identity":"cd5b34b6-4daf-42e7-9b8e-1290b4c8c5fe","added_by":"auto","created_at":"2020-03-09 14:26:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106396,"visible":true,"origin":"","legend":"Figure 4. miR-19a influences AKT signaling in HLE-B3 cells. (A) AKT, pAKT, and PTEN levels in miR-19a-transfected HLE-B3 and NC cells were assessed via western blotting; (B) AKT, pAKT, and PTEN levels were quantified. *P\u003c0.05. miR, microRNA; NC, negative control.","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397/v1/Fig 4.jpg"},{"id":13472741,"identity":"e8726a74-95ff-4aef-836f-67e72168985e","added_by":"auto","created_at":"2021-09-16 21:16:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":498249,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4397/v1/94dfed9b-30d4-470c-b856-3f97968efb4c.pdf"}],"financialInterests":"","formattedTitle":"miR-19a promotes proliferation and migration of lens epithelial cells by regulating AKT/pAKT signaling","fulltext":[{"header":"Background","content":"\u003cp\u003ePosterior capsule opacification (PCO) is a significant adverse event that occurs following surgical treatment of cataracts, impairing the vision of as many as 28% of individuals within 5 years of intraocular lens (IOL) implantation [1].\u003c/p\u003e\n\u003cp\u003ePCO develops as a result of tissue fibrosis that occurs as a result of some combination of inflammation induced by the surgical operation and an immunological response to the IOL. During PCO development and progression, lens epithelial cells (LECs), which are normally present in a single layer on the inner side of the anterior lens capsule, transdifferentiate, proliferate, and migrate in a pathogenic manner [2].\u003c/p\u003e\n\u003cp\u003eMicroRNAs (miRNAs) are short RNAs that lack coding potential but which play broad roles in the post-transcriptional regulation of a wide array of target genes, influencing processes including but not limited to proliferation, apoptotic cell death, and differentiation [3]. The miR–17–92 cluster encodes for 6 distinct oncogenic miRNAs with significant roles in regulating genes linked to cellular proliferation (miR–17, miR–18a, miR–19a, miR–19b, miR–20a, and miR–92) [4]. Of these, miR–19a overexpression has been detected in the context of colorectal cancer [5], and this miRNA is known to be induced in colorectal cancer cells by PRL3 whereupon it can target TG2 and thereby drive their metastasis and proliferation [6, 7].\u003c/p\u003e\n\u003cp\u003emiRNAs have been shown to play tissue-specific roles within the eye [8], with unique roles for multiple miRNAs including miR–19 in this setting [9, 10]. However, at present the specific understanding of the role of individual miRNAs in the eye is limited, and further investigation is thus warranted. \u003c/p\u003e\n\u003cp\u003eIn this study, we assessed the role of miR–19 in regulating the ability of LECs to proliferate and migrate and we explored the underlying molecular mechanisms in an effort to highlight new potential targets for treating PCO. \u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eThe human HLE-B3 LC line was from Shanghai Genechem (Shanghai, China). Cells were grown in DMEM (Gibco, MA, USA) containing 15% FBS (Zhejiang Tianhang Biotechnology, Hangzhou, China), along with penicillin and streptomycin in a 37°C 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003ch3\u003eCell Transduction\u003c/h3\u003e\n\u003cp\u003eHLE-B3 cells were transduced with control or miR–19-encoding lentiviral particles (Shanghai Genechem) in a 96-well plate, with 5x10\u003csup\u003e4\u003c/sup\u003e cells/mL in 100 uL per well. Lentiviral particles were added to cells at a range of multiplicities of infection (MOIs), and then after 72 h the GFP expression in transduces cells was assessed via fluorescent microscope (Leica DMI6000B).