MicroRNAs as Regulators of Radiation-Induced Oxidative Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The introduction should clearly define oxidative stress and its sources in a more concise manner.

Can you provide more detailed mechanisms by which specific miRNAs influence particular pathways, possibly with clear diagrams to illustrate these interactions?

Ensure the manuscript includes the most recent studies from 2022 to 2024 to offer the latest insights into miRNA research related to radiation-induced oxidative stress.

Could you provide a more comprehensive discussion on how miRNAs can be targeted therapeutically, including potential delivery methods and any clinical trials?

Make sure the terminology, especially terms like ROS, RNS, and oxidative stress, is used consistently throughout the manuscript.

Could you expand on the experimental evidence supporting the roles of specific miRNAs, with more detailed descriptions of the study designs and results?

It would be beneficial to discuss any known instances where miRNA modulation did not yield the expected outcomes, to provide a balanced view.

Can you differentiate more clearly between the direct and indirect pathways of miRNA action on redox-sensitive genes?

Address potential side effects and challenges of targeting miRNAs therapeutically, including any off-target effects and delivery challenges.

Comments on the Quality of English Language

fine 

Author Response

Dear reviewer.

Thank you very much for your review and comments on the manuscript. We greatly appreciate your valuable time that you devoted to the assessment and improvement of our manuscript and therefore we will try to modify our manuscript according to your remarks and comments. We hope that this modified manuscript will meet your expectations

On behalf of the collective of authors of the manuscript,

Branislav Kura

 

Reviewer 1:

The introduction should clearly define oxidative stress and its sources in a more concise manner.

We have modified the introduction section according to reviewer´s comment.

Can you provide more detailed mechanisms by which specific miRNAs influence particular pathways, possibly with clear diagrams to illustrate these interactions?

In general, miRNAs exert their function through suppressing the expression of their target genes. It means that miRNA binds to mRNA of a target gene, causing repression of mRNA translation or mRNA degradation (as is illustrated it the newly added figure 1). Whenever the text mentions that specific miRNA directly targets a particular gene, it means this basic mechanism of their action. miRNA may also act indirectly; it means that miRNA targets mRNAs encoding proteins that control the level and activity of redox-sensitive genes (e. g. Keap 1 controlling the activity of Nrf2). In some cases, the exact mechanism was not clear from the article, e.g. only up-/downregulation of a gene was demonstrated and not direct binding to miRNA. Also in some cases, only abstract of the article was available, so we could not check if the information about the specific mechanism is present in the article or not. Considering these aspects, we added/complemented relevant information to relevant parts of our review where possible according to the reviewer´s recommendations.

Ensure the manuscript includes the most recent studies from 2022 to 2024 to offer the latest insights into miRNA research related to radiation-induced oxidative stress.

We checked the entire manuscript and where we managed to find newer works, we supplemented them as recommended. To the best of our knowledge, all relevant studies are already listed in our review.

Could you provide a more comprehensive discussion on how miRNAs can be targeted therapeutically, including potential delivery methods and any clinical trials?

This is a great comment. Based on this we have included the information about targeting miRNAs, delivery methods, and clinical trials in the new chapter named “miRNAs as potential therapeutics” on page 10.

Make sure the terminology, especially terms like ROS, RNS, and oxidative stress, is used consistently throughout the manuscript.

We carefully checked the manuscript for consistent terminology and corrected it where necessary.

Could you expand on the experimental evidence supporting the roles of specific miRNAs, with more detailed descriptions of the study designs and results?

More detailed description of studies was included where possible.

It would be beneficial to discuss any known instances where miRNA modulation did not yield the expected outcomes, to provide a balanced view.

This information was included in the new chapter “miRNAs as potential therapeutics” on page 10.

Can you differentiate more clearly between the direct and indirect pathways of miRNA action on redox-sensitive genes?

Differentiation between direct and indirect effects of miRNA action was reflected in the introduction as well as in chapters no. 3 (MiRNAs affect ROS production and elimination) and no. 4 (Evidence for miRNA action in radiation-induced oxidative stress). Previous figure no. 2 (now figure no. 3) was modified to distinguish direct and indirect miRNA effects for clearer understanding for the reader.

