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Dear authors,
Thank you for submitting the modified version of the manuscript. The Reviewer agrees with the alterations introduced.
Congratulations on the acceptance of your work!
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
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Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. The author added to the experiment after his own discovery of the error. Through analysis, I agree with the author to replace with new experimental data, and believe that the conclusion of this study is still well-documented.
Dear authors,
The modifications you intend to do in the figures must be assessed by the reviewers and have an extra reviewer round.
Please submit a new version of the Ms and figures clearly indicating the modifications done in the figures and justifying the changes.
Thank you
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Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. The paper is improved and most concerned raised by the reviewer have been addressed.
Dear Authors,
Thank you for the improvements done in the manuscript. However, it will need additional modifications according to reviewer 2's criticisms.
Please fully address the important concerns raised by the Reviewer
Improved by revisions.
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Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. Although the author answered all of the questions, I still have some questions about the responses, which need further answers from the authors.
Major concerns:
1. “Rebuttal letter – Major Q1” Figure 3E to H. In the two experiments of Ctrl and CDS, CDS and CDS+ Mir-875-5p, are the processing factors of the CDS group the same? In Figure 3F, the clone number of CDS group is about 300. In Figure 3H, the clone formation number of CDS group is about 200. Why is there such a big difference in the number of clones generated in the same experimental treatment? Is there a statistical difference? Is it repeatable? The author must answer this question carefully, or it may give the reader the impression that experimental results in this study are very unstable.
2. “Rebuttal letter – Major Q2” Although the author makes two group comparisons, it is not possible to use the t-test. In the same repeated experiment, if more than two groups (multi-group) are grouped, ANOVA test and post test (eg. Tukey test) should be used for two groups comparison. The author should re-analyze the relevant data according to the statistics tutorial.
Dear Authors,
As you will see, the reviewers identified major concerns in your work that need to be addressed before consideration of publication. Some of the concerns will imply additional experiments and analysis. Some of the flaws identified by the reviewers must be convincingly addressed in order to be able to consider your work further.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
[# PeerJ Staff Note: It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors are in agreement that they are relevant and useful #]
Basic reporting
The paper assesses the tumor-promoting function of miR-875-5p via down-regulation of CAPZA1 in esophageal squamous cell carcinoma
It addresses a current and relevant topic since there is a growing interest in miR-875-5p in several malignancies.
The manuscript is quite well written and organized.
Figures and tables are comprehensive and clear.
The introduction explains in a clear and coherent manner the background of this study.
Overall, the discussion includes articles of historical importance in this setting. Nevertheless, we recommend the authors to include results and references related to the following recent papers, in order to enrich the discussion:
• Essadi I, Lalya I, Mansouri H. Esophageal carcinoma. N Engl J Med. 2015 Apr 9;372(15):1470-1. doi: 10.1056/NEJMc1500692. PMID: 25853758
• Rizzo A, Mollica V, Ricci AD, Maggio I, Massucci M, Rojas Limpe FL, Fabio FD, Ardizzoni A. Third- and later-line treatment in advanced or metastatic gastric cancer: a systematic review and meta-analysis. Future Oncol. 2020 Jan;16(2):4409-4418. doi: 10.2217/fon-2019-0429.
General comments
We believe this article is suitable for publication in the journal although minor revisions are needed.
The main strengths of this paper are that it addresses an interesting and timely question and provides a clear answer, with some limitations.
We suggest linguistic revision.
Please have a look at Minor concerns: 1 to 4.
Please have a look at Major concerns: 1 to 7.
No comment.
Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. Although the current study is interesting, there are some major concerns that need to be addressed before consideration of publication.
Major concerns:
1. In general, the authors found that the SNP of CAPZA1 gene 3’URT (rs373245753 T or G allele) could influence the interaction of miR-875-5p and thus have a surprising effect on the malignant phenotype of esophageal cancer. This is a very interesting result, but a key positive control is missing. I strongly recommend the authors to add the CAPZA1-CDS overexpression group as a positive control to observe whether the overexpression of CAPZA1-CDS can directly inhibit the malignant phenotype of esophageal cancer and is not reversed by the minic of miR-875-5p.