\u003c/p\u003e\n\u003ch3\u003eRT-qPCR\u003c/h3\u003e\n\u003cp\u003eThe RNAiso for Small RNA kit (TaKaRa, Dalian, China) was used to extract total RNA from WT or lentivirally-transduced cells, with a BioSpectrometer (Eppendorf, Germany) used to gauge RNA quality. A total of 0.25–8 μg RNA in a 3.75 μl volume was then used with the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa) for reverse transcription based on provided directions, and the Step One Plus Real-time PCR System (Applied Biosystems) was used to measure relative miR–19a expression. TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa) was used for all reactions, which consisted of a total of 25 µl made up of 12.5 µl TB Green Advanced premix, 0.5 µl ROX, 0.5 µl of the appropriate miR–19a primer, 0.5 µl of the mRQ 3′ primer, 2 µl cDNA, and 9 µl dd H\u003csub\u003e2\u003c/sub\u003eO. Thermocycler settings were: 95°C for 30 sec; 40 cycles of 95°C for 5 sec, 60°C for 30 sec. PCR products were confirmed via melting curve analyses. Relative miR–19a levels were normalized to levels of the U6 RNA, with the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method used for quantification. miR–19a primer sequences were as follows: Forward - 5′-GGAACGATACAGAGAAGATTAGC–3′; Reverse - 5′-TGGAACGCTTCACGAATTTGCG –3′ (TaKaRa). \u003c/p\u003e\n\u003ch3\u003eCell proliferation assay\u003c/h3\u003e\n\u003cp\u003eThe MTT assay approach was used to quantify the proliferation of HLE-B3 cells, which were plated into 96-well plates (2×10\u003csup\u003e4 \u003c/sup\u003ecells/ml, 100uL/well). After 24 h post-plating, cells were lentivirally transfected. After an additional 24, 48, 72, or 96 h, 20 μl MTT (5 mg/ml; Biofroxx, Germany) was added per well, and cells were incubated at 37°C for 4 h. Media was then exchanged for 150 µl DMSO (Beijing Solarbio, Beijing, China). A microplate reader (SepectraMax M3, Molecular Devices, Shanghai, China) was then used to assess absorbance values at 490 nm.\u003c/p\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eAt 72 h post-lentiviral transduction, HLE-B3 cells were replated into 12-well plates at 3.5×10\u003csup\u003e5\u003c/sup\u003e cells/well. After an additional 24 h, a wound was generated in the monolayer within each well using a sterile 20µl pipette tip, and PBS was used to wash away non-adherent cells. After washing, fresh media was added and cells were imaged for 24 h, with the size of the wound being imaged at 0 and 24 h via an inverted microscope (Leica DMI6000B), with Image J used to quantify wound size changes over time. \u003c/p\u003e\n\u003ch3\u003eCell Migration Assay\u003c/h3\u003e\n\u003cp\u003eAt 72 h post-lentiviral transduction, HLE-B3 cells (5x10\u003csup\u003e4\u003c/sup\u003e) were resuspended into 200 µl of serum-free DMEM, and were then added to the upper portion of a Transwell insert (Corning, High Wycombe, UK). This insert was then inserted into a well, with media supplemented with 20% FBS added to the lower chamber. Cells were incubated for 24 h, after which 4% paraformaldehyde was used to fix membranes prior to crystal violet (Beyotime) staining and counting of cells via Axio vert A\u003csub\u003e1\u003c/sub\u003e inverted fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eAfter a 30 minute lysis step on ice, HLE-B3 cell lysates were spun at 12,000 rpm for 10 minutes at 4°C, with supernatants being collected and protein levels therein being quantified via a BCA assay. After boiling for 5 min in sample buffer, proteins from each sample were separated via SDS-PAGE, transferred to PVDF membranes, blocked for 4 h using 5% non-fat dried milk at room temperature, and probed overnight with primary antibodies specific for AKT, pAKT, and PTEN (1:1000, Wanlei Biotechnology, China) at 4°C. Secondary HRP-conjugated antibodies were then used for antigen detection for 1 h at room temperature. Blots were then washed using TBST, and an ECL detection kit (Beyotime) was used to quantify protein levels. Actin served as a loading control\u003c/p\u003e\n\u003ch3\u003eStatistical analysis\u003c/h3\u003e\n\u003cp\u003eData are means ± SD. Values were compared via one-way ANOVAs with Bonferroni\u003cem\u003e post-hoc\u003c/em\u003e testing for multiplecomparisons using SPSS. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was the significance threshold.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003eOverexpression of miR–19a in HLE-B3 cells \u003c/h3\u003e\n\u003cp\u003eWe observed significantly enhanced GFP expression in HLE-B3 cells lentivirally transduced with viral particles encoding miR–19a, consistent with elevated miR–19a expression relative to control cells (Figure 1A), and this was confirmed via RT-qPCR (Figure 1B). We found an optimal MOI of infection to be 60, and as such this MOI was used for subsequent experiments.