LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis

Meng Han; Pathuama P; Jinhai Tian; Chen Wang; Shengnan Zhou; Lina Fu; Libin Wang; Na Tian

doi:10.1371/journal.pone.0322989

Peer Review History

Original SubmissionJune 12, 2024
12 Jun 2024 Author Response https://doi.org/10.1371/journal.pone.0322989.r001
16 Sep 2024 Decision Letter - Yash Gupta, Editor PONE-D-24-22267LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axisPLOS ONE Dear Dr. Na, 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. 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'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have indicated that there are restrictions to data sharing for this study. For studies involving human research participant data or other sensitive data, we encourage authors to share de-identified or anonymized data. However, when data cannot be publicly shared for ethical reasons, we allow authors to make their data sets available upon request. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. 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PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Additional Editor Comments: The figure quality is not appropriate. Reviewers have raised critical concerns regarding experiment set up and conclusions drawn. Authors need to adress these concerns and add a limitations section to the conclusions. In the current form this study is not of publication standard. [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: Yes 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: This study aims to investigate the mechanism of lncRNA Nuclear Enriched Abundant Transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury using Caco-2 and HEK293 cells. The findings demonstrate that lncRNA NEAT1 protects against uremic toxin-induced intestinal epithelial barrier injury by regulating the miR-122-5p/Occludin axis. However, this study has several limitations. It does not provide practical guidance on selecting urea treatment at 144 mg/dL for 48 hours, nor does it identify an optimal model for testing CKD recipient matches. Additionally, it lacks an in-depth analysis of the mechanisms underlying uremic toxin-induced intestinal epithelial injury. 1. The immunofluorescence images presented in this manuscript lack the clarity and resolution necessary for occludin staining (Figures 2G, 2H, 2I, 4F, and 5C). 2. In Figure 1, Western blot images D and H appear to be identical, particularly for ZO-1, claudin-1, and GAPDH. Could you clarify why these images seem to be duplicated? 3. Finally, the conclusion should address the debatable reduction of the tight junction proteins Occludin, ZO-1, and Claudin-1. Additionally, the permeability of sodium fluorescein does not appear to be significantly increased in the model group, which raises questions about the validity of the proposed mechanism involving the miR-122-5p/Occludin axis in supporting the observed injury. Reviewer #2: This manuscript investigates the role of LncRNA NEAT1 in urea induced Intestinal Epithelial barrier. Authors further explore the interaction between NEAT1 and miRNA-122-5p. This manuscript requires major revision before it could be accepted for publication. The language is not very easy to comprehend and would require significant change. Authors have provided complete data that they have discussed in the paper. However, their final conclusion that "NEAT1 acts as a sponge of miRNA-1225p to regulate Occludin expression" need more investigation. Figure5B shows that overexpression of LV-NEAT1 in the presence of miR-122-5p mimic and urea fails to rescue expression of Occludin, which doesnt support their conclusion. I recommend that authors should evaluate expression of Occludin in a dose-dependent response for both miR-122-5p mimic and urea independently, In the presence of a fixed concentration of other molecule and overexpression of LV-NEAT1. ****** 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: No Reviewer #2: Yes: Shwetank ******** [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". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0322989.r002
Revision 1
6 Nov 2024 Author Response Dear reviewer, Thank you very much for your comments and professional -advice. These opinions help to improve academic rigor of our article. Based on your suggestion and-request, we have made corrected modifications on the revised manuscript. Furthermore, we would like to show the details as follows Question1 When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file? id=wjVg/PLOSOne_formatting_sample_main_body.pdfandhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. The author’s answer: We have revised our manuscript as requested by PLOS One. Question2 We note that you have indicated that there are restrictions to data sharing for this study. For studies involving human research participant data or other sensitive data, we encourage authors to share de-identified or anonymized data. However, when data cannot be publicly shared for ethical reasons,we allow authors to make their data sets available upon request. For information on unacceptable data access restrictions, please seehttp://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. The author’s answer: We have chosen to upload the raw data to PLOS One as supporting information. Question3 Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. The author’s answer: We have included the full ethics statement in the Methods section of the manuscript. Question4 Please include a separate caption for each figure in your manuscript. The author’s answer: We have included a separate caption for each figure in our manuscript. Question5 PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation arehttps://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data:https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. The