HNF-4α inhibits hepatocellular carcinoma cell proliferation through mir-122-adam17 pathway

Guang Yang; Min Zhang; Yawei Zhao; Yue Pan; Mujie Kan; Jing Li; Kan He; Xuewen Zhang

doi:10.1371/journal.pone.0230450

Peer Review History

Original SubmissionNovember 27, 2019
27 Jan 2020 Decision Letter - Yi-Hsien Hsieh, Editor PONE-D-19-32902 HNF-4α inhibits hepatocellular carcinoma cell proliferation by suppression mir-122-adam17 pathway PLOS ONE Dear Dr. He, 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. We would appreciate receiving your revised manuscript by Mar 12 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed: (1) http://www.oncotarget.com/index.php?journal=oncotarget&op=view&page=article&path%5B%5D=9728&path%5B%5D=30481 (2) https://www.ncbi.nlm.nih.gov/pubmed/19296470 (3) https://link.springer.com/article/10.1007%2Fs13277-014-2546-0 (4) https://www.journal-of-hepatology.eu/article/S0168-8278(14)00731-4/fulltext The text that needs to be addressed involves the 1st paragraph of the Introduction section (1,2), the 2nd and 3rd paragraph of the Introduction section (3) and the 3rd and 4th paragraphs of the Discussion section. In your revision please ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed 5. Please provide additional information about each of the cell lines used in this work, including any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines 6. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. 7. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: No Reviewer #3: Partly Reviewer #4: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: No Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: The study is based on the potential function of HNF-4α and miR-122 on proliferation of hepatocellular carcinoma. It is a comprehensive study of miR-122 regulation and its involvement in cell cancer proliferation, Results and conclusions are coherent. N.ew insights are provided in the HCC field Minor changes: 1)Reagent section could be deleted since it is practically repeated in next two method sections and moreover, some incoherencies has been found as for example the kit used in miRNA extraction (miRNeasy Mini Kit was purchased from Qiagen (Hilden, Germany) vs. MiRNA extraction was performed using the miRNA Extraction Kit (Tiangen, Beijing, China). I suggest providing the manufacture at the moment the reactive is first written to avoid mistakes. 2)Include in methods the transfection methodology in an independent section . What mimic was used (please include sequence)? What transfection controls were used? Where they were purchased? What test was performed to calculate the transfection efficiency? 3)Is mir-122 3p or 5p? 4)Please, define Up-regulated and down-regulated genes of figure 1 in the text of results section 5)In figure 2, the relative quantification of ADAM17 (C), EGFR (D), p-AKT/AKT (E), and cleaved caspase 3/caspase 3 (F) levels expressed relative to control, are from western results? It is not clear in methods and results. 6)What is “con” and NC? 7)Why figure 2A lacks SD lines? And Fig 3H? MTT is only performed once? 8)According to the results, miR-122 mimic down-regulated ADAM17, EGFR, p-AKT but increased the cleaved Caspase 3. Why authors only consider ADAM17 in the title? I9)N figure 3, Relative quantification of HNF-4α (C), ADAM17 (D), EGFR (E), p-AKT/AKT (F), and cleaved caspase 3/caspase 3 (G) levels expressed relative to control, is from western results or from qPCR? In case of qPCR, please include which probes are used and methodologies used in methods section 10)It is not clear why two GEO databases are used to explore the DGE. 11)Include a limitation statement, in the discussion, regarding to normal hepatocyte cells used in this study which are not adult cells but rather they are a stablished line embryonic cells, and gene expression could differ from normal adult cells. Moreover, Other target genes of HNF-4α could be involved in the hepatocellular carcinoma proliferation and other target genes of miR.122 regardless of ADAM17 could also be involved. 