Regulation of corneal epithelial differentiation: miR-141-3p promotes the arrest of cell proliferation and enhances the expression of terminal phenotype

María Teresa Ortiz-Melo; Jorge E. Campos; Erika Sánchez-Guzmán; María Esther Herrera-Aguirre; Federico Castro-Muñozledo

doi:10.1371/journal.pone.0315296

Peer Review History

Original SubmissionJuly 24, 2024
16 Sep 2024 Decision Letter - Alfred S Lewin, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-24-30995Regulation of corneal epithelial differentiation: miR-141-3p promotes the arrest of cell proliferation and enhances the expression of terminal phenotypePLOS ONE Dear Dr. Castro-Muñozledo, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. You will note that the expert reviewers have made substantive recommendations, with which I agree. Both reviewers requested that you justify the use of the rabbit RCE1(5T5) cell line as a model as opposed to human corneal cell lines. Both reviewers requested that you justify the use of KRT3 expression as a marker of differentiation, and reviewer 2 recommended that KRT12 should also be used. Reviewer 1 points out a contradiction with respect to knock down of miR-141-3p and KRT15, and this should be resolved. Reviewer 2, demands a better characterization of EMT in your model. Please submit your revised manuscript by Oct 31 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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[Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have explored the role of miR-141-3p in the regulation of corneal epithelial differentiation and demonstrated that it promotes the arrest cell proliferation and enhances the expression of terminal phenotype using rabbit corneal epithelial cell line. The authors need to address the following points of concern for the manuscript to be considered for publication. 1. The authors need to justify the use of rabbit corneal epithelial cell line while there are several human corneal epithelial cell lines. They need to introduce the cell line and their previous work on the same as a model to study the corneal epithelial differentiation. A brief note on the different stages will also enable the readers to understand the significance of the current study. 2. Details on the markers used in the study: a. K3 – it is a corneal epithelial cell marker expressed at protein level by suprabasal limbal epithelium and central corneal epithelium in humans. In this study, K3 is defined as a terminal differentiation marker. The authors need to justify this as well as specify the percentage of RCE1 (5T5) cells that express K3? In a primary human limbal explant culture, almost all cells express K3. In contrast, in figure 4A, more than 85% of the cells were negative for K3. To discuss. b. ΔNp63α – is a corneal epithelial stem cell marker. In has been defined in this manuscript as i. P63 – epithelial stem cell marker – line no. 262 ii. Marker for proliferative early differentiating cells - line no. 267 iii. Related to early precursor/proliferative cells – line no. 293 iv. Proposed as a stem/proliferative cell marker – line nos. 345,346 v. Proliferating corneal epithelial cell marker – line no. 439 vi. Proliferative/progenitor cell marker – line nos. 484,485 3. The authors specify in line nos. 346-348 that “…knockdown of miR-141-3p promotes the expression of a corneal epithelial precursors-like phenotype..”. But in line nos. 474-475 they specify “…knock down of miR-141-3p decreased KRT15…. a biomarker for corneal epithelial stem and transient amplifying cells.” Both are contradictory. 4. Line 289 - … Figure 2G represents PAX6 expression and Figure 2H – KRT3 and not KRT3 and vimentin as specified in the manuscript. 5. Transfection with antagomiR-141 resulted only in 25% increase in vimentin mRNA level which was not significant. Similarly flow cytometry data also indicates only a minor increase in the vimentin expression in cells at protein level. Justify how this is associated with EMT. Reviewer #2: This study investigates the role of human miR-141-3p in corneal epithelial differentiation using the rabbit RCE1(5T5) cell line. The research provides insights into the miRNA-mediated molecular mechanisms regulating corneal epithelial cell differentiation. However, some issues need to be addressed. 1. The authors use the rabbit RCE1(5T5) cell line as a model, but a justification for choosing this model over human corneal epithelial cells or limbal stem cells would be helpful, as these might more accurately represent human corneal biology. Additionally, it is important to clarify whether the miR-141-3p sequence is fully conserved between rabbits and humans. 2. To comprehensively assess miR-141-3p's impact on corneal epithelial cell differentiation, examining only KRT3 expression is insufficient. KRT12 should also be included, as it is a key marker in conjunction with KRT3 for corneal epithelial differentiation. 3. Regarding the epithelial-mesenchymal transition (EMT) analysis, further evaluation is required. The expression of E-cadherin, a key epithelial marker, should be assessed since its reduction is a hallmark of EMT. Similarly, N-cadherin expression should be measured, as an increase indicates a mesenchymal transition. Additionally, the analysis of Snail or Twist, crucial transcription factors in EMT, would provide more comprehensive insight. 