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Thank for fully addressing all remaining comments and uploading the datasets. I now happy to accept this work.
[# PeerJ Staff Note - this decision was reviewed and approved by Sonia Oliveira, a PeerJ Section Editor covering this Section #]
The reviewers each make some valid points which you need to address in full and explain any changes in a rebuttal letter. In particular, point A of reviewer-2 and 3 of reviewer-3 will require further experimental work. I deem these to be essential.
Two other key issues must be addressed.
(1) Please include a clear statement in EVERY figure legend regarding the number of technical and biological replicates performed for every panel/graph/image etc. This is essential.
(2) You must then upload every biological replicate immunoblot, figures and datasets (e.g. for the qPCR data) for all experiments, clearly labelled, so that readers can look at the primary data themselves. This is essential. Simply showing one immunoblot in the supplemental files is insufficient - the experiment must have been replicated at least three times for validity, so show all data please.
No comment
1. The MiR-101-3p you choose requires more detailed supporting evidence. The 23rd reference you cited, which identifies 26 miRNAs were significantly different in PBMCs of TAO patients (14 miRNAs were down-regulated and 12 miRNAs were up-regulated). Why did you choose MiR-101-3p?
2. More experiments and markers are needed to verify the effect of MiR-101-3p on the proliferation of orbital fibroblasts.
In figure 3B, why are there two graphs of CCK8 results?
No additional comments
The authors present a nice overview of thyroid associated ophthalmopathy (TAO) pathology and the importance of orbital fibroblasts (OFs) in disease. The introduction is referenced properly, including showing previous studies highlighting the role of miRNAs in TAO and that miR-101-3p was previously identified as a differentially expressed miRNA in disease.
See General comments
The authors have accurately presented their findings.
This manuscript investigates the role of miR-101-3p in TAO OFs. The authors find that the miRNA is reduced in TAO OFs compared to non-TAO fibroblasts. The findings are interesting and show that pentraxin 3 (PTX3) levels are regulated by miR-101-3p levels. They also observe that increased PTX3 levels promote proliferation. The experimental design is clear and proper controls are done in most cases. While these studies are interesting, a few important points should be addressed.
A.) A key point in this manuscript is that miR-101-3p levels are low in TAO OFs and that low miR-101-3p allows for elevated PTX3 protein expression, and thus increased proliferation. However, PTX3 protein levels are not shown for control, non-TAO fibroblasts. It is important to determine if there are differences in PTX3 protein levels in the non-TAO vs. TAO fibroblasts. Do PTX3 protein levels correlate with miR-101-3p levels in fibroblasts?
B.) In the methods section lines 177-188, primer sequences for the mRNAs analyzed by RT-qPCR should be listed. Catalog numbers for the U6 and miR-101-3p qPCR assays should be included. U6 should be described as U6 snRNA.
C.) The catalog numbers of the antibodies for Pentraxin and actin should be listed.
D.) A positive control for apoptosis in the flow cytometry assay in figure 3E would be important to show the assay’s sensitivity.
E.) The abstract lists that the authors are measuring the mRNA level of miR-101-3p, however this is inaccurate, and the term “mRNA” for messenger RNA should not be used for a microRNA.
F.) Line 327- it should be clarified what is meant by genetic variation of miRNAs in TAO patients. It may be better to say changes in miRNA levels instead.
G.) There should be some discussion about the source(s) of tissues for this study. TAO tissue is from orbital decompression while non-TAO tissue is from upper lid or eyelid tissue blepharoplasty. These are distinct tissues and thus the tissue and resultant fibroblasts from non-TAO tissue may not be optimal. Obtaining orbital tissue from non-TAO patients is likely very rare, thus blepharoplasty lid is a more feasible control.
This is a well written paper and the study added new insights to the pathogenesis of TED, where the role of PTX and miR-101-3p in TED providing novel insights into the pathological mechanisms underlying TED development. The topic is important and interesting, but prior to acceptance for publication in this journal, the following minor points should be considered.
1. Recent consensus in the field has led to a shift in the name of the eye complications that manifest in autoimmune thyroid diseases. The consensus is that the disease should be referred to as thyroid eye disease (TED).
2. What was the cause that authors choose this miRNA? It need more than one reference in the introduction part.
3. CCK-8 assay not the best choice to evaluate proliferation or cell number, because the conversion of tetrazolium salt into formazane is depended on the metabolic activity of the cells. The author should use BrdU or EdU assay to examine proliferation of cultures in additon to Ki-67 immunofluorescence staining. The BrdU assay is a more sensitive and quantifiable method for examining proliferation than Ki-67 staining.
no comment
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