Differential Modulation of miR-122 Transcription by TGFβ1/BMP6: Implications for Nonresolving Inflammation and Hepatocarcinogenesis

Round 1

Reviewer 1 Report

Dear Editor,

Thanks for your invitation. Manuscript titled " Differential Modulation of miR-122 Transcription by TGFβ1/BMP6: Implications for Nonresolving Inflammation and Hepatocarcinogenesis". This research sought to determine whether microRNAs might have a role in the development of hepatocarcinogenesis linked with persistent inflammation.

Minor corrections and explorations are needed to improve the quality of this review.

1.      The authors need to expand on the significance of microRNAs in the pathophysiology of chronic illnesses in the introduction.

2.      Some important publications in this area of study are missed in this manuscript. For example

A) miRNAs inspirations in hepatocellular carcinoma: detrimental and favorable aspects of key performers‏

B) The role of miRNAs in liver diseases: Potential therapeutic and clinical applications‏

3.      The authors need to revise the manuscripts for language and grammar checks.

4.      Italicize in vitro,  wherever they appear in the manuscript.

5.      I noticed That the word miR-122 was written in more than one way. For example

In abstract and Manusrcript: miR-122 and MIR122; In keywords: miRNA-122. Please standardize the way you write

6.      Please re-arrange the reference number. For example, Reference 53 follows Reference 13. Then 14, 15, 39, 37, ....

7.      The first image is a duplicate. Please remove the duplicate

8.      The authors created beautiful figures, but the figures in the manuscript are not clear enough. The original or high-definition figures should be provided

 

 

The authors need to revise the manuscripts for language and grammar checks.

Author Response

Reviewer 1

Dear Editor,

 

Thanks for your invitation. Manuscript titled " Differential Modulation of miR-122 Transcription by TGFβ1/BMP6: Implications for Nonresolving Inflammation and Hepatocarcinogenesis". This research sought to determine whether microRNAs might have a role in the development of hepatocarcinogenesis linked with persistent inflammation.

 

Minor corrections and explorations are needed to improve the quality of this review.

Response to reviewer:

We would like to thank the reviewer for taking the time to assess our manuscript. We appreciate the reviewer's valuable comments and suggestions, which undoubtedly helped us to improve the quality of our work. We have addressed each of the reviewer's suggestions and comments in detail below.

 

 

1. The authors need to expand on the significance of microRNAs in the pathophysiology of chronic illnesses in the introduction.

A: We would like to thank the reviewer for the valuable feedback. We appreciate your suggestion and agree that the significance of microRNAs in the pathophysiology of chronic illnesses should be elaborated in the introduction. To better contextualize our study a more comprehensive overview of the role of microRNAs in chronic liver diseases (and relevant references), have been included in the revised version of our manuscript.

 

 

2. Some important publications in this area of study are missed in this manuscript. For example

 

1. A) miRNAs inspirations in hepatocellular carcinoma: detrimental and favorable aspects of key performers‏

 

1. B) The role of miRNAs in liver diseases: Potential therapeutic and clinical applications‏

 

A: We thank the reviewer for the constructive comment and we understand his/her concern about the omission of some important publications in this area of study. The reviewer's comment caught our attention, and while we agree that the work of Doghish et al could provide supporting information and was included in our manuscript, however we feel that the work of El-Mahdy et al serves no purpose or adds important information to our work.

 

 

3. The authors need to revise the manuscripts for language and grammar checks.

 

A: We have carefully considered the reviewers' comments and acknowledge the need for revisions regarding language and grammar. In response to these valuable suggestions, we have thoroughly revised the manuscript to ensure a higher standard of writing. Throughout the document, we have diligently addressed issues related to language clarity and grammatical correctness. We are now confident that the revised manuscript now meets these objectives.

 

 

4. Italicize in vitro, wherever they appear in the manuscript.

A: We would like to thank the reviewer for spotting this typo, all instances of "in vitro" were appropriately italicized throughout the manuscript.

 

 

5. I noticed That the word miR-122 was written in more than one way. For example

In abstract and Manuscript: miR-122 and MIR122; In keywords: miRNA-122. Please standardize the way you write

A: We would like to thank the reviewer for his/her valuable advice. Indeed, ours was an attempt to facilitate the understanding of the manuscript, where "miR-122" was to indicate the mature functional form of this miRNA, while "MIR122" was to indicate the gene product. Now we realized that this wording might increase confusion, and only "miR-122" was used in the revision of the manuscript.

