CRISPR-Cas9 genome and long non-coding RNAs as a novel diagnostic index for prostate cancer therapy via liposomal-coated compounds

Mai O. Kadry; Rehab M. Abdel-Megeed

doi:10.1371/journal.pone.0302264

Peer Review History

Original SubmissionSeptember 6, 2023
4 Jan 2024 Decision Letter - Laila Adel Ziko, Editor PONE-D-23-28438CRISPR-Cas9 genome as a novel prognostic tool for Prostate cancer: long non-coding RNAs the predictive diagnostic index of targeted liposomal-coated compounds in prostate cancer therapyPLOS ONE Dear Dr. Kadry, 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 17 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. 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Thank you for stating the following financial disclosure: "Mai O Kadry received funding from National research Centre, project grants, awards number 13020251" Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 6. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option. 7. Please include a separate caption for each figure in your manuscript. 8. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: Please address the reviewers’ comments thoroughly and send it back to us for the second revision. [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: Yes Reviewer #2: Yes ******** 2. 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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: Yes ****** 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: It's an interesting study. The author used the liposomal loaded DXM,DOX,and TUR to treat the DAE-induced PC, and shows a reduced dosage for the drugs and good efficacy on the biomarkers of PC, including PSA, NOX, as well as the MALAT1 and apoptosis signaling, such as AKT,PI3K and GSK-3. However, it will be better if they author could complete the last Figure HE staining with all control groups (without liposomal loaded DXM,DOX and TUR) and the protein expression of ATK.PI3K and GSK-3. Reviewer #2: 1. What’s the current techniques to defeat prostate cell carcinoma? What’s the five-year survival rate for prostate cell carcinoma? 2. What’s the obstacles for the clinical application of liposomal glucocorticoids in prostate cancer? 3. What’s the individual effect of CRISPR/Cs9 on PC-associated miRNAs? Which PC-associated miRNA is affected mostly by CRISPR/Cas9? 4. In the method of mRNA gene expression of prostate AKT, PI3K and GSK-3, the volume should be �l rather than L. 5. In Fig.1, what’s the reason for no difference between DOX and LIP-DOX? 6. In Fig.1, 2, 3, 4, 5 & 6, the authors should indicate the P-value number rather than the different letters. 7. In Fig.7, the authors should select other long-noncoding genes as positive and negative controls. ****** 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: Ping Han It's an interesting study. The author used the liposomal loaded DXM,DOX,and TUR to treat the DAE-induced PC, and shows a reduced dosage for the drugs and good efficacy on the biomarkers of PC, including PSA, NOX, as well as the MALAT1 and apoptosis signaling, such as AKT,PI3K and GSK-3. However, it will be better if they author could complete the last Figure HE staining with all control groups (without liposomal loaded DXM,DOX and TUR) and the protein expression of ATK.PI3K and GSK-3. 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". 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.0302264.r001
Revision 1
5 Feb 2024 Author Response Editorial comments ETHICS NUMBER WAS REMOVED and kept only in the materialand methods. Tables was inserted inthe manuscript. funder financially supported the work but had No role in study design ,data collection and analysis ,decision to publish or preparation of the manuscript. please I encountered a problem inmy labtop so kindly add this tothe cover letter. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming Adjusted as required We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar Adjusted as required To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Adjusted as required Please state what role the funders took in the study. NRC is financially supported the current work Please include a separate caption for each figure in your manuscript. Adjusted as required Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information Adjusted as required Reviewers comments What’s the current techniques to defeat prostate cell carcinoma? Different strategies such as liposomes, solid dispersion, complex, emulsion, micelles, nanogels and microspheres have been employed to overcome poor absorption and other limitations of curcumin [1]. DOX’s mechanism of action on cancer cells begins with its passive diffusion through the phospholipid bilayer membrane of malignant cells into the cytoplasm, where DOX is converted into a semiquinone and generates reactive oxygen species (ROS), causing free radical formation and oxidative stress. In the cytosol, DOX enters the mitochondria causing DNA damage and energetic stress. As a result, the mitochondria release the cytochrome C protein, triggering the caspase cascade leading to cell death. From the cytosol, DOX translocates into the nucleus where it intercalates between double-stranded DNA helices and inhibits the enzymes topoisomerases I and II. The resulting damage to DNA leads to free radical generation, alkylation, and activation of the p53 pathway However, the potential therapeutic benefits of DOX are limited by the risk of cardiotoxicity, which has been evidently related to its lifetime cumulative dose. To overcome this hurdle, the liposomal DOX (L-DOX) formulation was developed in order to reduce DOX-associated cardiotoxicity while preserving its antitumor efficacy.10 The L-DOX formulation encapsulates DOX within a phospholipid bilayer that is coated with methoxypolyethylene glycol. The PEGylation protects the liposomes from recognition by the mononuclear phagocyte system (MPS) and allows a longer circulation time in the bloodstream while reducing the exposure of free DOX circulating in the plasma. 1-Feng T, Wei Y, Lee RJ, Zhao L. Liposomal curcumin and its application in cancer. International journal of nanomedicine. 2017 Aug 21:6027-44. 2- Franco YL, Vaidya TR, Ait-Oudhia S. Anticancer and cardio-protective effects of liposomal doxorubicin in the treatment of breast cancer. Breast Cancer (Dove Med Press). 2018 Sep 11;10:131-141. doi: 10.2147/BCTT.S170239. PMID: 30237735; PMCID: PMC6138971. What’s the five-year survival rate for prostate cell carcinoma? The 5-year relative survival rate for most people with local or regional prostate cancer is nearly 100%. For people diagnosed with prostate cancer that has spread to other parts of the body, the 5-year relative survival rate is 90%. What’s the obstacles for the clinical application of liposomal glucocorticoids in prostate cancer? Glucocorticoids have been used widely in conjunction with other treatment for patients with cancer because they have potent proapoptotic properties in lymphoid cells, can reduce nausea, and alleviate acute toxic effects in healthy tissue. However, glucocorticoids are used in a supportive-care role, even though to our knowledge no prospective clinical studies have assessed the effect of these steroids on the growth of solid tumours. Data from preclinical and, to some extent, clinical studies, suggest that glucocorticoids induce treatment resistance in solid tumours, including prostate cancer. Research has focussed on disseminated cells that have been shed by the tumour: the potential of glucocorticoids to render these cells resistant to apoptosis--and to downregulate the immune response--might contribute to tumour metastasis. Here, we review the benefits of glucocorticoids and their negative effects, such as induction of resistance in tumour cells and concomitant induction of apoptosis in immune cells, with particular emphasis on prostate cancer. Reference: Herr, I., & Pfitzenmaier, J. (2006). Glucocorticoid use in prostate cancer and other solid tumours: implications for effectiveness of cytotoxic treatment and metastases. The Lancet. Oncology, 7(5), 425–430. https://doi.org/10.1016/S1470-2045(06)70694-5 What’s the individual effect of CRISPR/Cs9 on PC-associated miRNAs? Which PC-associated miRNA is affected mostly by CRISPR/Cas9? Prostate cancer (PCa) has a distinctive miRNA expression profile, which has been the basis for the functional study of miRNA in PCa (1). Porkka et al (1) demonstrated that 37 miRNAs were downregulated and 14 upregulated in PCa tissues compared with benign tissues. A previous study obtained differential expression data of miRNAs in PCa determined by miRNA microarray analyses and reported that 11 miRNAs were upregulated and 17 miRNAs were downregulated in PCa (2). However, these previous gain-of-function studies did not include loss-of-function analyses using miRNA knockout and applying the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (Cas) 9 system, which can effectively, specifically