12)First paragraph of the discussion usually summarizes main objectives and results of the study. Reviewer #2: check the attached file Reviewer #3: In this manuscript, Yang et al. aimed to study the antitumor mechanism of miR-122 in hepatocellular carcinoma (HCC). It is well documented that miR-122 behaves as tumor suppressor repressing HCC by targeting cell proliferation, migration, differentiation, angiogenesis and apoptosis genes. However, it is not well understood how such tumor suppressor role is regulated. In addition, Yang et al. explored the causative role that the hepatocyte nuclear factor HNF4A might have in regulating the levels of miR-122. By using qRT-PCR analysis, they found reduced levels of miR-122 in samples of HCC compared to adjacent non-cancerous tissue, as well as in the human hepatocellular carcinoma cell lines HepG2, Bel-7402 and Bel-7404 compared with the normal human hepatocyte cell line HL-7702. By using an MTT cell viability assay, they found that the transfection of a miR-122 mimic reduced the viability of HepG2 cells, which correlated with a reduction in the levels of the proteins ADAM17, EGFR, phospho-AKT, and an increase in the levels of cleaved Caspase 3. Interestingly, they found that the overexpression of HNF4A, also in HepG2 cells, resulted in similar effects in the levels of those proteins, which correlated with an increase in the levels of miR-122 and a decrease in cell viability. They compared the levels of the proteins HNF4A, ADAM17 and EGFR in the three hepatocellular carcinoma cell lines to that of HL-7702 cells and found an inverse correlation: higher levels of HNF4A and lower levels of ADAM17 and EGFR in HL-7702. Finally, they identified that HNF4A is differentially down-regulated in hepatocellular carcinoma tissue compared with adjacent non-cancerous tissue. Overall, the manuscript presents data of good standard. However, the major conclusion that HNF4A inhibits hepatocellular carcinoma cell proliferation by suppressing the miR-122-ADAM17 pathway is not supported by the data provided. In addition, a number of major and minor issues do not warrant publication of the manuscript as it is. Major issues: 1) As mentioned, the main conclusion of the manuscript has no experimental support. Although the HNF4A overexpression experiment shows a significant increase in the levels of miR-122, it also could have resulted in the increase of other miRNAs. The causal effects of HNF4A via miR-122 should be tested using an inhibitor of miR-122. 2) The authors nicely show that the levels of miR-122 are lower in all three hepatocellular carcinoma cell lines tested compared to that of the normal hepatic cell line. However, they only used HepG2 cells to test the effect of increasing the levels of miR-122 and of HNF4A on cell viability and on the levels of ADAM17, EGFR, phospho-AKT and cleaved Caspase 3. To make a more compelling case of the role of HNF4A and miR-122 in hepatocellular carcinoma cells, it should be tested the effect of increasing their levels in all three cell lines initially analyzed. 3) It would also greatly add to the story the testing in HL-7702 cells of decreasing the levels of HNF4A evaluating the effects on cell viability and the levels of miR-122, ADAM17, EGFR, phospho-AKT and cleaved Caspase 3. Minor issues: 4) Although overall the manuscript is read and understood, it is advised an additional professional scientific proofreading. 5) The "Abstract" is too redundant; please revise. Some suggested modifications: 6) Lines 48-49: Sentence needs reference. 7) Line 63: Replace "suggested" with "suggesting". 8) Line 66: Replace "investigate" with "investigated". 9) Line 67: Replace "reveal" with "revealed". 10) Line 80: Replace "metrixs" with "metrix". 11) Line 82: Replace "we" with "We". 12) Line 86: Replace "metrixs" with "metrix". 13) Line 102: Replace "ammonium, persulfate" with "ammonium persulfate" (remove comma). 14) Line 154: For "Tris buffered saline with Tween (TBST)" inform concentration of Tween. 15) In Figure 1A: Replace "Relative Micor 122" with "Relative miR-122". Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 16) Figure 1B: Replace "Relative Micro 122" with "Relative miR-122". Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 17) Figure 2A: Replace "Micro 122 mimic" with "miR-122 mimic". Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 18) Figure 2C,D,E,F: Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 19) Line 194: Replace "Over-expressions" with "Over-expression"; "inhibit" with "inhibits". 20) Line 197: Replace "transfection in" with "transfect". 21) Line 199: Replace "expressions" with "expression"; "protein" with "proteins". 22) Line 201-202: Remove the sentence "Similar with...(Fig.3G)" because the previous sentence stated the same. 23) Line 206: Replace "over-expressions" with "over-expression"; "inhibit" with "inhibits". 24) Line 211: Replace "genes" with "gene". 25) In Fig 4F: the position of HNF4A is not visualized in the plot: use an arrow or a different color. 26) Lines 234-236: Sentence needs revision. 27) Line 245: Add last name: (Natalia) Lazarevich et al. elucidated... 28) Lines 260-262: Sentence needs references. 29) Lines 268-269: Change the sentence because is not warranted by experiments performed/results obtained. Reviewer #4: The result stated that up-regulated miR-122 inhibited hepatocellular carcinoma cells proliferation through regulating ADAM17. Major revision: The authors need to demonstrate the luciferase reporter assay to clarify whether ADAM17 is a target of miR-122. ****** 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: Natalia Garcia-Giralt Reviewer #2: No Reviewer #3: No Reviewer #4: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Reject(1).docx https://doi.org/10.1371/journal.pone.0230450.r001
Revision 1
13 Feb 2020 Author Response Dear Editor, Thank you for the careful reading and constructive suggestions on our manuscript entitled “HNF-4α inhibits hepatocellular carcinoma cell proliferation by suppression mir-122-adam17 pathway” (Manuscript ID PONE-D-19-32902). We have fully addressed all the concerns from the editor and the reviewers and carefully revised the manuscript. And a thorough check of typos and grammar was done by a native English speaker. All the changes in the text are highlighted in red (submitted as a separate file), and a point-by-point response is detailed below. We would hope that the revised manuscript is now acceptable to Plos One. Sincerely, Kan He, Ph.D Associate professor Department of Pharmacology, College of Basic Medical Sciences, Jilin University Academic editor’s comments: 1) We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed: Response: Thank you for the kind suggestions. We have rephrased and rewritten duplicated text and all the changes in the text are highlighted in red. 2) Please provide additional information about each of the cell lines used in this work, including any quality control testing procedures (authentication, characterisation, and mycoplasma testing). Response: Thank you for the kind suggestions. We have added the additional information about the cell line in the Cell culture section in the revised manuscript. 3) please provide the name of the colleague or the details of the professional service that edited your manuscript Response: Thank you for the kind suggestions. The language editing during manuscript revision was received the help form Dr. Wei Qiu, who currently is holding a tenure track assistant professor title in Loyola University and received several funding supports from NIH, including RO1. 4) Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type of consent you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. Response: Thank you for the kind suggestions. This ethics statement was included in the Methods part of revised manuscript: The written informed consent was obtained from all patients. 5) To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. Response: Thank you for the kind suggestions. We revised the Statistical analysis part in revised manuscript according to PLOS ONE submission guidelines online. Reviewer’s comments: Reviewer #1: 1) Reagent section could be deleted since it is practically repeated in next two method sections and moreover, some incoherencies has been found as for example the kit used in miRNA extraction (miRNeasy Mini Kit was purchased from Qiagen (Hilden, Germany) vs. MiRNA extraction was performed using the miRNA Extraction Kit (Tiangen, Beijing, China). I suggest providing the manufacture at the moment the reactive is first written to avoid mistakes. Response: Thank you for the kind suggestions and we apologize for the mistake. We have deleted the Reagent section and incorporated relative information into the methods and corrected the incoherencies in the revised manuscript. 2) Include in methods the transfection methodology in an independent section. What mimic was used (please include sequence)? What transfection controls were used? Where they were purchased? What test was performed to calculate the transfection efficiency? Response: Thank you for the kind suggestions. We have added the transfection methodology in the Cell transfection with miRNA section. 