Therapeutic Potential of miR-4711-5p in Pancreatic Cancer: Antitumor Activity and Mechanistic Insights

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled 'Therapeutic Potential of miR-4711-5p in Pancreatic Cancer: Antitumor Activity and Mechanistic Insights' authored by Yokoyama et al. assesses the prospective antitumor efficacy of miR-4711-5p in pancreatic cancer cells. However, several significant concerns must be resolved prior to consideration for publication.

• The study relies only on two pancreatic cancer cell lines, with no representation of different molecular subtypes of pancreatic cancer.

 

• Although the authors reported antitumor activity in vitro, no in vivo cancer models were examined, thereby restricting the validity of the reported activity of miR-4711-5p.While the authors reported antitumor activity in vitro, no in vivo cancer model was investigated, which limits the validity of the miR-4711-5p-reported activity.

 

• The manuscript offers limited information concerning the formulation of the sCA delivery system utilized for miR-4711-5p. Detailed data regarding formulation, physicochemical characterization, stability, and biodistribution. This is a critical point for further assessments.

 

• Additional methodological details concerning the flow cytometry analysis, including biological replicates and statistical analysis, should be provided.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper of Yokoyama et al. put light on miR-4711-5p role in in pancreatic cancer progression, being yet another paper focusing on one miRNA in human cancer development. As such, it is interesting and contribute to general knowledge of miRNA role in human cancer. However, there are some aspects that must be imporved or clarified before the manuscript is published: 

1. The paper lacks novelty, as even the Authors claim that have shown the important role of mir-4711 in pancreatic cancer. The paper lacks clearly claruified purpose. For example, the addition of the safety pre-clinical assay in primate animal model is not clear. Apart from that, the paper seems to focus on only two cell lines being treated with miR-4711 mimics.
2. Where are the results of RNA sequencing which was mentioned in the methnods section?
3. The data presenttation need corrections - in case mean is shown, SEM must be added, not SD. Moreover, some plots and data does not suggest statistical significance stated on them, i.e. Fig. 2B-right; 3A-right/top) - the differences are simply too small to be significant, especially when there are only n=3 as stated below. Thus, I would reccomend to show each of the data point on all of the plots to support the claimed differences. 
4. FACS analysis must be impoved - example plots (Fig. 3A) are clearly un-compensated.
5. There are several abbreviations that must be clarified, such as  FOLFIRINOX, PARP, WST-1 assay, etc.
6. There are missing cictations, i.e. for ΔΔCt method.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “Therapeutic Potential of miR-4711-5p in Pancreatic Cancer: Antitumor Activity and Mechanistic Insights” they evaluate miR-4711-5p like a potencial therapy for pancreatic cancer, looking at both molecular effects and preclinical safety in non-human primates. The results shows that miR-4711-5p works in many ways: it supress the stemness in cancer cells, slows down proliferation and cell cycle, induce apoptosis and also reduce invasive ability. Still, i think the authors need clarify some points:

1. Which exact quantitative criterias was used to choose SUIT-2 and BxPC-3 over Panc-1?
2. Did they use normalized and replicated densitometry for KLF5 protein, and any load control beside actin/GAPDH?
3. Was the reduction of CSC markers measured by flow cytometry or just qPCR and western blot?
4. In the sphere assay, did they count number of spheres >50 μm or only the total area?
5. The G1-S blockage, was it check by cell synchronization or only BrdU, and any checkpoint or stress markers analysed to explain the cycle arrest?
6. In apoptosis, did they clearly separate early and late apoptosis to calculate total percentage?
7. In invasion assay, how was basal migration and initial cell density controlled to make sure results are reproducible?
8. Did they perform functional validation of MET, CTSA and ANO1 to confirm their supression really contributes to antitumor effect, like with rescue by overexpression?
9. The dose in primates, does it correspond to mg/kg and expected plasma levels in humans, and was any off-target gene expression checked in key organs for subclinical toxicity?
10. Has the stability of miR-4711-5p in plasma against nucleases been checked, and sCA compared with other non-viral vectors for delivery and safety?
11. I think it could be useful to add dose-response curves, functional validation of MET, CTSA and ANO1, molecular toxicity analysis in primates and checkpoint marker evaluation, to make the findings and the mechanism explanation stronger.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

-Figure 2 does not show what the reader is looking at. Panels A and B must be labeled (y-axis) as "Proliferation (OD)" and "BrdU incorporation (OD)", respectively.

-Provide a mechanistic link among miR-4711-5p and MET, CTSA, and ANO1 in the Discussion and speculate on a possible signaling pathway/axis.

-Table 1: Either use Pre- and Post-treatment or Before and After. Pre and After is unacceptable.

-Table 1 and Table 2 must show statistical analysis (P values) to make sense of the data. The authors must test their data for normality (most likely skewed) then apply appropriate paired tests (t or MU) to compare before and after measurements.

-In every figure legend, mention the number of experiments performed and how many technical replicates were run and present as n = x. Also mention the name of the statistical test used.

-Provide a justification for using monkeys instead of mice.

-Provide a justification of the very low number of biological replicates used (only two). Mention in the Discussion as a limitation and provide future directions.

-The Discussion leaves out numerous studies on miR-4711-5p which must be included.

-Reference every method.

-Mention the manufacturer of every instrument (e.g., spectral analyzer missing).

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have provided reasonable justification for their raised point. 

Reviewer 2 Report

Comments and Suggestions for Authors

I am satisfied with the revision.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of the manuscript "Therapeutic Potential of miR-4711-5p in Pancreatic Cancer: Antitumor Activity and Mechanistic Insights" have adequately addressed the suggested revisions. The manuscript shows a notable improvement in both clarity and scientific rigour, and I consider it suitable for publication in its current form on the journal’s platform.

Reviewer 4 Report

Comments and Suggestions for Authors

Comments adequately addressed.