author’s answer: We provide raw uncropped and unadjusted images of all blot or gel results in the Supporting Information file. Additional questions The figure quality is not appropriate. Reviewers have raised critical concerns regarding experiment set up and conclusions drawn. Authors need to adress these concerns and add a limitations section to the conclusions.In the current form this study is not of publication standard. The author’s answer: We have modified the quality of the figure. Reviewer #1: This study aims to investigate the mechanism of lncRNA Nuclear Enriched Abundant Transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury using Caco-2 and HEK293 cells. The findings demonstrate that lncRNA NEAT1 protects against uremic toxin-induced intestinal epithelial barrier injury by regulating the miR-122-5p/Occludin axis. However, this study has several limitations. It does not provide practical guidance on selecting urea treatment at 144 mg/dL for 48 hours, nor does it identify an optimal model for testing CKD recipient matches. Additionally, it lacks an in-depth analysis of the mechanisms underlying uremic toxin-induced intestinal epithelial injury. 1. The immunofluorescence images presented in this manuscript lack the clarity and resolution necessary for occludin staining (Figures 2G, 2H, 2I, 4F, and 5C). 2. In Figure 1, Western blot images D and H appear to be identical, particularly for ZO-1, claudin-1, and GAPDH. Could you clarify why these images seem to be duplicated? 3. Finally, the conclusion should address the debatable reduction of the tight junction proteins Occludin, ZO-1, and Claudin-1. Additionally, the permeability of sodium fluorescein does not appear to be significantly increased in the model group, which raises questions about the validity of the proposed mechanism involving the miR-122-5p/Occludin axis in supporting the observed injury. Attachments Attachment Submitted filename: Response letter.docx https://doi.org/10.1371/journal.pone.0322989.r003
19 Jan 2025 Decision Letter - Yash Gupta, Editor PONE-D-24-22267R1LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axisPLOS ONE Dear Dr. Na, 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. Please submit your revised manuscript by Feb 23 2025 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'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Yash Gupta, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. 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: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #3: I Don't Know ****** 4. 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 #3: Yes ****** 5. 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 #3: Yes ****** 6. 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: I still have some concerns regarding the manuscript. For the Western blots, please ensure the molecular weight (kDa) is specified for each protein. Additionally, clarify how the Western blot bands were quantified, and ensure this is clearly described. Specify the number of experiments conducted. Lastly, there are additional references missing in the manuscript that should be included in the final version. Reviewer #3: The author has carefully considered the comments and revised the manuscript to enhance its scientific validity and transparency. ****** 7. 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: No Reviewer #3: 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". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0322989.r004
Revision 2
18 Feb 2025 Author Response Dear reviewer, Thank you very much for your comments and professional -advice. These opinions help to improve academic rigor of our article. Based on your suggestion and-request, we have made corrected modifications on the revised manuscript. Furthermore, we would like to show the details as follows� As some references were retracted, we have replaced them to ensure the accuracy of the article, which has been standardized in the article. Reviewer #1: I still have some concerns regarding the manuscript. For the Western blots, please ensure the molecular weight (kDa) is specified for each protein. Additionally, clarify how the Western blot bands were quantified, and ensure this is clearly described. Specify the number of experiments conducted. Lastly, there are additional references missing in the manuscript that should be included in the final version. My answer:Thanks for your suggestion. We have annotated the molecular weight of each protein in the figures of the article.The gray value of the target protein bands was quantitatively analyzed by imageJ, and each experiment was repeated 3 times to determine the expression level of the target protein.AdditionIy, we have refined and revised the references for completeness and accuracy. Reviewer #3: The author has carefully considered the comments and revised the manuscript to enhance its scientific validity and transparency. My answer:Thank you for your review. We tried our best to improve the manuscript and made some changes to the manuscript. These changes will not influence the content and framework of the paper. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0322989.r005
1 Apr 2025 Decision Letter - Yash Gupta, Editor LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis PONE-D-24-22267R2 Dear Dr. Na, 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. 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, Yash Gupta, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0322989.r006
Formally Accepted
Acceptance Letter - Yash Gupta, Editor PONE-D-24-22267R2 PLOS ONE Dear Dr. Tian, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. Yash Gupta Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0322989.r007

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