Address potential side effects and challenges of targeting miRNAs therapeutically, including any off-target effects and delivery challenges.

Challenges of targeting miRNAs were included in the new chapter “miRNAs as potential therapeutics” on page 10.

Reviewer 2 Report

Comments and Suggestions for Authors

The present review article summarizes involvement of miRNAs in a wide variety of gene regulation regarding ROS and antioxidation. The manuscript is well written and readable. This review is unique and novel, since the concept, miRNAs of ROS ant antioxidation, are significantly different from those of previously published reviews about the role of miRNA in various physiological and pathological functions. This review covers a wide range of important research fields such as molecular biology, chemical biology, and cell biology. I think that this interesting review article would certainly advance our understanding of the molecular biology of miRNA associated with ROS generation and antioxidation.  

 

I strongly recommend this great review to be published in the Journal. However, I raise minor concerns that need to be addressed before publication. If those concerns should be adequately addressed in the revised manuscript, this superb review would be further significantly strengthened.

 

[1]-1 I suggest the authors to make a list of abbreviations which would be before/after “1. Introduction” on the first page or after “5. Conclusions”.  For example, please define ROS, NOX, COX, RNS and others, if those words appear more than twice in the text. Especially, the list is essential for Review articles. Please consult with Assistant Editor about the list so as to follow the Journal policy.

 

-2 The legends for Figures 1 and 2 again define ROS, .OH, SOD et al. even they are defined in the main text previously. Legends of tables also define SOD, NOX and other words again, while the main text already define those words. Once the list of abbreviation is made, such duplication can be avoided.

 

-3 Line 33 describes …. reactive oxygen species (ROS)……. Line 82 describes …superoxide dismutase (SOD), catalase (CAT). Other abbreviations are defined in this way. But, lines 107 and 108 describes … SOD – superoxide dismutase, CAT – catalase. I found many other similar cases. Thus, description way is opposite. Please remedy this by unifying the order. 

 

-4 I was very impressed to see many references are new and were recently published in just last 5 years (after 2019) (46 references out of total 121references), demonstrating that this review describes really novel and cutting-edge research results and is worthy for publication. 

 

[2]-1 It is not clear how irradiation generates ROS and .OH.  Figure 1 does not clearly explain how nucleus be involved in the chemical reactions in accordance with sentences lines 92-120.  Lines 34-35, 122-131 mention that NOX, LOX, NOS, and COX of mitochondria generate ROS, but those words are not seen in Figure 1.  Please remedy those.

 

-2 Figure 2 describes that irradiation directly generates ROS. Please explain more in detail molecular mechanism of the effect of radiation. General readers would be confused.

 

[3] ROS is produced first by radiation and then ROS is deprived by antioxidant enzymes. Thus, the order of Tables 1 and 2 should be reversed, namely, ROS-producing enzymes are described first, then antioxidant enzymes are described next. Then, the main text should be revised accordingly. 

 

[4] Lines 48-60: miRNA, pri-miRNA and pre-miRNA are explained.  Could the authors make a new graphical figure which explicitly explains those?  This review emphasizes miRNA, thus from this new graphical figure, general readers can easily grasp the points of miRNA. 

 

[5] Lines 137-172: Nrf2, p53, NF-kB, NOX, MARK, SIRT and Keap1 are mentioned. Please cite Figure 2 here. Those proteins are described in Figure 2.

 

[6] miRNA-23a, 24, 122 are associated with p53 at lines 182-192. miRNA-506, 26a-5p are associated with NF-kB at lines 193-203.  miRNA-1991, 34a, 29b, 217 are associated with SIRT at lines 204-212. miRNA-146a, 30a-5p are associated with MAPK at lines 213-222. In addition, Nrf2 and FOXO are discussed at lines 259-278 and lines 314-328, respectively.  But those are not described in Tables 1 and 2. How come? Do the authors emphasize only enzymes, but not transcription factor proteins? But SIRT and MAPK are enzymes. 