4. To better understand miR-141-3p's impact on the migratory ability of corneal epithelial cells, functional assays such as wound healing or transwell migration should be conducted. Moreover, demonstrating whether overexpression of miR-141-3p can reverse the EMT phenotype induced by its inhibition would provide stronger evidence of its regulatory role. 5. There are areas in the manuscript that could be improved for better clarity and flow: • The introduction lacks a clearly defined hypothesis or research question. Explicitly stating the research objective would better frame the study. • In the methods section, related experimental details (e.g., cell culture, RNA extraction, and sequencing) should be grouped into cohesive paragraphs for improved readability. • The results section could benefit from smoother transitions between experiments. Providing context or linking sentences would help illustrate how the findings build on each other. • The rationale for selecting specific markers (e.g., KRT3, Vimentin, ZEB1) as indicators of differentiation or EMT should be detailed to clarify their relevance to miR-141-3p's function. 6. Finally, minor grammatical issues, such as sentence structure and punctuation, should be corrected to enhance overall readability. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Gowri Priya Chidambaranathan Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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Revision 1
21 Nov 2024 Author Response November 20, 2024. Alfred S. Lewin, Ph.D. University of Florida Section editor. PLoS ONE Dear Dr.Lewin: We are grateful for the valuable comments raised by the reviewers and you, after reviewing our manuscript entitled “Regulation of corneal epithelial differentiation: miR-141-3p promotes the arrest of cell proliferation and enhances the expression of terminal phenotype.” by María Teresa Ortiz-Melo, Jorge E. Campos, Erika Sánchez-Guzmán, María Esther Herrera-Aguirre and Federico Castro-Muñozledo. Based on these comments, we have made modifications, added new results, and carried out text clarifications, as follows: 1. A common concern shared by the reviewers and you, is the use of a rabbit corneal epithelial cell line instead of the use of cells from human origin. To clarify this point, we must remark that our cell line was obtained by serial transfer and avoiding gene manipulation, using an experimental approach similar to that followed by Todaro and Green to generate the 3T3 fibroblast cell line (J Cell Biol. 1963, 17:299-313). This contrasts with those cell lines obtained from human cornea generated by expression of viral sequences (see Araki et al., 1993, Invest Ophthalmol Vis Sci. 34(9):2665-2671, and Kahn et al., 1993, Invest Ophthalmol Vis Sci. 34:3429-3441; Kim et al., 2016, J Pharmacol Toxicol Methods. 78:52-57). The published literature on these cell lines shows that, although they display specific markers of the corneal epithelium, in most cases, their expression pattern is characterized by levels lower than those found in the tissue of origin (see Gipson et al., 2003, Invest Ophthalmol Vis Sci. 44:2496-2506; Rubelowski et al., 2020, Graefes Arch Clin Exp Ophthalmol. 258:565-575; Latta et al., 2022, Graefes Arch Clin Exp Ophthalmol 260:4019-4020). Some of these alterations are associated with the insertion site of those sequences used to generate the immortalized cells. In addition, it was reported that they undergo genomic alterations, becoming heterogeneous cell populations, and in some cases these cell lines are tumorigenic (Yamasaki et al., 2009, Invest Ophthalmol Vis Sci. 50:604-613). Other human cell lines have been immortalized by the use of telomerase (Robertson et al., 2005, Invest Ophthalmol Vis Sci. 46:470-478), and more recently, a human cell line was obtained by spontaneous immortalization (Notara and Daniels, 2010, Brain Res Bull. 81:279-286), however, these cell populations have not been extensively characterized. Other cell lines, such as SIRC cells, have an uncertain origin and have been poorly characterized (Leerhoy, 1965, Science, 149:633-634). In contrast with these cell lines, the RCE1(5T5) cells have been extensively studied, showing a sequential expression of markers similar to that described for primary cultures of rabbit corneal epithelial cells. These cells express the keratin pair K5/K14 as described for primary cultures during early growth, and in basal cells when they have constituted a stratified epithelium (Castro-Muñozledo, 1994, J Cell Sci 107: 2343-2351; Castro-Muñozledo et al., 2017, J Cell Physiol 232(4):818-830): Later, during the exponential growth phase, suprabasal layers express the keratin pair K6/K16, and only when cultures constitute a confluent epithelium suprabasal cells become K3/K12 positive showing similar levels to primary cultures. Such an expression pattern that was previously described, as we said before, is comparable to that found in primary cultures. To illustrate this point, below we have included a figure composed by our results (taken from figure 7 in Castro-Muñozledo, 1994, J Cell Sci 107: 2343-2351) and those obtained by Schermer et al (Fig 2, 1989, Differentiation. 42:103-110). Moreover, to our knowledge, the RCE1(5T5) cell line has been fully characterized, on the basis of a day by day expression of differentiation markers, both at protein and mRNA expression, as well as the establishment of tight junctions and development of trans epithelial resistance, and mechanisms that regulate tight junctions’ assembly (Martínez-Rendón et al.,2017, J. Cell. Physiol. 