Non-canonical miRNA-RNA base-pairing impedes tumor suppressor activity of miR-16

  1. Anaïs M Quéméner
  2. Laura Bachelot
  3. Marc Aubry
  4. Stéphane Avner
  5. Delphine Leclerc
  6. Gilles Salbert
  7. Florian Cabillic
  8. Didier Decaudin
  9. Bernard Mari
  10. Frédéric Mouriaux
  11. Marie-Dominique Galibert
  12. David Gilot

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Abstract

Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 yr. Despite the discovery of gene signatures, the molecular actors driving cancer aggressiveness are not fully understood, and UM is still associated with very poor overall survival (OS) at the metastatic stage. By defining the miR-16 interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels. Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes, such as cyclin D3 , is impaired. This non-canonical base-pairing can explain both the derepression of miR-16 targets and the promotion of oncogene expression observed in patients with poor OS in two cohorts. miR-16 activity, assessment using our RNA signature, discriminates the patient’s OS as effectively as current methods. To the best of our knowledge, this is the first time that a predictive signature has been composed of genes belonging to the same mechanism (miR-16) in UM. Altogether, our results strongly suggest that UM is a miR-16 disease.

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  1. Version published to 10.26508/lsa.202201643
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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    Uveal melanoma is the most common primary intraocular malignancy in adults. About 50% of patients develop metastases, being the liver the most common place for them. Despite over 50 years of study, little progress has been done in efficacious treatments. In this report, the authors aimed at a better understanding of the mechanistic drivers for cancer aggressiveness and poor overall survival at the metastatic stage. To accomplish this, the authors performed a series of elegant genomics and transcriptomics analyses and identified molecular aberrations in miR16, which has been previously associated with other malignancies. The authors demonstrated that high level of miR16 sponges inversely correlate with poor overall survival. As a reference and validation, they are using the TCGA data analysis. Lastly, the authors generated a signature for survival prediction based on 4 genes, which was confirmed using an independent study.

    The methodology was very elegant. The appropriate analyses were done.

    Significance

    This manuscript provides incremental knowledge to the field. In the last 5 years there are many manuscripts addressing different transcription factors or miRNA molecules and their role in different cancers. Uveal melanoma is an orphan disease with high unmet need. Prognostication is highly valuable, however; it is the treatment where we need the most attention.

    The authors did elegant studies to demonstrate the relevance of miR16. This is not part of the standard of care, but the prognostic tool of the selected 4 genes, could be very helpful. I wish they could have included a sentence on the impact in the field.

    The response to the following questions can make the manuscript more robust: Description of the TCGA - how many of the primary tumors had clinically detectable metastases? This is important as you are describing a potential companion diagnostic testing to predict OS. We need additional information on the UM cells, which can be found in literature. It is necessary for the audience to understand which of these cell lines come from patients that die from metastases, which ones had additional malignancies. There are UM cell lines - commercially available ? for which the primary and metastatic line were developed from the same patient. Those are very helpful, especially if one of the objectives is demonstrating poor OS due to metastatic disease. That was not clear from the manuscript. Levels of miR16 - What were the copy numbers in healthy patients? Do we need them relative to 501 Mel? or should we compare to a system that is not dysregulated? Why were the DROSHA KO be lung cancer cells? HCT116? Why choosing this cell line?

    Overall, I consider this a good manuscript and with some tweaking it can be better.

    Referees cross-commenting

    Based on the different reviews and the added note, we all agree the manuscript needs work and is not ready yet to be accepted. We all agreed that needs to be re-structured as we all mentioned about key missing pieces in the writing. It will be of help to the authors to go back to the guidelines to know the max words and extend their manuscript.

    The timing needed is might not be too relevant, as we all agreed it needs work.

    Thank you to my reviewers/colleagues as they pointed out things very comprehensively.

    Agreed with their comments.