\u003c/p\u003e\n\n\u003cp\u003eFigure 1.miR–19a in lentivirally transduced HLEB3 cells. (A) GFP was used to assess miR–19a expression in HLE-B3 cells following lentiviral transduction. (B) miR–19a expression was assessed by RT-qPCR following lentiviral transduction. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. NC. miR, microRNA; NC, negative control\u003c/p\u003e\n\u003ch3\u003emiR–19a regulates HLEB3 cell proliferation\u003c/h3\u003e\n\u003cp\u003eWe next used an MTT assay approach to assess how miR–19a impacted HLE-B3 cell proliferation. We observed a significant increase in the proliferation of these cells upon mIR–19a overexpression, with a significant increase in proliferation at 96 h post-transduction relative to NC cells (Figure 2).\u003c/p\u003e\n\n\u003cp\u003eFigure 2. miR–19a impacts the proliferation of HLEB3 cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. NC.\u003c/p\u003e\n\u003cp\u003emiR, microRNA; NC, negative control\u003c/p\u003e\n\u003ch3\u003emiR–19a regulates HLEB3 cell migration\u003c/h3\u003e\n\u003cp\u003eWe further explored the impact of miR–19a overexpression on HLEB3 cell migration using wound healing and Transwell assay approaches. We observed significantly enhanced rates of migration following miR–19a overexpression relative to NC cells in the wound healing assay (Figure 3A, 3B), and consistent with this the Transwell assay similarly confirmed enhanced migration of cells overexpressing miR–19a (Figure 3C, 3D).\u003c/p\u003e\n\n\u003cp\u003eFigure 3. miR–19a regulates HLEB3 cell migration. (A) Images and (B) quantification of relative cell migration in a wound healing assay. (C) Images and (D) quantification of cell migration and invasion in a Transwell assay. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. miR, microRNA; NC, negative control.\u003c/p\u003e\n\u003ch2\u003e \u003c/h2\u003e\n\u003ch3\u003emiR–19a influences AKT signaling in HLE-B3 cells\u003c/h3\u003e\n\u003cp\u003eWe next measured the levels of proteins in HLE-B3 cells following lentiviral transduction via western blotting, revealing that miR–19a overexpression did not significantly impact PTEN levels, whereas it did significantly increase pAKT levels relative to NC cells (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; Figure 4).\u003c/p\u003e\n\n\u003cp\u003eFigure 4. miR–19a influences AKT signaling in HLE-B3 cells. (A) AKT, pAKT, and PTEN levels in miR–19a-transfected HLE-B3 and NC cells were assessed via western blotting; (B) AKT, pAKT, and PTEN levels were quantified. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. miR, microRNA; NC, negative control.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePCO frequently arises following surgical treatment of cataracts through a fibrotic process wherein LECs undergo proliferation, migration, and transdifferentiation within the lens capsule. While surgeons generally attempt to remove as many of these cells as possible during surgery, it is inevitable that some remain within the capsular bag. At 3–4 days post-surgery, these cells reach their maximal rate of proliferation in a manner that is age-dependent, such that younger individuals are at a higher risk of PCO. LECs are also able to contribute to PCO via migrating over the posterior lens capsule and towards the posterior visual axis through the use of a range of adhesion molecules, including integrins and intracellular adhesion molecule 1 (ICAM1), which allow them to adhere to other cells and to the extracellular matrix [11]. The specific mechanisms that initiate this LEC proliferation and migration, however, remain uncertain. \u003c/p\u003e\n\u003cp\u003e miR–19a is a member of the miR–17–92 family, which consists of a polycistronic locus encoding miRNAs that play key roles in regulating the survival, proliferation, and differentiation of many different cell types [12]. Multiple recent studies have detected both physiological and pathological roles for miRNAs within the eye [13]. In this report, we provided direct evidence of the role of miR–19a in LECs, suggesting it may contribute to their proliferation