 

 

6. Please re-arrange the reference number. For example, Reference 53 follows Reference 13. Then 14, 15, 39, 37, ....

A: We thank the reviewer for identifying the problem with the inconsistent numbering of references. The reason is that in the uploaded version of our manuscript the material and method section was located after the discussion, but upon conversion of the text to the Cells format the material and method section was moved after the introduction. In revising the manuscript, we took care to follow a stricter order for the different parts of the manuscript and now the numbering of the references should be more consistent.

 

 

7. The first image is a duplicate. Please remove the duplicate

A: We would like to assure that the duplication of Figure 1 puzzled us as much as it puzzled the reviewer. We think that this was an error in converting the uploaded text into the Cells format, since in the uploaded manuscript Figure 1 and its legend appear only once. If and when our manuscript is accepted for publication, we will check for these kinds of errors in the final proof.

 

8. The authors created beautiful figures, but the figures in the manuscript are not clear enough. The original or high-definition figures should be provided

A: The authors appreciate the reviewer's feedback and his/her recognition of our work. We also agree with the reviewer that some figure axis names and numbering are indeed difficult to read. In the revised version of the manuscript, we have taken action to increase the font size and include figures with higher resolution in our revised manuscript.

Reviewer 2 Report

Overall, it is a valuable research that provides new insights into the regulatory role of miR-122 in the onset and progression of hepatocellular carcinoma. Through a variety of animal models and in vitro experiments, Paluschinski and their colleagues have found that miR-122 transcription is differentially regulated by TGFβ1 and BMP6, and the TGFβ1-mediated reduction of MiR-122 transcription may contribute to inflammation-associated hepatocarcinogenesis. The finding has important implications for understanding the role of miR-122 in inflammatory liver disease. Due to the integrative approaches, the study is rich in content, reliable in data and reasonable in conclusion. I have the following suggestions for the manuscript:

 

1. Abstract

“The results of this study strongly suggest that prolonged activation of signaling pathways, ...” here prolonged activation of what signaling pathways?

 

2. There appear to be repeated Fig. 1 and the corresponding figure legend in the manuscript

 

3. Page 7, 3.3 MIR122 transcription is differentially regulated by TGFβ1 and BMP6

“The observed alterations in miR-122 expression in the livers of Tmprss6-/- and Hjv-/- mice (Figure 2A), ...” Figure 2A here should be Figure 2B

 

4. Page 8, 6th line from the bottom

“Importantly, TGFβ1 effect on MIR122 was blocked in...” should be read as “Importantly, the effect of TGFβ1 on MIR122 was blocked in...”

 

5. Page 8, 8th line from the bottom

“Confirmation of Smad signaling attenuation in siRNA-transfected cells can be inferred through the significant reduction in hepcidin and Smad7 activation in response to TGFβ1 stimulation. ” However, the corresponding Figure 3B shows a decline in “Smad4”, which I think is a clerical error.

 

6. Page 12, 3.6. miR-122 is downregulated in the livers of LCMV infected and in CCl4 treated mice

“Furthermore, we provided evidence that inflammatory and fibrotic signals associated with CCl4 administration lead to significant downregulation of miR-122 in the absence of liver cirrhosis or cancer.” What about the corresponding graph?

 

7. As for figure, maybe I didn’t see the HD version. The horizontal and vertical names on some bar charts are not clear enough for publication, such as Fig. 1F, Fig. 5B, Fig. 6A, B, D, Fig. 8A, as well as the proposed mechanism diagram

 

8. The analyses of some indicators in this study are missing in the methodological part, such as histopathological detection (H&E, Sinus red staining, etc.), qPCR, ALT, AST, GLDH, Bilirubin, etc. In addition, what is the full spelling of GLDH?

 

9. Page 13 “which suggested that these genes are potentially involved in control-ling the G2/M transition of the mitotic cell cycle (Figure 6C) ” here “are” should be “were”

NA

Author Response

Reviewer 2

Overall, it is a valuable research that provides new insights into the regulatory role of miR-122 in the onset and progression of hepatocellular carcinoma. Through a variety of animal models and in vitro experiments, Paluschinski and their colleagues have found that miR-122 transcription is differentially regulated by TGFβ1 and BMP6, and the TGFβ1-mediated reduction of MiR-122 transcription may contribute to inflammation-associated hepatocarcinogenesis. The finding has important implications for understanding the role of miR-122 in inflammatory liver disease. Due to the integrative approaches, the study is rich in content, reliable in data and reasonable in conclusion. I have the following suggestions for the manuscript:

 

Response to reviewer:

We thank the reviewer for his/her valuable feedback on our research. We are pleased to know that the study has been well-received and found to be a valuable contribution to understanding the regulatory role of miR-122 in in inflammatory liver disease. Regarding the suggestions for the manuscript, we have carefully considered each of them and made the necessary revisions accordingly. Thank you once again for your insightful comments, which have undoubtedly improved the quality and clarity of our work.