 

[7] Lines 223-250, Section 4. Evidence ….:  This section is well written and readable. This is the point of this interesting review article. Could the authors make a new graphical figure (cartoon) which explicitly explain Section 4? 

 

[8] Table 2 mentions eNOS. But it don’t find eNOS anywhere in the main text. Am I wrong?

 

[9] Even if the first word sets in the sentence, small letter should be used for miRNA and p53. For example, miRNA at lines 137, 141 and p53 at lines 182, 304 should be remedied.

 

In summary, I strongly recommend this great review for publication, after concerns are adequately addressed in the revised manuscript. 

Author Response

Reviewer´s comments

Dear reviewer.

Thank you very much for your review and comments on the manuscript. We greatly appreciate your valuable time that you devoted to the assessment and improvement of our manuscript and therefore we will try to modify our manuscript according to your remarks and comments. We hope that this modified manuscript will meet your expectations.

On behalf of the collective of authors of the manuscript,

 

Branislav Kura

 

Reviewer 2:

The present review article summarizes involvement of miRNAs in a wide variety of gene regulation regarding ROS and antioxidation. The manuscript is well written and readable. This review is unique and novel, since the concept, miRNAs of ROS ant antioxidation, are significantly different from those of previously published reviews about the role of miRNA in various physiological and pathological functions. This review covers a wide range of important research fields such as molecular biology, chemical biology, and cell biology. I think that this interesting review article would certainly advance our understanding of the molecular biology of miRNA associated with ROS generation and antioxidation. 

I strongly recommend this great review to be published in the Journal. However, I raise minor concerns that need to be addressed before publication. If those concerns should be adequately addressed in the revised manuscript, this superb review would be further significantly strengthened.

[1]-1 I suggest the authors to make a list of abbreviations which would be before/after “1. Introduction” on the first page or after “5. Conclusions”.  For example, please define ROS, NOX, COX, RNS and others, if those words appear more than twice in the text. Especially, the list is essential for Review articles. Please consult with Assistant Editor about the list so as to follow the Journal policy.

This is a great comment. After the consultation with the Assistant Editor, we have included a list of abbreviations after the “Conflict of interest” part, on pages 11-13.

-2 The legends for Figures 1 and 2 again define ROS, .OH, SOD et al. even they are defined in the main text previously. Legends of tables also define SOD, NOX and other words again, while the main text already define those words. Once the list of abbreviation is made, such duplication can be avoided.

Thank you for this suggestion. After consultation with the journal editor, we have kept explanations of figure abbreviations in the relevant legends. The editor explained to us that explaining abbreviations used in images or tables, regardless of their previous introduction in the text, is stated in the journal policy.

-3 Line 33 describes …. reactive oxygen species (ROS)……. Line 82 describes …superoxide dismutase (SOD), catalase (CAT). Other abbreviations are defined in this way. But, lines 107 and 108 describes … SOD – superoxide dismutase, CAT – catalase. I found many other similar cases. Thus, description way is opposite. Please remedy this by unifying the order.

Based on this recommendation, we carefully checked the whole manuscript and unified the use of abbreviations across the whole manuscript.

 -4 I was very impressed to see many references are new and were recently published in just last 5 years (after 2019) (46 references out of total 121references), demonstrating that this review describes really novel and cutting-edge research results and is worthy for publication.

 

 [2]-1 It is not clear how irradiation generates ROS and .OH.  Figure 1 does not clearly explain how nucleus be involved in the chemical reactions in accordance with sentences lines 92-120.  Lines 34-35, 122-131 mention that NOX, LOX, NOS, and COX of mitochondria generate ROS, but those words are not seen in Figure 1.  Please remedy those.

In figure 1 (now in figure 2), ^.OH radicals are illustrated to be generated via radiolysis of water after irradiation. This is the main way of ROS generation after irradiation (as is also described in lines 105-107). Involvement of nucleus in the radiation damage is via direct damage to DNA, including crosslinks, single-strand, and double-strand breaks (as is illustrated in figure 1 (now figure 2) and described in lines 119-121). As the mitochondrial DNA might also be damaged after irradiation, we complemented this information into figure 1 (now figure 2) too. Information about upregulation of different ROS-producing enzymes (LOX, COX, NOX, NOS) after irradiation was included in figure 1 (now figure 2), according to reviewer´s recommendation.