3) Is mir-122 3p or 5p? Response: Thank you for the kind suggestions. Mir-122 3p was used in our study. And we’ve added this information into the Method section. 4) Please, define Up-regulated and down-regulated genes of figure 1 in the text of results section Response: Thank you for the kind suggestions. The 2 up-regulated genes were hsa-miR-17 and hsa-miR-106a, and the 3 down-regulated genes were hsa-miR-1274b, hsa-miR-122 and hsa-miR-223. We have added the descriptions of these genes in the result section. 5) In figure 2, the relative quantification of ADAM17 (C), EGFR (D), p-AKT/AKT (E), and cleaved caspase 3/caspase 3 (F) levels expressed relative to control, are from western results? It is not clear in methods and results. Response: Thank you for the kind suggestions and we apologize for the confusion. The quantification of ADAM17 (C), EGFR (D), p-AKT/AKT (E), and cleaved caspase 3/caspase 3 (F) were from the western blot results. We have added the information in the method section and figure legend of Figure 2. 6) What is “con” and NC? Response: Thank you for the kind suggestions and we apologize for the use of unspecified abbreviations. CON stands for control and NC stands for negative control. We have added the definition of the abbreviations we used in the legends of each figure respectively. 7) Why figure 2A lacks SD lines? And Fig 3H? MTT is only performed once? Response: Thank you for the comments. Each MTT assay in current study was repeated for 3 times. Data were expressed as the mean ± standard deviation (SD). We apologize the oversight during figure processing, SD lines were added into Fig. 2A and Fig. 3H in the revised figures. 8)According to the results, miR-122 mimic down-regulated ADAM17, EGFR, p-AKT but increased the cleaved Caspase 3. Why authors only consider ADAM17 in the title? Response: Thank you for the comments. The EGFR/PI3K/AKT pathway is a well-established signaling which is regulated by ADAM17. Therefore, we detected the EGFR and p-AKT expression in this pathway as a down-stream effector of ADAM17. Since this regulation of EGFR and AKT have been confirmed by other studies, we mainly focused on studying the down-regulation of ADAM17 miR-122 in our research. 9)IN figure 3, Relative quantification of HNF-4α (C), ADAM17 (D), EGFR (E), p-AKT/AKT (F), and cleaved caspase 3/caspase 3 (G) levels expressed relative to control, is from western results or from qPCR? In case of qPCR, please include which probes are used and methodologies used in methods section Response: Thank you for the kind suggestions and we apologize for the confusion. The quantification of ADAM17 (C), EGFR (D), p-AKT/AKT (E), and cleaved caspase 3/caspase 3 (F) were from the western blot results. We have added the information in the legend of Figure 3. 10)It is not clear why two GEO databases are used to explore the DGE. Response: Thank you for the comments. In our study, the GSE54751 dataset contains information of microRNA profiles while the GSE84402 dataset contains information on gene expression rather than microRNA levels. Therefore, two GEO databases were used based on different research preferences. 11)Include a limitation statement, in the discussion, regarding to normal hepatocyte cells used in this study which are not adult cells but rather they are a stablished line embryonic cells, and gene expression could differ from normal adult cells. Response: Thank you for the kind suggestions. We have added the limitation of the normal hepatocyte cell line we used in the discussion section as follows: In our study, regarding to the normal hepatocyte cells we used were not adult hepatocyte cells but an established cell line of embryonic cells, the gene expression could differ from normal adult cells. Moreover, Other target genes of HNF-4α could be involved in the hepatocellular carcinoma proliferation and other target genes of miR.122 regardless of ADAM17 could also be involved. Response: Thank you for the comments. Indeed, there have been studies demonstrating the role of other genes like HNF-4α being involved in regulating the proliferation of HCC. And some other targets of miR122 such as HIF-1α and VEGF were reported to be regulated by miR-122. The crosstalk and network effects of signalings are complicated to fully study. In this work, we mainly focused on the role of miR-122 based on the bioinformatic analysis of the differently expressed genes between HCC and normal tissue in the clinical databases, as well as its regulation of ADAM17 in HCC. Our results did confirm their role although there were other proteins involved in this process which are worth for further exploration in our future studies. 