and migration after surgical treatment of cataracts. \u003c/p\u003e\n\u003cp\u003eLEC expression of miR–19a at baseline is very low, and as such to examine its function in this study we transduced HLE-B3 LECs with a lentivirus encoding this miRNA. We found that miR–19a overexpression significantly enhanced the migration and proliferation of these LECs, although the specific underlying mechanism still requires further clarification. \u003c/p\u003e\n\u003cp\u003eAKT proteins are kinases that play essential roles in central signaling events necessary to mediate the growth and differentiation of cells, such that the PI3K/AKT signaling pathway is central to cellular survival, proliferation, and migration [14]. Cancer cells with enhanced proliferative and migratory activity typically also exhibit increased AKT activation, such that suppressing AKT activity is a promising approach to suppressing the aberrant proliferation and invasion of tumor cells in a variety of cancers [15].\u003c/p\u003e\n\u003cp\u003ePTEN serves to negatively regulate PI3K/AKT signaling in humans [16], and it is known to be regulated by specific miRNAs. For example, miR–17–5p was recently found to be expressed at elevated levels in ovarian cancer cells, and to therein target and suppress PTEN signaling, thereby promoting the epithelial-mesenchymal transition process within these tumor cells [17]. Similarly, in lung cancer cells it has been shown that miR–10a overexpression can promote both proliferation and metastasis through activation of PTEN/AKT/ERK signaling \u003cem\u003ein vitro [\u003c/em\u003e18]. In this report, we determined that miR–19a overexpression is linked to enhanced LEC proliferation and migration through a mechanism that correlates with enhanced AKT phosphorylation but without any corresponding change in PTEN levels, suggesting miR–19a does not target PTEN directly. \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTogether these findings clearly demonstrate that overexpressing miR–19a can drive enhanced proliferation and migration of LECs through a mechanism potentially linked with regulation of AKT signaling. This thus suggests that targeting miR–19a may be a viable therapeutic strategy for treating PCO.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003ePCO: Posterior capsule opacification; miR: microRNA; LECs: lens epithelial cells; PTEN: phosphatase and tensin homolog.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis study was supported by grants from the Yantai Technology Development Plan (No. 2017ws109) and Shandong Provincial Natural Science Foundation (No. ZR2017MH068, ZR2017LH043). The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on request.\u003c/p\u003e\n\u003ch3\u003eAuthors’ contributions\u003c/h3\u003e\n\u003cp\u003eYBL, SJL and FLZ conceived and designed the study, YFW and FLZ drafted the manuscript. YFW, YBL, FLZ, SJL and YDP performed the experiments. YFW and FLZ analyzed the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003eLu C, Yu S, Song H, Zhao Y, Xie S, et al. Posterior capsular opacification comparison between morphology and objective visual function. BMC Ophthalmol. 2019; 19: 40.\u003c/li\u003e\u003cli\u003eNibourg LM, Gelens E, Kuijer R, Hooymans JM, van Kooten TG, et al. Prevention of posterior capsular opacification. Exp Eye Res. 2015; 136: 100–15.\u003c/li\u003e\u003cli\u003eAmbros V. The functions of animal microRNAs. Nature. 2004; 431: 350–5.\u003c/li\u003e\u003cli\u003eKhan AA, Penny LA, Yuzefpolskiy Y, Sarkar S, Kalia V. MicroRNA–17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections. Blood. 2013; 121: 4473–83.\u003c/li\u003e\u003cli\u003eLiu Y, Liu R, Yang F, Cheng R, Chen X, et al. miR–19a promotes colorectal cancer proliferation and migration by targeting TIA1. Mol Cancer. 2017; 16: 53.\u003c/li\u003e\u003cli\u003eZhang J, Xiao Z, Lai D, Sun J, He C, et al. miR–21, miR–17 and miR–19a induced by phosphatase of regenerating liver–3 promote the proliferation and metastasis of colon cancer. Br J Cancer. 2012; 107: 352–9.\u003c/li\u003e\u003cli\u003eCellura D, Pickard K, Quaratino S, Parker H, Strefford JC, et al. miR–19-Mediated Inhibition of Transglutaminase–2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer. Mol Cancer Res. 2015; 13: 1095–105.