PONE-D-24-49209tsRNA-49-73-Glu-CTC: A Promising Serum Biomarker in Non-Small Cell Lung CancerPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 75: Sentence 'Research has demonstrated that exhibit' appears not clear, please clarify.

Line 77: 's' is omitted in stress

Abstract: Each sections i.e. Objectives, Methods....should start on a new line to enable readability

Line 106: The clause 'which included from' is better written as 'which were gotten from' in my opinion

Sample size: Sample size 3 patients and 3 controls was reported in Line 93. However, 32 patients and 20 controls were reported in line 106. Please clarify if the pair of 3 was used as pre-test, otherwise, sample sizes aren't coherent.

Also, formula for determining sample size was not stated. Could the figure had been arbitrarily arrived at? If yes, let it be stated as a limitation in the study.

Sampling technique used to select samples was not stated.

I consider the use of personal pronoun 'we' in Lines 178 and 293 inappropriate. A passive sentence could have been better.

Line 167: No need for 'date' after 'on', to avoid tautology. It should simply be 'on' or 'dated'

The statement 'Lung cancer is the leading cause of both incidence and mortality rates worldwide' seems to be over used, as it appears in Abstract, Introduction and Discussion.

Line 289: The active statement 'aims' is better written as 'aimed', once it is the discussion section.

Reviewer #2: The authors report their finding of using the tsRNA-49:73-Glu-CTC as a molecular diagnostic marker for the patients with non-small cell lung cancer (NSCLC) and this would have a significant role in the biological processes associated with NSCLC proliferation. In this study, they performed high-throughput sequencing analysis of serum samples from NSCLC patients and healthy individuals (3 for each) and identified 3,522 types of tsRNAs, while 1,014 tsRNAs were specifically expressed in the serum of NSCLC patients. Subsequently, out of the five most expressed tsRNAs, they focused on only 3 that were significantly upregulated in NSCLC patients compared to healthy individuals (P < 0.05) as follows: tsRNA-49:73- Glu-CTC, tsRNA-19:32-Lys-CTT, and tsRNA-18:32-Lys-CTT and eventually they selected the longer and more structurally stable tsRNA-49:73-Glu-CTC as the target for further investigation. To confirm, they conducted RT-qPCR analysis on the serum of 32 patients with non-small cell lung cancer (NSCLC) and 20 healthy controls and found that tsRNA-49:73- Glu-CTC was significantly upregulated in NSCLC patients. Finally, using normal lung epithelial cells (Beas-2b) and lung adenocarcinoma cells (A549), they found that tsRNA-49:73-Glu-CTC was significantly more expressed in A549 cells compared to Beas-2b cells, and using the tsRNA-49:73-Glu-CTC inhibitor decreased the cell proliferation using the CCK-8 proliferation kit and also the wound healing using the cell scratch assay.

This manuscript represents a high contribution to a better understanding of the importance of the tsRNA-49:73-Glu-CTC as a valid marker for NSCLC. This comprehensive finding is vital to provide a scientific foundation for the potential of effective early diagnostic methods for lung cancer, which improves the treatment outcomes. However, the following comments would improve the manuscript:

- The discussion and the introduction are rather similar and convey the same facts, so please rephrase the discussion section to emphasize the importance of the findings in relation to the literature and the controls.

L51: In the introduction, you should have mentioned the two types of lung cancer: small and non-small. Then, you ought to focus on the non-small varieties.

L54: You should mention the known diagnostic markers as well as the general pros and cons.

L62: Please add a transition sentence for clarity (such as in this study).

L98: Please explain what these acronyms mean, similar to what you did in L239, and then use the same abbreviation in the remaining sections as you did in L249.

L106: Why are there different numbers of samples? Using 32 non-small cell lung cancer samples and 20 healthy controls is not comparable, which would affect the significance of most of these results.

L122: Why 3 samples only for each?

L138: Please mention the name of the inhibitor, whether it is widely available, and any additional information you may have designed.

L77: stress>stres.

L6: In the title page, please remove the space before the comma.

Table S1:

- Why did you only include one patient with adenosquamous carcinoma and thirty-one with adenocarcinoma? This cannot be compared. Does this have an impact on tsRNA-49:73-Glu-CTC expression?

- Would the results be affected by the fact that lung cancer of the patients includedin the studty was at different stages according to the TNM staging system?

- Since you have already clarified what SD stands for, could you also clarify that the TNM is a formal method of describing the stage of lung cancer?

Reviewer #3: Dear authors,

Below are all of my comments.

English proofreading is highly recommended.

Typho spotted: line 77, please check thoroughly the whole manuscript again.

2.1: line 106-Methodology begin with this - What is the purpose of mentioning samples no. include 32 non-small cell lung cancer 107 (NSCLC) patients and 20 healthy controls from 20/09/2024 to 30/10/2024, but this manuscript start the results and discussion on the finding from three untreated NSCLC patients and three healthy only (line 122-123, 178-179). Description on the samples nombers with the objective not really clear here.

BEAS-2B and A549- add the purpose and justification of using these cell lines in the methodology section.

2.3:

No information on the target location for amplification using RT-qPCR. Primers detail? Protocol for this section no citation at all. Lack information here compare to other similar articles.

2.6 subtitle is not quite approriate although the assay use to see the function of wound healing. Replace with migration analysis or use directly Scracth Assay

Perhaps 2.4,2.5 and 2.6 can be combined in one section under functional analysis subtitle.

References of ROC curves analysis has to be citation in text (line 157-159).

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Reviewer #1: Yes:  Sunday Charles Adeyemo

Reviewer #2: No

Reviewer #3: No

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tsRNA-49-73-Glu-CTC: A Promising Serum Biomarker in Non-Small Cell Lung Cancer

PONE-D-24-49209R1

Dear Dr. Mu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Jun Hyeok Lim, M.D.

Academic Editor

PLOS ONE

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