Reviewer ’s report  
 
Title: The contribution of miR -122 to the innate immunity by regulating Toll -like receptor 4 in 
hepatoma cells  
 
Version: 2 Date: 22 Mar 2019  
Reviewer: Ahmed Mohamed Elsharkawy  
Reviewer's report:  
In this interesting study, Shi et al study the correlation betwee n miR -122 and TLR -4 in the 
context of HCC. They first show that miR -122 is down -regulated in HCC compared to the 
normal liver and that this is reasonably strongly correlated with the disease. They then go on to 
show an inverse regulation link between miR -122 and TLR -4 in 3 different cell lines using a 
variety of in vitro assays. This is related to a miR -122 binding sequence in the 3' -UTR of TLR -4 
which they show through mutation analysis and reporter assays.  
 
I have a number of points that I think should be addressed - 
1. What effect does miR -122 over -expression or inhibition have on the proliferation rates of the 
HCC cell lines? It should be relatively straight forwards to perform these experiments.  
 
2. The quality of English especially in the abstract should  be improved.  
 
3. The Western blot in figure 2C is not very clear and an alternative image should be provided.  
 
4. The authors have not explained why there is a need for addition of LPS in LO2 cells to see 
the effects of miR -122 on TLR4 whereas this is not n ecessary in HepG2 cells. What happens 
in LO2 cells to TLR4 levels with only miR -122 transfection without LPS?  
 
5. Why were experiments with AMO transfection not performed in LO2 cells as they were in 
HepG2 cells?  
 
6. In the experiments used in figure 5 it is  not clear to me why two different cell lines were used 
in panels A and B. Also protein expression should have been included - not only mRNA 
levels.  
  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the da ta shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what  aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain o r lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your r eply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ).  I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: The contribution of miR -122 to the innate immunity by regulating Toll -like receptor 4 in 
hepatoma cells  
 
Version: 2 Date: 22 Mar 2019  
Reviewer: Ahmed Mohamed Elsharkawy  
Reviewer's report:  
In this interesting study, Shi et al study the correlation betwee n miR -122 and TLR -4 in the 
context of HCC. They first show that miR -122 is down -regulated in HCC compared to the 
normal liver and that this is reasonably strongly correlated with the disease. They then go on to 
show an inverse regulation link between miR -122 and TLR -4 in 3 different cell lines using a 
variety of in vitro assays. This is related to a miR -122 binding sequence in the 3' -UTR of TLR -4 
which they show through mutation analysis and reporter assays.  
 
I have a number of points that I think should be addressed - 
1. What effect does miR -122 over -expression or inhibition have on the proliferation rates of the 
HCC cell lines? It should be relatively straight forwards to perform these experiments.  
 
2. The quality of English especially in the abstract should  be improved.  
 
3. The Western blot in figure 2C is not very clear and an alternative image should be provided.  
 
4. The authors have not explained why there is a need for addition of LPS in LO2 cells to see 
the effects of miR -122 on TLR4 whereas this is not n ecessary in HepG2 cells. What happens 
in LO2 cells to TLR4 levels with only miR -122 transfection without LPS?  
 
5. Why were experiments with AMO transfection not performed in LO2 cells as they were in 
HepG2 cells?  
 
6. In the experiments used in figure 5 it is  not clear to me why two different cell lines were used 
in panels A and B. Also protein expression should have been included - not only mRNA 
levels.  
  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the da ta shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what  aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain o r lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your r eply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ).  I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: The contribution of miR -122 to the innate immunity by regulating Toll -like receptor 4 in 
hepatoma cells  
 
Version: 2 Date: 22 Mar 2019  
Reviewer: Ahmed Mohamed Elsharkawy  
Reviewer's report:  
In this interesting study, Shi et al study the correlation betwee n miR -122 and TLR -4 in the 
context of HCC. They first show that miR -122 is down -regulated in HCC compared to the 
normal liver and that this is reasonably strongly correlated with the disease. They then go on to 
show an inverse regulation link between miR -122 and TLR -4 in 3 different cell lines using a 
variety of in vitro assays. This is related to a miR -122 binding sequence in the 3' -UTR of TLR -4 
which they show through mutation analysis and reporter assays.  
 
I have a number of points that I think should be addressed - 
1. What effect does miR -122 over -expression or inhibition have on the proliferation rates of the 
HCC cell lines? It should be relatively straight forwards to perform these experiments.  
 
2. The quality of English especially in the abstract should  be improved.  
 
3. The Western blot in figure 2C is not very clear and an alternative image should be provided.  
 
4. The authors have not explained why there is a need for addition of LPS in LO2 cells to see 
the effects of miR -122 on TLR4 whereas this is not n ecessary in HepG2 cells. What happens 
in LO2 cells to TLR4 levels with only miR -122 transfection without LPS?  
 
5. Why were experiments with AMO transfection not performed in LO2 cells as they were in 
HepG2 cells?  
 
6. In the experiments used in figure 5 it is  not clear to me why two different cell lines were used 
in panels A and B. Also protein expression should have been included - not only mRNA 
levels.  
  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the da ta shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what  aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain o r lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your r eply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ).  I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant