Targeting Non-Coding RNAs for the Development of Novel Hepatocellular Carcinoma Therapeutic Approaches

Round 1

Reviewer 1 Report

This review presents the detailed contents about the non-coding RNAs in HCC.  The article was well written, and I have no other constructive suggestions. It can be accepted in its present form. It will greatly stimulate more discussion about the ongoing novel ncRNA-targeting therapeutics in a variety of cancers. Of course, it will be more convenient for readers to provide a table summarizing the current categories of ncRNA-targeting therapeutics for HCC. 

Author Response

Reviewer 1

This review presents the detailed contents about the non-coding RNAs in HCC.  The article was well written, and I have no other constructive suggestions. It can be accepted in its present form. It will greatly stimulate more discussion about the ongoing novel ncRNA-targeting therapeutics in a variety of cancers. Of course, it will be more convenient for readers to provide a table summarizing the current categories of ncRNA-targeting therapeutics for HCC.

We wish to thank the reviewer for the appreciation of our work.

We believe that a table with the therapeutics usable to target ncRNAs would not be too informative. Indeed, the only possibility is to use siRNAs against the oncogenic ncRNA or simply increase the intracellular level of the down-regulated anti-oncogenic ncRNAs. In the revised version of the manuscript, this concept has been made clear on page 7 lines 256-261.

Reviewer 2 Report

This review provides a comprehensive overview of the role of non-coding RNAs in hepatocellular carcinoma. Tanja Jesenko et al. have synthesized related studies of the past five years, and the literature is also very informative, and the regulatory network related ncRNAs are clearly described. There is no problem with the logic of the entire article, except that it mainly focuses on ncRNAs and their targeted genes against hepatocellular carcinoma, but both have therapeutic effects as mentioned in the title and are not directly expressed, and the associated delivery process is not well described. Some parts of the paper are a bit difficult to understand, and I have some concerns before considering it for publication.

1. When describing the available treatments for HCC on page 3, the section on immune checkpoint therapy should clearly state that first-line and second-line treatments are based on different stages of tumor progression and then list the differences in use of each.

2. The format of the table in the whole article should be Adjusted the content of the first line of the rest except Table 1 The table is not aligned with the table title.

3.In describing the potential of ncRNA-targeted therapy for HCC, most of the ncRNAs and target genes listed work by regulating cell growth, migration, and apoptosis, and you can integrate these ncRNAs into a single graph instead of individually list the mechanisms of regulatory map ncRNAs? Mapping reviews are very important, and maps of ncRNA mechanism of action alone do not give the whole picture. It should incorporate the ncRNAs mentioned in a single figure, otherwise it's too much for the reader to read, since you cite too many and don't show the picture directly.

4. It is possible to screen out lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA to make an interactive network diagram, showing the target non-coding RNA, so that the interaction between them can be better explained. While the article collates targeted gene therapy LOCI, it is time to describe how delivery is performed.

5.In the section on ncRNAs and drug resistance, I found many repetitions and considered in describing the targeting potential of miRNAs, lncRNAs and circRNAs.

Author Response

Reviewer 2

This review provides a comprehensive overview of the role of non-coding RNAs in hepatocellular carcinoma. Tanja Jesenko et al. have synthesized related studies of the past five years, and the literature is also very informative, and the regulatory network related ncRNAs are clearly described. There is no problem with the logic of the entire article, except that it mainly focuses on ncRNAs and their targeted genes against hepatocellular carcinoma, but both have therapeutic effects as mentioned in the title and are not directly expressed, and the associated delivery process is not well described. Some parts of the paper are a bit difficult to understand, and I have some concerns before considering it for publication.

 

 

1. When describing the available treatments for HCC on page 3, the section on immune checkpoint therapy should clearly state that first-line and second-line treatments are based on different stages of tumor progression and then list the differences in use of each.

Based on the referee comment, in the revised manuscript we have made clear this concept on page 3 lines 102-3. However, we prefer not to go into excessive details about the use of immune therapies in HCC, as this is not the focus of the review; despite this, interested readers can find details in ref 16.

 

2. The format of the table in the whole article should be Adjusted the content of the first line of the rest except Table 1 The table is not aligned with the table title.

We do not exactly understand what the referee is referring to: to prepare the table we have used the format provided by the journal without introducing any style modification.

 

3.In describing the potential of ncRNA-targeted therapy for HCC, most of the ncRNAs and target genes listed work by regulating cell growth, migration, and apoptosis, and you can integrate these ncRNAs into a single graph instead of individually list the mechanisms of regulatory map ncRNAs? Mapping reviews are very important, and maps of ncRNA mechanism of action alone do not give the whole picture. It should incorporate the ncRNAs mentioned in a single figure, otherwise it's too much for the reader to read, since you cite too many and don't show the picture directly.

We wish to thank the reviewer for rising this important aspect, which we recognize as very true. The problem is that the role of ncRNAs in HCC/LF is very complex and only partially understood. Thus, it is not possible to draw a general simplified picture of their mode of action and interconnection. However, with the aim to try to simplify the complex scenario for the readers, in the revised manuscript we have introduced some explanatory sentences on page 7 lines 246-256. In particular, we propose the subdivision of ncRNA into “oncogenic“ when they promote cancer or “tumor suppressor “when they prevent cancer development. In the first case, ncRNAs typically inhibit tumor suppressor molecules able to down-regulate cell proliferation/migration or to promote cell apoptosis. In the second case, they down regulate pro-proliferative/migratory molecules and/or inhibit anti-apoptotic molecules. To easily catch the oncogenic/tumor suppressor nature of the ncRNAs, in the revised manuscript, in each table reassuming the ncRNAs effects, we have added the oncogenic (O) or the tumor suppressor (Ts) role.

 

 

4a. It is possible to screen out lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA to make an interactive network diagram, showing the target non-coding RNA, so that the interaction between them can be better explained.

For the reasons reported in response to point three, it is not possible to draw a simple and clear interactive network diagram. This is mainly because the known interconnection among the various ncRNAs are very limited. The only known networks have been discussed in the conclusion section (page 28, lines 1189-1200). Thus, we believe that the presentation of the interconnection in form of tables is more understandable for the readers.

 

 

4b. While the article collates targeted gene therapy LOCI, it is time to describe how delivery is performed.

We are not sure about the meaning of the request of the referee. If he refers to the delivery approaches for anti ncRNA molecules, we have stated (page 3 section 2, lines 128-129) that this is not the topic of the review and that this aspect has been recently discussed by us and others elsewhere (see refs 18-23).

 

 

5.In the section on ncRNAs and drug resistance, I found many repetitions and considered in describing the targeting potential of miRNAs, lncRNAs and circRNAs.

With regard to the presence of ncRNAs in table 4 and in other tables, there is only one case (miR-23-a-3p) present also in table 1. Notably, however, the targets of miR-23-a-3p in table 1 (PCDH17) and table 4 (ACSL4) are different. Additionally, the miRNA targets reported in table 4 do not appear in the other tables, thus, any explanation about target function is unique and not repeated. Finally, no circRNAs are presented in table 4. The only repetition we see regards ref [122]/[125] were vimentin might be the miRNA target: in both cases the concept of EMT in HCC is discussed

Reviewer 3 Report

The present manuscript reviewed the biological significance of non-coding RNA (ncRNA) in hepatocellular carcinoma (HCC) and considered their possible availability as a therapeutic target of HCC. Many studies have identified the altered expressions of various ncRNAs in HCC cells and/or tissues and suggested the important roles of ncRNAs in HCC behavior. The reviewer considers the present manuscript is well-constructed and contains sufficient amount of information regarding the biological function of ncRNA in HCC. The reviewer would like to ask some queries to the authors as described below.

 

1.    The reviewer can understand the ncRNAs have various biological functions in HCC cells and contribute to the change of cancer behavior. On the other hand, the reviewer can insufficiently understand the effectiveness of ncRNAs as therapeutic targets. The authors described in the part of conclusion that the papers presented here invariably indicate that the possibility to target oncogenic ncRNAs is at the moment restricted to the use of molecules such as siRNAs that interact with ncRNAs via specific base pairing. Although the reviewer understands the authors’ description, the reviewer would request the evidence that investigate this possibility. Because the title of this review manuscript is ‘Targeting non-coding RNAs for the development of novel hepatocellular carcinoma therapeutic approaches’ (line 1178-1180 in main text), the reviewer hope to contain more specific information regarding the therapeutic effectiveness of ncRNA. If there is few evidence regarding the therapeutic effectiveness of ncRNA, the reviewer would request the authors to describe the authors’ consideration regarding the difference among ncRNA, mRNAs and proteins as therapeutic targets.

2.    There are several typographical and grammatical errors in the main text. So, the reviewer considers the proofreading should be performed again.