and stably suppress gene expression in vitro and in vivo (3,4). CRISPR/Cas9 is a recently discovered genome editing system, which has markedly changed the way that researchers study genes and their functions in mammalian systems (4,5). It is derived from the CRISPR/Cas bacterial-acquired immune system and Cas9 is directed by guide (g)RNAs, which match the DNA targeted in cleavage to modify the respective gene (3,5,6). References: 1-Porkka KP, Pfeiffer MJ, Waltering KK, Vessella RL, Tammela TL and Visakorpi T: MicroRNA expression profiling in prostate cancer. Cancer Res 167: 6130-6135, 2007 2- He HC, Han ZD, Dai QS, Ling XH, Fu X, Lin ZY, Deng YH, Qin GQ, Cai C, Chen JH, et al: Global analysis of the differentially expressed miRNAs of prostate cancer in Chinese patients. BMC Genomics 14: 757, 2013. 3-Cho SW, Kim S, Kim JM and Kim JS: Targeted genome engineering in human cells with the Cas9 RNA guided endonuclease. Nat Biotechnol 31: 230-232, 2013. 4- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA and Zhang F: Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823, 2013 5- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE and Church GM: RNA guided human genome engineering via Cas9. Science 339: 823-826, 2013 6- Jiang, F. N., Liang, Y. X., Wei, W., Zou, C. Y., Chen, G. X., Wan, Y. P., ... & Zhong, W. D. (2020). Functional classification of prostate cancer associated miRNAs through CRISPR/Cas9 mediated gene knockout. Molecular Medicine Reports, 22(5), 3777-3784. l rather than L.�4. In the method of mRNA gene expression of prostate AKT, PI3K and GSK-3, the volume should be corrected 5. In Fig.1, what’s the reason for no difference between DOX and LIP-DOX? In Fig.1, 2, 3, 4, 5 & 6, the authors should indicate the P-value number rather than the different letters. Added to figure caption It will be better if they author could complete the last Figure HE staining with all control groups (without liposomal loaded DXM,DOX and TUR) and the protein expression of ATK.PI3K and GSK-3. Added as required Attachments Attachment Submitted filename: PLOS PROSTATE Response to comments.docx https://doi.org/10.1371/journal.pone.0302264.r002
8 Feb 2024 Decision Letter - Laila Adel Ziko, Editor PONE-D-23-28438R1CRISPR-Cas9 genome as a novel prognostic tool for Prostate cancer: long non-coding RNAs the predictive diagnostic index of targeted liposomal-coated compounds in prostate cancer therapyPLOS ONE Dear Dr. Kadry, 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. The reviewers' comments were sufficiently addressed, however the title needs to be grammatically proofread and shortened preferably. Also, there are several grammatical typos throughout the manuscript with words without spaces between them, this needs to be corrected before it can be further accepted. Please submit your revised manuscript by Mar 24 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. 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, Laila Adel Ziko 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. Additional Editor Comments: The reviewers' comments were sufficiently addressed, however the title needs to be grammatically proofread and shortened preferably. Also, there are several grammatical typos throughout the manuscript with words without spaces between them, this sneedes to be corrected before it can be further accepted. [Note: HTML markup is below. Please do not edit.] [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.0302264.r003
Revision 2
15 Mar 2024 Author Response DOI LINK for Data availability was supplied Title was adjusted as required Typo writing and sticky words were adjusted as required cover letter the role of funder was mentioned Attachments Attachment Submitted filename: PLOS PROSTATE Responses.docx https://doi.org/10.1371/journal.pone.0302264.r004
1 Apr 2024 Decision Letter - Laila Adel Ziko, Editor CRISPR-Cas9 genome and long non-coding RNAs as a novel diagnostic index for Prostate cancer therapy via liposomal-coated compounds PONE-D-23-28438R2 Dear Dr. Kadry, 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, Laila Adel Ziko Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0302264.r005
Formally Accepted
29 Apr 2024 Acceptance Letter - Laila Adel Ziko, Editor PONE-D-23-28438R2 PLOS ONE Dear Dr. Kadry, 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. Laila Adel Ziko Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0302264.r006

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