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    Referee #2

    Evidence, reproducibility and clarity

    This is an extremely short report that identified a potentially new mechanism as to how miR-16 may be involved in uveal melanoma (UM). In this report the authors used previous data, that identified that miR-16 is involved in UM, to gain a more comprehensive understanding of the mechanism involved. miR-16 was selected as a candidate due to its location in chromosome 3, which UM patients of the have chromosome 3 monosomy. The group evaluated the transcriptome following miR-16 overexpression in a single cell line, and as others often find with miRNAs, there was a cohort of up- and down-regulated RNAs. Figure 1 is a summary of the workflow, indicating the number of up- and down-regulated RNAs with validations performed. In figure 2 the authors identified 2 cohorts based on the previous expression data, one of which defines a more at-risk group. While the date presented in Figures 1 and 2 is interesting overall, the conclusions are mostly drawn from a supplemental figure. The data in this manuscript needs to be revaluated and the most critical included in the main figures. If done soe, and rewritten appropriately this study could have a bigger impact. As is, this study would be of low to moderate interest. There are also some instances, early on, where conclusions are overstated. Overall the text is lacking in description to conduct a thorough review, the authors fail to provide an introduction to put the study in the context of the field, and they provide a one-sentence discussion. Some of these issues are defined below, but due to the lack of description of the studies, and overstatements made, this is not a thorough review.

    Major issues:

    The main text and Figure 1 inappropriately referred to upregulated RNAs as miR-16 sponges. At this point in the manuscript, these are nothing more that upregulated RNAs.

    The text is often vague and lacks discretion that is essential for the Reviewer to understand the study. Some sentences are fragments and are not clear. Some (but not all) examples of difficulties encountered while attempting the review are indicated below as well.

    Figure 1E legend. "in function of the experimental workflow detailed". "In function" does not make sense in this sentence. It is not clear what the authors are referring to. Similarly, "In function of their expression...." In function aging is inappropriate and the sentence is not clear. Also "MRE for miRNA response element". Perhaps the authors mean "MRE = miRNA response element." Also, the text "10arbouring" is included int this legend. In brief, Figure 1E is extremely difficult to evaluate with the poor text in the legend.

    Multiple figures have odd wording to indicate biological replicates. This needs to be clarified in better, complete sentences.

    For Figure S2, Fold induction is indicated as a %. This is not appropriate. It is either a fold change or a change in %, but not both.

    How is the experiment done in Figure S3A a "kinetic" experiment? There is no kinetic analysis here.

    Details of the critical biotinylating study are completely lacking. And since this is the most critical experiment that gets to the main point of the study, this should be well defined and part of the main figures in the manuscript. It should not be in the supplemental information. All of Figure S2 should be in the main part of the manuscript.

    All axis in Figure S2B are not labeled appropriately. Enrichment is used, however not all RNAs are enriched. Perhaps fold change would be a better and more accurate name. Those on the left side (in blue) are depleted, not enriched.

    For Figure S3C, the authors should change "blue ones" to "solid blue bars indicate". Same for S3E

    For Figure S3D, "logo" is inappropriate and not the correct term. This is a consensus sequence, not a logo.

    How did the authors make the conclusion that the upregulated RNAs are targets of miR-16 if they do not have a canonical miR-16 binding sites? They could easily be indirect RNAs that are elevated post miR-16 exposure. The authors do not validate that the cohort of RNAs upregulated are indeed miR-16 targets. Thus, the overstatement of "sponge" RNAs (Figure 1H) or even "target" RNAs (Figure 1F) without appropriate validation is overstated. Simply doing the biotinylating study is still not enough to conclude direct interactions of these RNAs with miR-16. These can be false positives, that are not well controlled for due to poor selection of a control RNA.

    Figure 1G is not large enough to see, nor is the inclusion of it clear in the text of the manuscript.

    Figure 1H, referring to upregulated RNAs, post miR-16 expression as sponges is inappropriate unless they have all been validated as miR-16 sponges. These could merely be RNAs that are indirectly upregulated following miR-16 transfection, and their upregulating following miR-16 overexpression has been validated. However their miR-16 sponging activity has not been validated. Similarly for Figure 2A.

    Figure 2 is poorly defined. This needs clarification and the font should be increased. There are also "..." in the figure legend which is inappropriate. Many things are not defined such as "CN" which the reviewer is assuming means copy number. Also the colors and description for "Yes/Dead/Male" are not clear. What are these? How are they relevant?

    For Figure 2B legend, what is meant by miR-16 "in function"?

    The authors should show the level of upregulation of miR-16 following transfection for all experiments where miR-16 is transfected.

    For all figures where qRT-PCR was conducted, what are RNAs normalized to. This should be indicated on the axis and/or in the figure legend. While in the methods section, this should also be present in the main body of text (ie. figures).

    For Sup Figure 2A the authors indicate that RNA levels were compared to 501Mel. They should show the 501mel levels in the same graph. They also state that the absolutely copy number was determined from Norther Blot. As the authors likely know, quantification using qRT-PCR is much more quantitative than Northern. They should conduct qRT-PCR for the main cell line they are comparing to. The Northern is also not shown and the reference provided for it is for a Nature Review article, not for a study that shows a Northern blot.