Author Response

Reviewer 3

The present manuscript reviewed the biological significance of non-coding RNA (ncRNA) in hepatocellular carcinoma (HCC) and considered their possible availability as a therapeutic target of HCC. Many studies have identified the altered expressions of various ncRNAs in HCC cells and/or tissues and suggested the important roles of ncRNAs in HCC behavior. The reviewer considers the present manuscript is well-constructed and contains sufficient amount of information regarding the biological function of ncRNA in HCC. The reviewer would like to ask some queries to the authors as described below.

 

We wish to thank the reviewer for the appreciation of our work.

 

 

1. The reviewer can understand the ncRNAs have various biological functions in HCC cells and contribute to the change of cancer behavior. On the other hand, the reviewer can insufficiently understand the effectiveness of ncRNAs as therapeutic targets. The authors described in the part of conclusion that the papers presented here invariably indicate that the possibility to target oncogenic ncRNAs is at the moment restricted to the use of molecules such as siRNAs that interact with ncRNAs via specific base pairing. Although the reviewer understands the authors’ description, the reviewer would request the evidence that investigate this possibility. Because the title of this review manuscript is ‘Targeting non-coding RNAs for the development of novel hepatocellular carcinoma therapeutic approaches’ (line 1178-1180 in main text), the reviewer hope to contain more specific information regarding the therapeutic effectiveness of ncRNA. If there is few evidence regarding the therapeutic effectiveness of ncRNA, the reviewer would request the authors to describe the authors’ consideration regarding the difference among ncRNA, mRNAs and proteins as therapeutic targets.

We thank the referee for rising this point. In the revised manuscript, we have inserted some sentences to make clear the therapeutic potential of ncRNAs silencing by siRNAs (page 7 lines 256-261). Additionally, this aspect comes clearly out from the many works presented in our review where the siRNA-silencing of oncogenic ncRNAs (“O” ncRNAs) results in the down regulation of  HCC/LF development in many vitro and in vivo models.

 

2. There are several typographical and grammatical errors in the main text. So, the reviewer considers the proofreading should be performed again.

We wish to thank the referee for rising this aspect. The revised manuscript has been proofread again.

Reviewer 4 Report

ibution to the state of the art in the treatment of hepatocellular carcinoma, a global health challenge. This work brings together the scientific knowledge underlying approaches that target molecules, non-coding RNAs, whose aberrant expression is seen in tumour cells, and the strategies that can be used in their treatment. The authors have carefully selected topics that illuminate new trends and opportunities for more advanced and effective HCC treatment, as well as scientific ambiguities that need further investigation.

 

I have only a few concerns.

 

Lines 131-138. While I understand the authors reviewing data from papers published in the last four years, this may have lost some valuable contributions published before that time. It would improve the report if you published a table of milestones highlighting the process of developing therapeutic advances based on non-coding RNAs, mainly microRNAs.

 

Fig. 2. change protein "inretaction" in the figure to "interaction".

Fig. 3. Add "s" to get "Translation..." in the figure.

Tables 2, 3, 4, 5. Check hyphenation in raw 2.

 

Author Response

Reviewer 4

ibution to the state of the art in the treatment of hepatocellular carcinoma, a global health challenge. This work brings together the scientific knowledge underlying approaches that target molecules, non-coding RNAs, whose aberrant expression is seen in tumour cells, and the strategies that can be used in their treatment. The authors have carefully selected topics that illuminate new trends and opportunities for more advanced and effective HCC treatment, as well as scientific ambiguities that need further investigation.

We wish to thank the reviewer for the appreciation of our work.

 

 

1) Lines 131-138. While I understand the authors reviewing data from papers published in the last four years, this may have lost some valuable contributions published before that time. It would improve the report if you published a table of milestones highlighting the process of developing therapeutic advances based on non-coding RNAs, mainly microRNAs.

According to the review request, in the revised manuscript we have inserted some sentences where the main milestones in the area of ncRNAs and HCC are reported (page 3 lines 140-145). We have also added two novel refs [24,25] where are described the main papers published in the ncRNAs/HHC field before 2020.

 

2) Fig. 2. change protein "inretaction" in the figure to "interaction".

In the revised manuscript, the mistake has been corrected.

 

3) Fig. 3. Add "s" to get "Translation..." in the figure.

In the revised manuscript, the mistake has been corrected.

 

4) Tables 2, 3, 4, 5. Check hyphenation in raw 2.

Check done