-2 Figure 2 describes that irradiation directly generates ROS. Please explain more in detail molecular mechanism of the effect of radiation. General readers would be confused.

Generation of ROS after irradiation is already explained earlier in this review, concretely in chapter 2 (Radiation and oxidative stress) and in figure 1 (now in figure 2). To make the figure 2 (now figure 3) more clearer, generation of ROS via radiolysis of water and upregulation of specific ROS-producing enzymes was included.

 [3] ROS is produced first by radiation and then ROS is deprived by antioxidant enzymes. Thus, the order of Tables 1 and 2 should be reversed, namely, ROS-producing enzymes are described first, then antioxidant enzymes are described next. Then, the main text should be revised accordingly.

We have rearranged the tables and text, according to reviewer´s comment.

 [4] Lines 48-60: miRNA, pri-miRNA and pre-miRNA are explained.  Could the authors make a new graphical figure which explicitly explains those?  This review emphasizes miRNA, thus from this new graphical figure, general readers can easily grasp the points of miRNA.

New figure 1 was included in the review, describing schema of individual steps of miRNA biogenesis.

 [5] Lines 137-172: Nrf2, p53, NF-kB, NOX, MARK, SIRT and Keap1 are mentioned. Please cite Figure 2 here. Those proteins are described in Figure 2.

Proteins like Nrf2, p53, NF-kB, NOX, MARK, SIRT, and Keap1 are described in figure 2 (now in figure 3) in connection with their regulation by miRNAs after irradiation. However, lines 137-172 described regulation of these proteins by miRNAs under general oxidative stress conditions. Therefore, we consider it better to cite figure 2 (now figure 3) in chapter 4 (Evidence for miRNA action in radiation-induced oxidative stress), as this figure reflects the content of chapter 4.

 [6] miRNA-23a, 24, 122 are associated with p53 at lines 182-192. miRNA-506, 26a-5p are associated with NF-kB at lines 193-203.  miRNA-1991, 34a, 29b, 217 are associated with SIRT at lines 204-212. miRNA-146a, 30a-5p are associated with MAPK at lines 213-222. In addition, Nrf2 and FOXO are discussed at lines 259-278 and lines 314-328, respectively.  But those are not described in Tables 1 and 2. How come? Do the authors emphasize only enzymes, but not transcription factor proteins? But SIRT and MAPK are enzymes.

The information in tables 1 and 2 was not intended to summarize what was already described in the main text of the review. Some information was only included in the form of table and not described in the main text (like information on ROS-producing or ROS-eliminating enzymes). Other information was included in the text only. This is why the information from the text do not appear in tables. We did not want to duplicate the information, if not needed.

 [7] Lines 223-250, Section 4. Evidence ….:  This section is well written and readable. This is the point of this interesting review article. Could the authors make a new graphical figure (cartoon) which explicitly explain Section 4?

Figure 2 (now Figure 3) belongs to the mentioned chapter, in which we tried to illustrate as many mechanisms as possible during the action of oxidative stress caused by irradiation. Signaling pathways of oxidative stress, inflammation, and apoptosis are captured in the figure, and we have also added a distinction of miRNAs that act directly or indirectly on these signaling pathways, so we think it now appropriately reflects the main content of Chapter 4.

[8] Table 2 mentions eNOS. But it don’t find eNOS anywhere in the main text. Am I wrong?

Yes, as explained previously, information from tables was not included in the text and vice versa.

 [9] Even if the first word sets in the sentence, small letter should be used for miRNA and p53. For example, miRNA at lines 137, 141 and p53 at lines 182, 304 should be remedied.

We checked and corrected the whole manuscript according to reviewer´s recommendation.

In summary, I strongly recommend this great review for publication, after concerns are adequately addressed in the revised manuscript.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the revision.