\u003c/li\u003e\u003cli\u003eKarali M, Peluso I, Marigo V, Banfi S. Identification and characterization of microRNAs expressed in the mouse eye. Invest Ophthalmol Vis Sci. 2007; 48: 509–15.\u003c/li\u003e\u003cli\u003eHughes AE, Bradley DT, Campbell M, Lechner J, Dash DP, et al. Mutation altering the miR–184 seed region causes familial keratoconus with cataract. Am J Hum Genet. 2011; 89: 628–33.\u003c/li\u003e\u003cli\u003eLee SK, Teng Y, Wong HK, Ng TK, Huang L, et al. MicroRNA–145 regulates human corneal epithelial differentiation. PLoS One. 2011; 6: e21249.\u003c/li\u003e\u003cli\u003eNibourg LM, Gelens E, Kuijer R, Hooymans JM, van Kooten TG, et al. Prevention of posterior capsular opacification. Exp Eye Res. 2015; 136: 100–15.\u003c/li\u003e\u003cli\u003eHayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, et al. A polycistronic microRNA cluster, miR–17–92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res. 2005; 65: 9628–32.\u003c/li\u003e\u003cli\u003eRaghunath A, Perumal E. Micro-RNAs and their roles in eye disorders. Ophthalmic Res. 2015; 53: 169–86.\u003c/li\u003e\u003cli\u003eFumarola C, Bonelli MA, Petronini PG, Alfieri RR. Targeting PI3K/AKT/mTOR pathway in non small cell lung cancer. Biochem Pharmacol. 2014; 90: 197–207.\u003c/li\u003e\u003cli\u003eRevathidevi S, Munirajan AK. Akt in cancer: Mediator and more. Semin Cancer Biol. 2019; pii: S1044–579X: 30062–2.\u003c/li\u003e\u003cli\u003eZhang X, Chen Y, Zhao P, Zang L, Zhang Z, et al. MicroRNA–19a functions as an oncogene by regulating PTEN/AKT/pAKT pathway in myeloma. Leuk Lymphoma. 2017; 58: 932–40.\u003c/li\u003e\u003cli\u003eFang Y, Xu C, Fu Y. MicroRNA–17–5p induces drug resistance and invasion of ovarian carcinoma cells by targeting PTEN signaling. J Biol Res (Thessalon). 2015; 22: 12.\u003c/li\u003e\u003cli\u003eYu T, Liu L, Li J, Yan M, Lin H, et al. MiRNA–10a is upregulated in NSCLC and may promote cancer by targeting PTEN. Oncotarget. 2015; 6: 30239–50.\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-19a, Lens epithelial cells, Proliferation, Migration","lastPublishedDoi":"10.21203/rs.2.13691/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.13691/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background: Posterior capsule opacification (PCO) occurs frequently following surgical treatment of cataracts, representing a common adverse outcome. PCO develops as a result of lens epithelial cell (LEC) transdifferentiation, migration, and proliferation. In the present report we sought to explore the role of microRNA (miR)-19a in this process and to establish the underlying molecular mechanisms. \nMethods: miR-19a was transduced into HLE-B3 LECs, with microscopy and RT-qPCR used to confirm transfection efficiency. We then employed MTT, wound healing, and transwell assay approaches to monitor the proliferation and migration of these LECs. We further assessed levels of the proteins PTEN, AKT, and phosphorylated AKT (pAKT) via western blotting in WT and miRNA-transfected cells.\nResults: HLE‑B3 proliferation was markedly enhanced by miR-19a transduction, as well the migration activity of these cells (both P\u003c0.01). Furthermore, overexpressing miR‑19a failed to reduce PTEN expression whereas it did enhance pAKT levels within these LECs (P\u003c0.05).\nConclusions: This suggests that miR-19a can enhance LEC proliferation and migratory activity through a mechanism that may be linked with regulating AKT activation and signaling, thus highlighting a potential avenue for therapeutic treatment of PCO patients.","manuscriptTitle":"miR-19a promotes proliferation and migration of lens epithelial cells by regulating AKT/pAKT signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-08-31 01:18:15","doi":"10.21203/rs.2.13691/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"794cecb8-db79-4ff7-8a4f-f722a7d91ff8","owner":[],"postedDate":"August 31st, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":23518,"name":"Ophthalmology"},{"id":23519,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-08-31 01:18:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4397","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-4397","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}