PONE-D-21-15478

Loss of miR-1469 expression mediates melanoma cell migration and invasion

PLOS ONE

Dear Dr. Carson,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Clarify the introduction section as suggested by both referees

Explain why you have employed hsa-miR-16 as control

Importantly: Explain the divergent fold-change values obtained by the two methods Nanostring and qPCR

provide more details on the statistics, as suggested by referee 2

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: DiVincenzo and colleagues proposed a research article aimed at elucidating the role of miR-1469 in cutaneous melanoma unveiling its role in tumor invasiveness and in the development of ulcerating neoplasms. For this purpose, the authors have performed different functional experiments evaluating miR-1496 expression, cell function alterations and protein expression. Overall, the manuscript is interesting, however, it should be described better and some parts should be improved. Below are reported some comments:

1) Please provide references supporting the following sentences: “Cutaneous melanoma can sometimes present with ulceration of the tumor surface. Tumor ulceration is defined as the full thickness loss of epidermis overlying the tumor to the level of the basement membrane, with evidence of a host inflammatory response and associated thinning, effacement, or reactive hyperplasia of the adjacent epidermis. Tumor ulceration has been correlated with both decreased disease-free and overall survival in melanoma patients with otherwise similar staging criteria. Therefore, recognition of gross or histologic evidence of ulceration in cutaneous melanoma tissue has been incorporated into melanoma staging to better stratify melanoma patients. While ulceration is a feature that provides prognostic information to clinicians, few studies have explored the molecular features that explain its poor prognosis. Therefore, further studies are needed to characterize the molecular pathways that drive the ulcerated phenotype.”. For this purpose, please see:

– PMID: 29532857

- PMID: 27123116

– PMID: 25220403

– PMID: 29461778

- PMID: 28350549

2) In the Introduction the authors state: “Reduced expression of miR-1469 was identified in our assessment of miR expression patterns in ulcerated melanomas when compared to non-ulcerated tumors.”. In the results section they describe again the results of miR-1469 obtained for ulcerated and non-ulcerated melanomas. Probably the authors referred to nanostring results as previous results obtained and qPCR results in the present study, however, they have to better clarify this in the Introduction section;

3) Please provide references for this statement: “miR-1469 has also demonstrated consistent downregulated expression in other tumor types;”;

4) Please check the grammar in the following sentence: “however, a role for miR-1469 in melanoma has not been previously been reported.[7-10]”;

5) In the subheading “Patient samples and RNA isolation from formalin fixed paraffin embedded tissue”, How was the RNA extracted?;

6) Why the authors used hsa-miR-16 as endogenous control? For cell or tissue samples usually RNA U6 is generally preferred together with the analysis of an exogenous control like cel-miR-39. Please, clarify this aspect;

7) Were the data shown in Figure 1A obtained in previous studies? Please, clarify;

8) How do the authors explain the difference between the fold change value observed by Nanostring vs that obtained by qPCR (1.34 vs 11.81)?

9) Check the grammar of the following sentence: “As miR-1469 dysregulation was found to be a feature associated ulcerated primary cutaneous melanoma and limited studies have explored its role in cancer,”;

10) All these sentences should be moved into the Discussion section: “As a member of the BCL2 family of proteins, MCL1 is well recognized for its role as an inhibitor of apoptosis. Additionally, the expression of MCL1 has also been shown to affect the migratory and invasive capacity of tumor cells.[12] Increased MCL1 expression has also been demonstrated to occur in melanoma.[15] Furthermore, a recent study identified MCL1 as a confirmed target gene of miR-1469 in the context of laryngeal cancer by dual luciferase reporter assay.[9]”;

11) In the discussion section, the authors state :”However, the present results also stand in contrast to a recent study of miR-1469 as a promoter of cellular invasion in pancreatic cancer cell lines via inhibition of metastasis suppressor gene NDRG1 with subsequent activation of NF-κB.[25]”. Please better describe the role of NF-κB in melanoma invasiveness mediated by other factors including OPN and MMPs. For this purpose, please see:

– PMID: 28075446

– PMID: 32392801

Reviewer #2: The authors investigated the role of miR-1469 in ulcerated melanoma and found that this particular marker, if expressed, reduces the migratory and invasive capacity of melanoma cells in vitro, possibly by targeting MCL-1, a marker involved in apoptosis. The workflow is well thought and described and I believe the manuscript will have an impact in the field considering the molecular features associated with ulcerated melanoma that contribute to a poor prognosis are insufficiently explored.

However, minor revisions should be made and I advise the authors to spellcheck their manuscript.

The introduction section is too superficial. More information about miR-1469 and its roles should be given from the literature.

The number of patient samples included in this study should be given and information about patient informed consent should be included.

Statistical analysis is insufficiently described. Please describe the software used for statistical analysis and the algorithms used. Also, please indicate the number of experimental replicates used for the statistical analysis.

Line 45-54: a large section of the introduction is missing the reference(s).

Line 56, 66, 263, 307, 324, 360, 384: please check grammar

Line 70: the full name of the MCL-1 marker is not mentioned at all, only abbreviated, please amend this

Line 98: “24 hours or more”, please be more specific with the time frame.

Line 179, 372: it should be C instead of B, please amend this.

Fig 1C: How do the Authors explain the huge variation in data (standard deviation) in fold change of miR-1469 expression?

Fig.2A: The Authors are suggested to maintain the same scale for all 3 graphics, seeing as the values are similar.

Fig.2B: the scale bar is missing. The images corresponding to miR scramble in CHL1 and A375 cells seem to be insufficiently washed.

Fig.3B: the scale bar is missing.

Lines 249-250, 390-391: please check grammar, repetition of “following transfection”

Figure 4. Representative images should be provided for the trypan blue staining assay.

Line 269: “minimum p-value = 0.153, 0.117, and for CHL1”, I believe there is a missing value, please correct this.

Fig.5C: I suggest the authors place the cell lines’ names on the right of the corresponding immunoblot for an easier understanding and a better visual impact.

In the Discussion section I suggest the authors switch the paragraphs (326-332) and (334-348) with one another, as it will better link these portions with the rest of the text.

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Reviewer #1: No

Reviewer #2: No

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Loss of miR-1469 expression mediates melanoma cell migration and invasion

PONE-D-21-15478R1

Dear Dr. Carson,

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Klaus Roemer

Academic Editor

PLOS ONE

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