12)First paragraph of the discussion usually summarizes main objectives and results of the study. Response: Thank you for the kind suggestions. We have added the summary of our main results in the first paragraph of the discussion as follows: The prevalence and severity of HCC are increasing worldwide, and the prognosis of HCC patients remains unsatisfactory due to the high rate of recurrence and metastasis. Therefore, improved therapeutic strategies for HCC patients are critical for the management of HCC. In our study, we investigated the potential function of miR 122 in the development and progression of HCC. The relationship between HNF-4α and miR-122 and the molecular mechanisms of HNF-4α and miR-122 in inhibiting hepatocellular carcinoma were revealed. Reviewer #3: Major issues: 1) As mentioned, the main conclusion of the manuscript has no experimental support. Although the HNF4A overexpression experiment shows a significant increase in the levels of miR-122, it also could have resulted in the increase of other miRNAs. The causal effects of HNF4A via miR-122 should be tested using an inhibitor of miR-122. Response: Thank you for the comments. It has been reported that other miRNAs such as miR43a and miR21 were also the downstream targets of HNF4A. The crosstalk and network of the genes were extremely complicated to be thoroughly studied. In our study, miR-122 was confirmed to be regulated by HNF4A, although there are also other targets regulated by HNF4A. we have revised our conclusion on this experiment in the Abstract and Discussion section as follows. Thanks again for the suggestion, we found it very constructive. We will study the molecular mechanism of the regulation of miR-122 by HNF4A using inhibitor of miR-122 and luciferase reporter assay in our future studies. And miR-122 is one of the downstream effector of HNF-4α. 2) The authors nicely show that the levels of miR-122 are lower in all three hepatocellular carcinoma cell lines tested compared to that of the normal hepatic cell line. However, they only used HepG2 cells to test the effect of increasing the levels of miR-122 and of HNF4A on cell viability and on the levels of ADAM17, EGFR, phospho-AKT and cleaved Caspase 3. To make a more compelling case of the role of HNF4A and miR-122 in hepatocellular carcinoma cells, it should be tested the effect of increasing their levels in all three cell lines initially analyzed. Response: Thank you for the kind suggestions. In our study, the down-regulation of miR-122 was confirmed to be universal in the three hepatocellular carcinoma cell lines. Based on that we chose HepG2 cells as a representative and did the further experiments. Indeed, it would be more compelling to do the downstream experiments on all the 3 cell lines. But due to the outbreak of the coronavirus in China, it is now difficult for us to do long-term experiments during the quarantine. We will include these experiments in our future study and make a more compelling story. 3) It would also greatly add to the story the testing in HL-7702 cells of decreasing the levels of HNF4A evaluating the effects on cell viability and the levels of miR-122, ADAM17, EGFR, phospho-AKT and cleaved Caspase 3. Response: Thank you for the kind suggestions. It would indeed be a more interesting story if we also test the cell viability and the levels of miR-122, ADAM17, EGFR, phospho-AKT and cleaved Caspase 3 effected by HNF4A in normal hepatocyte cells. However, as mentioned above, it is now difficult for us to do long-term experiments during the quarantine. We thank you again for the great idea and we will do these experiments and dig deeper into the molecular mechanism in our future study. Minor issues: 4) Although overall the manuscript is read and understood, it is advised an additional professional scientific proofreading. Response: Thank you for the suggestions. A thorough check of typos and grammar was done by a native English speaker. And all the changes in the text are highlighted in red in the revised manuscript. 5) The "Abstract" is too redundant; please revise. Response: Thank you for the suggestions. We have rewritten and simplified the abstract as follows: Hepatocellular carcinoma (HCC) is one of the most common human cancers, its prevalence and severity need us to discover novel early diagnostic biomarkers and new therapeutic strategies. MicroRNA-122 is the most abundant microRNA in the liver, and acts as a tumor suppressor and represses HCC development. In our study we showed that HNF-4α and MiR-122 were down-regulated significantly in hepatocellular carcinoma. Over-expression of HNF-4α inhibit hepatocellular carcinoma cells proliferation. And miR-122 is one of the downstream effector of HNF-4α. Up-regulated miR-122 inhibited hepatocellular carcinoma cells proliferation through regulating ADAM17. Collectively, our results suggested that HNF-4α could inhibit hepatocellular carcinoma proliferation with miR-122 being a downstream target of it. And miR-122 would inhibit hepatocellular carcinoma proliferation by regulating ADAM17 signal pathway. Some suggested modifications: 6) Lines 48-49: Sentence needs reference. 7) Line 63: Replace "suggested" with "suggesting". 8) Line 66: Replace "investigate" with "investigated". 9) Line 67: Replace "reveal" with "revealed". 10) Line 80: Replace "metrixs" with "metrix". 11) Line 82: Replace "we" with "We". 12) Line 86: Replace "metrixs" with "metrix". 13) Line 102: Replace "ammonium, persulfate" with "ammonium persulfate" (remove comma). 14) Line 154: For "Tris buffered saline with Tween (TBST)" inform concentration of Tween. 15) In Figure 1A: Replace "Relative Micor 122" with "Relative miR-122". Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 16) Figure 1B: Replace "Relative Micro 122" with "Relative miR-122". Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 17) Figure 2A: Replace "Micro 122 mimic" with "miR-122 mimic". Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 18) Figure 2C,D,E,F: Remove "%" symbols in y-axis (is already indicated in parentheses in axis title). 19) Line 194: Replace "Over-expressions" with "Over-expression"; "inhibit" with "inhibits". 20) Line 197: Replace "transfection in" with "transfect". 21) Line 199: Replace "expressions" with "expression"; "protein" with "proteins". 22) Line 201-202: Remove the sentence "Similar with...(Fig.3G)" because the previous sentence stated the same. 23) Line 206: Replace "over-expressions" with "over-expression"; "inhibit" with "inhibits". 24) Line 211: Replace "genes" with "gene". 25) In Fig 4F: the position of HNF4A is not visualized in the plot: use an arrow or a different color. 26) Lines 234-236: Sentence needs revision. 27) Line 245: Add last name: (Natalia) Lazarevich et al. elucidated... 28) Lines 260-262: Sentence needs references. 29) Lines 268-269: Change the sentence because is not warranted by experiments performed/results obtained. Response: Thank you for the thorough review and constructive the suggestions, and we apologize for the mistakes. We have fixed all the mistakes one by one in our revised manuscript. Reviewer #4: Major revision: The authors need to demonstrate the luciferase reporter assay to clarify whether ADAM17 is a target of miR-122. Response: Thank you for the suggestions. The direct binding of ADAM17 and miR-122 was reported in other studies (Tsai W C et al. MicroRNA‐122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma[J]. Hepatology, 2009, 49(5): 1571-1582.). and in our study, although we didn’t do the luciferase reporter assay, we believe the reports of other studies can be a proof of miR-122 being a target of ADAM17. Indeed, it would make a more compelling story doing the luciferase reporter assay, thank you for the constructive suggestion, we will do these experiments and dig deeper into the molecular mechanism in our future study. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0230450.r002
26 Feb 2020 Decision Letter - Yi-Hsien Hsieh, Editor PONE-D-19-32902R1 HNF-4α inhibits hepatocellular carcinoma cell proliferation by suppression mir-122-adam17 pathway PLOS ONE Dear Dr. He, 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. We would appreciate receiving your revised manuscript by Apr 11 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Yi-Hsien Hsieh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (if provided): In reviewer1 : I cannot see the changes highlighted in red [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: (No Response) Reviewer #4: (No Response) ******** 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: (No Response) Reviewer #4: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #4: Yes ****** 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: (No Response) Reviewer #4: 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: (No Response) Reviewer #4: 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: (No Response) Reviewer #4: According to the title "Up-regulated miR-122 inhibited hepatocellular carcinoma cells proliferation 186 through regulating ADAM17" , it is supposed to show the mechanism how miR-122 regulates ADAM17. The results included in this ms are not adequate for supporting that mechanism. At least, the functional anaysis of ADAM17 needs to be addressed in this study. Otherwise, the authors may change the title that could be relevant to the data investigated. ****** 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: Yes: Natalia Garcia Giralt Reviewer #4: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0230450.r003
Revision 2
27 Feb 2020 Author Response Dear reviewers, Thank you for the careful reading and constructive suggestions on our manuscript entitled “HNF-4α inhibits hepatocellular carcinoma cell proliferation through mir-122-adam17 pathway” (Manuscript ID PONE-D-19-32902R1). We have fully addressed all the concerns from the editor and the reviewers and carefully revised the manuscript. Since reviewer 1 pointed that the changes of first revision were not observed. All the changes (including first and current revision in the text are highlighted in red (submitted as a separate file), and a point-by-point response is detailed below. We would hope that the revised manuscript is now acceptable to Plos One. Sincerely, Kan He, Ph.D Associate professor Department of Pharmacology, College of Basic Medical Sciences, Jilin University Reviewer’s comments: Reviewer #4: According to the title "Up-regulated miR-122 inhibited hepatocellular carcinoma cells proliferation through regulating ADAM17", it is supposed to show the mechanism how miR-122 regulates ADAM17. The results included in this ms are not adequate for supporting that mechanism. At least, the functional analysis of ADAM17 needs to be addressed in this study. Otherwise, the authors may change the title that could be relevant to the data investigated. Response: Thank you for the kind suggestions. The direct binding of ADAM17 and miR-122 was reported in other studies (Tsai W C et al. MicroRNA‐122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma[J]. Hepatology, 2009, 49(5): 1571-1582.). Since we still have another project undergoing to explore the tumor suppressor function of miR-122 in hepatocellular carcinoma, the detailed mechanism of how miR-122 regulates ADAM17 will be conducted further. Therefore, in current manuscript, we decided to change the title into “HNF-4α inhibits hepatocellular carcinoma cell proliferation through mir-122-adam17 pathway”. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0230450.r004
2 Mar 2020 Decision Letter - Yi-Hsien Hsieh, Editor HNF-4α inhibits hepatocellular carcinoma cell proliferation through mir-122-adam17 pathway PONE-D-19-32902R2 Dear Dr. He, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. 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With kind regards, Yi-Hsien Hsieh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 #4: 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 #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #4: Yes ****** 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 #4: 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 #4: 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: Re-write conclusions in more narrative manner and correct the sentence -In our study, 257 regarding to the normal hepatocyte cells we used were not adult hepatocyte cells but an 258 established cell line of embryonic cells, the gene expression could differ from normal 259 adult cells. which is difficult to understand Reviewer #4: I agreed with the revised title "HNF-4α inhibits hepatocellular carcinoma cell proliferation through mir-122-adam17 pathway." No more revision is needed. ****** 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 #4: No https://doi.org/10.1371/journal.pone.0230450.r005
Formally Accepted
10 Mar 2020 Acceptance Letter - Yi-Hsien Hsieh, Editor PONE-D-19-32902R2 HNF-4α inhibits hepatocellular carcinoma cell proliferation through mir-122-adam17 pathway Dear Dr. He: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Yi-Hsien Hsieh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230450.r006

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