    It is not clear what the control RNA was for all the studies. Specifically for the biotinylated studies, the authors should use another miRNA, not a non-specific control. Because a control miRNA will also binds AGO and other miRNA-associated factors, non-specific binding due to these factors could be better controlled for. The non-specific RNA will not account for these factors.

    Sup Fig 4 is missing details. What orientation is the consensus sequence shown in relative to the miRNA (5'-3' or 3'-5')? Other details are missing as well, this is just one example of many issues.

    For Sup Fig 5A the CT values should be included. That gives a better direct comparison than a graph of something that is indicated as not determined. You cannot graph something that is not determined.

    For Sup Fig 5C the font cannot be read it is too small.

    For Sup Fig 5D, again, how much miR-16 is present when overexpressed. Would this amount be physiologically achievable?

    The title is poor and not descriptive enough for the study. It reads more like the title for a review article.

    Methods for siRNAs indicated kinetic as well. Not clear what kinetic data were acquired during this study.

    Significance

    Referees cross-commenting

    I am in full agreement with the additional comments made by Reviewer #1. I however disagree with Reviewer #3 that the study was well conducted and "elegant". Based on multiple issues (many cooperated) between R#1 and R#2 I do not feel that this study is acceptable and will take an extraordinary amount of time to be acceptable for publication.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The article presents interesting data regarding the role of miR-16 in the development and progression of uveal melanoma. The authors propose the analysis of miR-16 activity as a marker for uveal melanoma progression instead of miR-16 expression. To analyze the activity of miR-16 they propose a risk model developed around a 4 genes signature that was shown to be able to stratify uveal melanoma patients into low and high-risk groups. Unfortunately, the current version of the manuscript is hard to understand and to carefully evaluate due to a lack of structure and clear presentation of the experimental design, methods, results, and discussions.

    Major Comments:

    • The manuscript needs to be restructured into clear sections including a detailed introduction, materials and methods, results, discussions, and conclusion. The present format is hard to follow with information and results being spread across multiple documents and parts of the manuscript.
    • The authors mention that the level of miR-16 reached after transfection is higher than that in the physio-pathological level, but there is no data to support this. I recommend adding a quantification of miR-16 levels achieved after transfection as part of the S2.
    • The way of using references is confusing as it is not clear what was done in the results section and what work is cited from the literature. The results section should focus strictly on presenting the results achieved by the experiments while delineating clearly the work that was done in other articles.
    • The discussion section needs to be extended to better present the role of specific investigated genes and proteins like PYGB and PTP4A3 in the development and progression of uveal melanoma.
    • The experiments analyzing the sequestration of miR-16 at non-canonical sites are performed using the cell line HCT116 WT and DROSHA KO. HCT116 is a human colon adenocarcinoma cell line, a tumor with a completely different histology. These experiments should be performed also on a human uveal melanoma cell line in order to ensure consistency of the results.

    Minor Comments:

    • The manuscript is lacking consistency regarding the usage of abbreviations. These should be defined the first time when used in the text.
    • The figure S2 needs to be adjusted. It is hard to understand in S2B how the statistical analysis was performed. I recommend representing each line with the two conditions side by side to increase clarity.
    • When presenting the miR-16 interactome the data are spread in three different sources Figures S2, 1D-E, and Table S1 which makes it difficult to follow the images. I recommend presenting these data in the same figure.
    • The manuscript requires English grammar and style editing. There are several words misspelled and phrases with a complicated syntax that makes it difficult to understand.
    • The method of presenting the data in Figures 1 F and H needs to be reorganized. The current version makes it very hard to understand how the gene expression changed after miR-16 exposure.

    Significance

    The article presents important results regarding the role of miR-16 in uveal melanoma by an innovative approach analyzing the activity of miR-16 instead of its expression. The authors focus on the relationship between miR-16 sponges and targets and through a set of elegantly designed experiments they identify a set of 4 genes whose expression can be used as a risk predictor model in uveal melanoma. The audience of this article can be represented by both clinicians and researchers that could take advantage of the results presented. Also, the approach of the article can open new directions for other cancers and miRNAs.

    Referees cross-commenting

    Would go with a revision of the manuscript according to the comments

  6. Version published to 10.1101/2021.11.30.470499v1 on bioRxiv