 

1. Abstract

 

“The results of this study strongly suggest that prolonged activation of signaling pathways, ...” here prolonged activation of what signaling pathways?

 

A: We would like to thank the reviewer for spotting the incomplete sentence, the phares has now been completed as: “The results of this study strongly suggest that prolonged activation of the pro-inflammatory signaling pathways”

 

2. There appear to be repeated Fig. 1 and the corresponding figure legend in the manuscript

 

A: We would like to assure that the duplication of Figure 1 puzzled us as much as it puzzled the reviewer. We think that this was an error in converting the uploaded text into the Cells format, since in the original version of the uploaded manuscript Figure 1 and its legend appear only once. If and when our manuscript is accepted for publication, we will check for these kinds of errors in the final proof.

 

 

3. Page 7, 3.3 MIR122 transcription is differentially regulated by TGFβ1 and BMP6

 

“The observed alterations in miR-122 expression in the livers of Tmprss6-/- and Hjv-/- mice (Figure 2A), ...” Figure 2A here should be Figure 2B

 

A: We are grateful to the reviewer for identifying that the reference to Figure 2A was inaccurate, this has now been corrected in the revised manuscript.

 

 

4. Page 8, 6th line from the bottom

 

“Importantly, TGFβ1 effect on MIR122 was blocked in...” should be read as “Importantly, the effect of TGFβ1 on MIR122 was blocked in...”

 

A: We would like to thank the reviewer for identifying the incorrect sentence, which has now been corrected and changed in the revised manuscript.

 

 

5. Page 8, 8th line from the bottom

 

“Confirmation of Smad signaling attenuation in siRNA-transfected cells can be inferred through the significant reduction in hepcidin and Smad7 activation in response to TGFβ1 stimulation. ” However, the corresponding Figure 3B shows a decline in “Smad4”, which I think is a clerical error.

 

A: We would like to thank the reviewer for identifying this discrepancy between the text and the figure; in fact, "Smad7" was not analyzed in this context, and the revised text now reads, " Confirmation of Smad signaling attenuation in cells transfected with anti-Smad4 siRNA can be inferred through the inability of TGFβ1 to stimulate hepcidin expression (Figure 3B, middle panel)".

 

 

6. Page 12, 3.6. miR-122 is downregulated in the livers of LCMV infected and in CCl4 treated mice

 

“Furthermore, we provided evidence that inflammatory and fibrotic signals associated with CCl4 administration lead to significant downregulation of miR-122 in the absence of liver cirrhosis or cancer.” What about the corresponding graph?

 

A: We are grateful to the reviewer for identifying this confusing sentence. Indeed, the phrase ““Furthermore, we provided evidence that inflammatory and fibrotic signals associated with CCl4 administration lead to significant downregulation of miR-122 in the absence of liver cirrhosis or cancer”, was from a previous version of the manuscript that we failed to remove from the finalized version of our work. This phrase has now been removed.

 

 

7. As for figure, maybe I didn’t see the HD version. The horizontal and vertical names on some bar charts are not clear enough for publication, such as Fig. 1F, Fig. 5B, Fig. 6A, B, D, Fig. 8A, as well as the proposed mechanism diagram

 

A: We agree with the reviewer that some figure axis names and numbering are indeed difficult to read. In the revised version of the manuscript, we have taken action to increase the font size and include figures with higher resolution in our revised manuscript.

 

 

8. The analyses of some indicators in this study are missing in the methodological part, such as histopathological detection (H&E, Sinus red staining, etc.), qPCR, ALT, AST, GLDH, Bilirubin, etc. In addition, what is the full spelling of GLDH?

 

A: We are grateful to the reviewer for identifying the missing information in the materials and methods section. We have now included descriptions of histopathologic staining for H&E and Sirius red, as well as descriptions of qPCR analysis and measurements of ALT, AST, GLDH, and bilirubin. We have also revised the list of abbreviations for missing descriptions.

 

 

9. Page 13 “which suggested that these genes are potentially involved in control-ling the G2/M transition of the mitotic cell cycle (Figure 6C) ” here “are” should be “were”

 

A: The reviewer's comment was addressed. On page 13, "are" was corrected to "could be" to accurately reflect the findings.