2. “Materials & Methods – Statistical analysis” Line 141-145. “Data differences were analyzed by Student’s t-test (two-tailed).” Some of the experiments (eg. Figure 1B) in this study were multi-group comparisons, and t-test was not applicable. The author should choose an appropriate statistical method to analyze the data again and explain it in detail.
3. “Results – miR-875-5p promoted cell proliferation and metastasis of ESCC cells” Line 152-154. “According to the TCGA database, we found that miR-875-5p was amplified 6% and 5% in EAC and ESCC, respectively, while approximately 1% were deleted in EAC and ESCC (Figure 1A).” In the TCGA database, the CNV information only gives the data of Level3, and the CNV of special gene cannot be directly obtained, so further analysis software is needed. The author should present the analytical method or the source of CNV of MIR875. In addition, miR-875-5p is the mature body of miRNA, which is an RNA-level concept, without the presence of CNV, so the authors should present CNV of MIR875 gene.
4. “Results – miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753” Line 169-172. “…employed a pair of PCR primers based on…” The author should give the sequence information of primers and probes in the method.
5. “Results – miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753” Line 179-181 and Figure 2E. “As shown in Figure 2E, for the pGL3-CAPZA1(T) construct, the miR-875-5p mimic significantly reduced luciferase activity compared with miR-NC…” In Figure 2E, the vertical axis is shown as the relative luciferase activity, so what is the reference for this experiment? The author should be shown on the chart and stated in the results.
6. “Results – CAPZA1(T) was negatively correlated with the proliferation of ESCC cells, while CAPZA1(G) attenuated such a negative effect” Line 188-190. “Cells were stably overexpressed with CAPZA1(T) and CAPZA1(G) by lentiviral infection…” What structures of the CAPZA1 gene are the authors overexpressing here? CZPZA1-5’UTR-CDS-3UTR? CZPZA1-CDS-3URT? Or CZPZA1-3URT? Please explain clearly in the corresponding position.
7. “Results – CAPZA1(T) was negatively correlated with the proliferation of ESCC cells, while CAPZA1(G) attenuated such a negative effect” Line 185-197. If CAPZA1 is a tumor suppressor gene, it is reasonable that its high expression has an effect on the malignant phenotype of esophageal cancer, but why does overexpression of CAPZA1(G) promote the growth of esophageal cancer cells instead (Figure 3A)? Therefore, I think it is necessary to overexpress CZPZA1-CDS alone to observe its effect on esophageal cancer cells.
Minor comments:
1. “Abstract” Line 22-33. In the abstract, the methods and results of this study are too simple. Please modify the abstract according to the requirements of the journal of PeerJ.
2. “Materials & Methods – Stable and transient transfection” Line 90-91. “Stable recombinant plasmids over-expressing CAPZA1(T) and CAPZA1(G) by lentiviral infection and matched control vectors were constructed by Obio Technology Co. Ltd…” The number or name of lentiviral vectos should be presented.
3. “Results – miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753” Line 166-167. “We used public databases and predicted that miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753 (Figure 2A)…” The author should introduce the predictive software which predicted the binding of miRNA and mRNA 3’UTR used in the method.
4. Figure 2B. The schematic diagram which author given is mRNA of CAPZA1, but "113162075", "113213865" and "113214241" are the physical locations of genomic DNA. Please do not mix them.
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I am afraid to say that, even if the experiments seem well carried out, this article does not rest on any foundation.
1 / Even if the gene is amplified, the authors must prove that this amplification is associated with increased expression, which has never been described in patients. In my opinion, the amlification is not specific and we find an entire region (including miRNAs) amplified, so the effect of miRNAs is an effect among hundreds / thousands of genes.
2 / miRNA is not expressed in any CCLE cell line "upper_aerodigestive" or "esophagus" or "head and neck".
3 / The rs373245753 which is described in the article is only found on pubmed but does not contain any frequency so it has never been described as real in the 1000 genome project.
So I cannot accept this article which for me is only a series of experiences based on artefactual hypotheses.
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