232, 1794-1807; Ortiz-Melo et al., 2013, Biology Open 2:132-143). These cells show an expression pattern of claudins similar to that reported for the corneal epithelium from different species (see Ortiz-Melo et al., 2013, Biology Open 2:132-143). This figure depicts, for comparison, the expression patterns of cytokeratins KRT5/KRT14, KRT6/KRT16 and KRT3/KRT12 reported along the growth and differentiation of cultured primary corneal epithelial cell, and RCE1(5T5) cells (LEFT: Schermer et al Fig 2, 1989, Differentiation. 42:103-110, primary cultures rabbit corneal epithelial cells. RIGHT: figure 7 in Castro-Muñozledo, 1994, J Cell Sci 107: 2343-2351, RCE1(5T5) cells.) Moreover, to our knowledge, the RCE1(5T5) cell line has been fully characterized, on basis to a day by day expression of markers, both at protein and mRNA expression, as well as establishment of tight junctions and trans epithelial resistance, and mechanisms that regulate tight junctions assembly (Martínez-Rendón et al.,2017, J. Cell. Physiol. 232, 1794-1807; Ortiz-Melo et al., 2013, Biology Open 2:132-143). On the basis of such analysis, we are confident that the results obtained in RCE1(5T5) cell cultures are representative of what happens in primary cell cultures. Moreover, we have used this cell culture model to design a treatment for corneal alkali burns, which afterwards, was successfully used for treatment of experimental chemical damage in mice (Gulias-Cañizo, et al., 2019, Burns. 45, 398-41), or for in vitro assays for the damage caused by Acanthamoeba on corneal surface (Coronado-Velázquez et al., 2020, Parasitol Int. 74: Art No. 102002). In addition, we must remark that in the first days after confluence, our model possesses a structure organization that makes it similar to the limbal epithelium, and only after more prolonged periods, the cultured epithelia show K3 keratin expression at the basal cell layer. Since we are interested in the regulation of tissue-specific gene expression, we are confident that our experimental model is ideal for this purpose. In addition, its sequential expression of differentiation markers enables us to analyze such regulatory mechanisms. Taking into account the above considerations we made modifications to the manuscript as follows: Reviewer 1. 1. We have modified the introduction to include a paragraph describing our previous work with the cell line, including the requested note which explains the three different stages that we have distinguished in the cell culture system. 2a. We consider that KRT3 keratin is linked to differentiation. Since it is expressed in suprabasal layers of the limbal epithelium, and in both basal and suprabasal cells of central cornea, it could be considered a marker of terminal differentiation due to the loss of proliferative abilities of those cells which express it. However, it may be expressed by cells that have started the differentiation program, which show a limited proliferative ability giving rise to paraclones/meroclones (abortive colonies in cell culture) (see Barrandon and Green, 1987, Proc Natl Acad Sci USA 84(8):2302-2306), and were also described in corneal epithelial cell cultures (Pellegrini et al., 1999, J Cell Biol 145(4):769-782). On the other hand, as requested by the reviewer, we have introduced an explanation about the low percentage of corneal epithelial cells which were detected as KRT3+/Vim+ and KRT3+/Vim- in the experiment that was originally described in Figure 4 (and now moved to Figure 5). The major reason for this result is that flow cytometry experiments as well as all experiments in which miR-141-3p was inhibited with the AntagomiR, were carried out in the lapse comprised between 4 to 6 days, a timing in which cultured cells were starting the expression of the differentiation process. In such sense, we tried to prevent the expression of miR-141-3p during the early stages of cell differentiation since we believe that the inhibition of this miRNA at later times, would not have any effect, since the miRNA began its expression from the fourth day in cell culture, and its rise had a delay of 48 hours in relation to the expression of PAX6. It is probable that the AntagomiR would not have any effect on cells expressing the differentiated phenotype, since at later differentiation stages, targets (at least all the predicted targets) such as ZEB1, ZEB2 and vimentin, would have decreased their expression. (In our experimental model, Vim, ZEB1 and ZEB2 show very low or null expression from the 7th day in cell culture). 2b. As requested, we have corrected the variability in how we defined the role of �Np63� in corneal epithelial cells. 3. We believe that the expression “corneal epithelial precursor-like phenotype” does not imply that cells are regressing their expression to become precursor cells, since miR-141-3p does not seem to have any target that could lead to “dedifferentiation” of cells. We used such term, in view that some characteristics were similar to those found in proliferative, non-differentiated cells. This fact is supported by our results showing that the transcriptome of AntagomiR treated cells is completely different from the profile of proliferative non-differentiated cells. Moreover, the decrease in KRT15 although non-significant as determined by qPCR, was also decreased when we analyzed the transcriptome of AntagomiR treated cells. Such results suggest that the number of stem cells/progenitors in our experimental model is decreasing and therefore miR-141-3p inhibition does not block differentiation. In this sense, we modified the manuscript to avoid confusion. 4. We have corrected the references to Figure 2, as noted in the manuscript by the reviewer. 5. Please, refer to answer 2a. It should be noted that differentiation does not occur synchronously. Reviewer2. 1. We have modified the introduction to include a paragraph that describes our previous work with the cell line, including the requested note which describes the three different stages that we have distinguished in the cell culture system. Moreover, in this rebuttal letter we justify the use of the rabbit corneal epithelial cell line instead of a human corneal cell line. Also, as requested by the reviewer, we have included in the miRNA inhibition section a text clarifying the full conservation of the miR-141-3p in human, mouse, rat, rabbit, bovine, goat, horse chimpanzee and in a bat species. 2. In our previous version of the manuscript we have not included KRT12 keratin since, in rabbit, as well as in human, such keratin shows an 8-10 hour delay in its expression, in relation to the expression of KRT3 keratin. It was previously demonstrated that within the corneal-type keratin pair, during differentiation, KRT3 precedes the appearance of KRT12, both in rabbit and in the chick (Chaloin-Dufau et al., 1990, Cell Diff Dev. 32:97-108) and that in human, the expression pattern of KRT3 is expressed from the 12-13 week of gestation, providing the earliest sign of the corneal epithelial differentiation (Rodriguez et al., 1987, Differentiation 34:60-67). Such results are in contrast with mice tissue, which only expresses KRT12 (Kao, W., 2020, Exp Eye Res. 200:108206). It is considered that in mice, KRT5/KRT12 corresponds to the corneal epithelial-type cytokeratin pair, and for such reason, in mice KRT12 is much more important than KRT3 as a corneal differentiation cell marker. In our case, we expected that KRT12 would be expressed to lower levels than KRT3. Nevertheless, as requested by the reviewer, we have included the quantification of KRT12 by qPCR, showing that KRT12 has higher expression levels in the cells transfected with the antagomiR-141 (although non-significant), than in control cultures that received the scrambled sequence. (see Fig. 2L). 3. As requested by the reviewer, we determined the expression of N-cadherin (CDH2), Snail and E-cadherin. See figure 2D, 2E, and 2M. The results suggest that effectively miR-141-3p inhibition leads to an EMT. However, since we observed an increase in the expression of PAX6, KRT3, KRT12, and considering that miR-141-3p increases about 48 hours after the expression of PAX6, we think that the increase in EMT and proliferation markers reflects the effect of the miRNA inhibition in the cell population that has started the differentiation process, and only these cells show the EMT, but not those that are more advanced in the differentiation process. This would be an explanation for detecting both an increase in EMT markers and in the differentiation markers (i.e. K3+/Vim+ cells). 4. As suggested by the reviewer, we have included in vitro wound healing experiments, and the results are included in a brand new Figure 3. In addition, we have modified Figure 4, to include some results from the old Figure 3 5. We have modified the manuscript as requested by the reviewer. Moreover, we have grouped related experimental details and made clearer the experimental rational trying to establish an adequate explanation to show the relevance of miR-141-3p in corneal epithelial cell differentiation. We think that our results could be relevant since the studies about the role of different miRNAs on the corneal epithelial differentiation are scarce. Moreover, we think that these could be of wide interest since they agree with results published by others in other tissue types and developmental processes. We hope that these modifications made our manuscript suitable to be accepted for publication in PLoS One. Thank you in advance, we look forward to hearing from you soon. Sincerely Federico Castro-Muñozledo. Please send all correspondence to: Federico Castro-Muñozledo Ph.D. Dept. of Cell Biology CINVESTAV-IPN. Apdo. Postal 14-740, Mexico City 07000, Mexico Phone: 52(55)5061-3985 Fax: 52(55)5061-3393 E-mail: federico.castro@cinvestav.mx Alternative e-mail: f.castromunozledo@gmail.com Attachments Attachment Submitted filename: Rebuttal letter november 20-2024.docx https://doi.org/10.1371/journal.pone.0315296.r002
25 Nov 2024 Decision Letter - Alfred S Lewin, Editor Regulation of corneal epithelial differentiation: miR-141-3p promotes the arrest of cell proliferation and enhances the expression of terminal phenotype PONE-D-24-30995R1 Dear Dr. Castro-Muñozledo, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Thank you for addressing the concerns of both expert reviewers thoroughly and for improving the writing style of the manuscript. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Alfred S Lewin, Ph.D. Section Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0315296.r003
Formally Accepted
27 Nov 2024 Acceptance Letter - Alfred S Lewin, Editor PONE-D-24-30995R1 PLOS ONE Dear Dr. Castro-Muñozledo, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Alfred S Lewin Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0315296.r004

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