The “LINC” between Δ40p53-miRNA Axis in the Regulation of Cellular Homeostasis

  1. Apala Pal
  2. Pritam Kumar Ghosh
  3. Saumitra Das

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Abstract

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  1. Version published to 10.1080/10985549.2023.2213147
  2. Version published to 10.1101/2022.04.28.489891v4 on bioRxiv
  3. Version published to 10.1101/2022.04.28.489891v3 on bioRxiv
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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The manuscript by Pal and Das has explored the regulation of LincRNAs by p53 and by the shorter p53 isoform 40p53. In particular, through a series of knockdown and overexpression studies (primarily in one cell line) they demonstrate that LINC00176 is regulated by 40p53 to a greater extent than full-length p53 and provide evidence that this is occurring at the transcriptional level as well as through direct modulation of the RNA stability. This study has provided novel insights into the role of ∆40p53 in regulating the lncRNA-miRNA axis and perhaps more importantly, demonstrates the functional differences between the full-length p53 and its smaller isoforms.

    Major Comments

    While the subject matter is extremely interesting and has provided new mechanistic insights into how the smaller forms of p53 modulate the functions of the full-length form, many of the results are overstated, have been performed in a single cell line and are not convincing in their current form.

    • It's completely unclear how the HCT 116 cell lines expressing only p53 or Δ40p53 were generated. It is stated that the expression of these isoforms is endogenous, yet there is not mention of siRNA to knock down either of the isoforms while maintaining high expression of the other isoform. I am a little confused by the approach used here to maintain endogenous expression of one or the other isoform specifically. Apologies if I have missed this.
    • Line 209: "which is analogous to WT p53 and Δ40p53 levels in cancer". Which studies are you referring to, most published studies seem to indicate that the opposite is true??
    • Line 211-213: "LINC00176 was positively correlated with p53 levels (Figure S1C, E, G). However, in tumor tissues, they were negatively correlated (Figure S1D, F, H)." This is completely overstated and untrue, needs to be reworded. The only correlation is in Supp Fig 1C. p53 is not significantly correlated with LINC00176 in any other figure (as demonstrated by your p-values and R values).
    • Line 225-26: "However, the fold change was highest in cells with Δ40p53 overexpression, suggesting that Δ40p53 might be a more important regulator than p53." Overstated - there is no statistically significant difference between p53 and Δ40p53.
    • Fig 1 doesn't make sense... If you transfect cells with siRNA to p53 (Fig 1E) wouldn't you expect an increase in LINC00176??
    • Western Blots are very cropped. Full length western blots should be provided.
    • Line 251-253- "In HCT116+/+ cells, LINC00176 was upregulated in the doxorubicin treated and glucose-deprived cells; however, in cells treated with thapsigargin, there was no significant change (Figure S2 A-F)." There is no increase in p53 expression in some of the figs shown in the supp figs- then are the levels really correlated with p53 expression??
    • "We observed a slight increase in the proportion of cells in G1 phase and a slight decrease in the proportion of cells in S phase with LINC00176 overexpression (Figure S5F)." This is not obvious at all- delete or reword.
    • "We found a decrease in the number of colonies after LINC00176 overexpression (Figure S5G) and an increase in the number of colonies after LINC00176 knockdown (Figure S5H)". Colony formation needs to be quantitated, otherwise, don't show it. The results are not obvious at all.
    • Almost all statistics reported in this manuscript need corrections for multiple comparisons. I am fairly certain if this is done, many of the comparisons will lose their significance.
    • All results should be validated in an additional cell line.
    • There is little insight given with respect to the literature regarding what the known functions of LINC00176 RNA are or with respect to the known functions of Δ40p53
    • Although Figure 5 starts to delve into the functional impacts of LINC00176, it doesn't really look at any particular function in enough detail for it to make a significant contribution to the results. For instance, the assessment of EMT genes in Figure 5 D-F doesn't really mean much if you haven't shown altered migration/invasion capacity. This needs to be demonstrated. There are no error bars on the proliferation rates (Fig 5G and H, Supp 5B-E). Cell cycle analysis- no stats and the changes look fairly minimal. Target genes such as p21 involved in p53 cell cycle function should be analysed. Colony formation assays need to be quantitated.
    • Figure 5 should be complemented by the examination of genes known to be involved in the p53 pathway or known to be regulated by Δ40p53. Additionally, Figure 4 should show if the expression of target genes regulated by the miRNAs is altered in these experiments.
    • In all figures (and in the methods section), it needs to be stated how many experiments and how many replicates the result is representing. Given the lack of error bars in 5G,H, I can only assume the experiment has been done once.

    Minor Comments

    • Cell cycle analysis can use much greater detail, both in the experimental and analysis details (page 7, 178-182). How were the results analysed? Gating examples should be shown in the supplementary data.
    • Line 104: siRNA directed to Δ40p53, in Figs 1/2. There are no details of the Δ40p53 siRNA used in these studies.
    • Line 124: "non-specific siRNA (Dharmacon) was used in the partial silencing of p53/Δ40p53 in the experiments as control. si" This doesn't make sense, the non-specific siRNA should not affect gene expression??
    • Line 219: "Given the distinctive functions of LINC00176 reported in the literature.......". Please qualify and provide appropriate references and examples.
    • I can't see the 14A construct in the materials and methods.
    • Fig 1, p-values need to be stated and keys to the ** need to be stated as well in the figure legend.
    • Line 233 "The levels of LINC00176 decreased in both cell lines after siRNA transfection (Figure 1E, G). However, we did not observe a significant decrease in LINC00176 in either cell line.....". Yet there is a star indicating significance in both figures?? Please correct this.
    • Western blots: It makes no sense to choose a housekeeping protein the same size as your protein of interest, as this can skew the results if your blot isn't properly stripped prior to re-probing and there may be some cross reactivity with your protein of interest if the same secondary is used for both that target and housekeeping. What was the rational for choosing B-actin?
    • Fig 2K- no mention of Bip or its relevance in the text.
    • Fig S3 Why is p53 MW so high (63kDa) compared to other figures throughout the manuscript.
    • A better description of the Actinomycin D experiments is needed. Why use this? What are you showing? It is not obvious to the reader and needs to be described.
    • The discussion is very poorly referenced overall and greater insight should be given with respect to the current literature and how this has advanced the field.
    • Software used to perform statistical analysis is not stated.
    • Figure 5 G and H needs error bars.
    • The English is poor throughout despite the statement that the manuscript has been checked by English language editors.

    Significance

    ∆40p53 is an important and often underappreciated isoform of p53. It's important regulatory functions are only beginning to be recognised and this study has provided some novel and exciting insights into the role of ∆40p53 in regulating the lncRNA-miRNA axis. However, overall, the experimental details are lacking and the results are overstated in several areas and require validation.

    This manuscript would particularly appeal to researchers in the p53 and lncRNA fields.

    My expertise is in the p53 field, cancer, cell and molecular biology.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors investigate the interaction between LINC00176 and the translational isoform Δ40p53, exploring how Δ40p53, separately from full-length p53, regulates the expression of LINC00176 and what effects the lncRNA ,ay have on miRNAs and on various cellular processes such as growth and proliferation. Although this is an interesting study, it lacks depth and it feels very preliminary. Even though the main focus of the study lies on the transcriptional regulation of LINC00176 by p53, this process still needs further investigation0 Moreover, the experimental design is messy which is reflected on the figures as well. There is also a lack of depth on the role of the lncRNA in the cells and in miRNA regulation.

    Major comments:

    1. Despite the title mentioning miRNAs, the results regarding miRNAs (when investigating LINC00176's mechanism of action) seem almost like they belong in another study and there isn't much expansion on these results beyond the fact that the miRNAs identified are potential targets of LINC00176. Additionally, there is no substantial background on miRNAs and the ceRNA theory in the introduction.
    2. The authors should show in a figure how the screen was done and what other lncRNAs were discovered
    3. The TCGA data in Figures S1A and S1B are contradictory regarding the lncRNA expression.
    4. Figure S1C-H do not show clear correlation and in some cases there is no statistical significance.
    5. On figure 1, the authors mention that the HCT116-/- express the Δ40p53 isoform. Is this based on the way that the cells were generated (e.g., only the first nucleotides deleted)? Or, is the Δ40p53 stably expressed after ectopic expression in the HCT116-/- cells? It should be clarified.
    6. An HCT116-/- cell line with no Δ40p53 should be used as a negative control in Figure 1.
    7. The results on Figure 1A and 1I are contradictory. Explain
    8. For Figures 1E and 1G, the authors state in the results section that the siRNA KD of either p53 isoform does not lead to a significant decrease in LINC00176, but there are significance markers (p < 0.05) on both graphs.
    9. The WB in Figure 2J should be repeated
    10. There is no description of Figure 2K in the text.
    11. Figure 3: A Luciferase assay with the lncRNA promoter should be conducted. The p53 response element should also be mutated to confirm direct regulation of p53 on the promoter. A schematic of the promoter with p53 response elements is required.
    12. Figure S3 is based on a prediction from an algorithm and is not validated. Is Δ40p53 directly binding to the lncRNA? EMSA assay with purified protein and in vitro transcribed RNA is required. There can be no claims for direct p53-RNA interactions otherwise. Δ40p53 may regulate stability indirectly, through a different regulator. Is the lncRNA expressed in the cytoplasm or in the nucleus?
    13. There is no ChIP assay description in the methods
    14. The potential miRNA targets of LINC00176 identified in Figure 4 have functions involved in the processes of apoptosis, senescence, and autophagy in addition to cell proliferation and cell cycle regulation. It would be nice if they had done phenotypic assays for these processes in Figures 5/S5 as well. How does LINC00176 affect those cellular processes if its potential targets are known to be involved in them?
    15. Figure 4: Are the miRNAs regulated by the lncRNA also regulated after p53 knockdown? Is lincRNA the mediator? A rescue experiment is required.
    16. Figure 4: Multiple siRNAs for the lncRNA are required to make sure that there is no off-target effect.
    17. Figure 5: Why is shRNA used instead of siRNA? In both cases, transient transfection is used.
    18. Figure 5G-H: show the average of 3 repeats and not each repeat separately.
    19. Figure S5A: shLINC00176 leads to lower Δ40p53. Why? The authors should discuss
    20. Figure S5F: How many repeats is the graph showing? There are no stats and no apparent change in cell cycle.
    21. Figure S5G should be quantified.
    22. There should be a greater focus on the role of the lncRNA in the phenotype of the cells. Is the lncRNA playing any role in the migration and invasion of the cells? A xenograft model would greatly strengthen the study.
    23. Is the p53 knockdown phenotype rescued after o/e of the lncRNA?

    Minor comments:

    1. There are few mistakes such as typos and structural mistakes throughout the text.
    2. The siRNA for Δ40p53 is not specific for this isoform. It should be mentioned in the main body of the text. 'Ns psi' should be changed to 'si Nsp' or 'Nsp siRNA'.
    3. How many replicates were conducted for each experiment?
    4. Figure 2F: How are the stats are compared?
    5. Figures 2 J and 2K should be flipped.
    6. The models in figures 3 and 4 don't really add much. Since there is a graphical abstract at the end to show an overview of the paper's findings, these earlier models don't seem necessary.
    7. The labeling of Fig 3D is distracting. The labels on both the x-axis and the legend for the same things are unnecessary. Also, the fold enrichment in 3D in the control samples seems mild, especially compared to the levels that it was in 3B.
    8. The nomenclature of the antibodies is very confusing. I would prefer to just call them p53 antibody and just specify in the methods what specific antibodies were used for each assay.

    Significance

    This is an interesting topic. It is important to decipher the role and regulation of lncRNAs in the p53 pathway so as to understand how it mediates its function. There are numerous similar studies focusing on the role of lncRNAs in cancer but not so many in the role of p53-regulated lncRNAs. The audience of the study is broad, targeting both p53 and cancer biology as well as RNA biology interest. My expertise lies on cancer biology and RNA biology

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    In this manuscript of title "The "LINC" in between 40p53-miRNA axis in the regulation of cellular processes" authors identify long noncoding RNA LINC00176 as a target of delta40p53 and also as an interactor of delta40p53 protein. Modulation of LINC00176 leads to altered levels of a panel of miRNAs and of some epithelial-mesenchymal markers. Moreover, LINC00176 negatively regulates proliferation/viability.

    Major comments:

    • Are the key conclusions convincing?

    Despite the findings presented in this study are novel and relevant with regard to p53 function in cancer cells, the study is very preliminary and opens different lines of investigation that remain incomplete. Some examples: Authors identify a panel of miRNAs that may interact with LINC00176 but do not provide any functional impact of these miRNAs on cancer cell functions. Authors show modulation of epithelial-mesenchymal markers but do not provide evidence for changes in cell behavior (motility for example) Authors show that LINC00176 impacts on D40p53 protein level, but no statistical significance of this result nor description of the mechanism are provided. Importantly, the study is basically carried out using HCT116 p53-/- cells for the majority of the experiments. It would be worth trying to prove the relevance of the identified axis in the context of a cancer type, by analyzing the expression of D40p53 protein by WB and, concomitantly, the level of LINC00176 expression on the same samples.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    I have indicated this below

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    I have indicated this below

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    I have indicated below some critical points in this regard

    There are several conclusions in the presented data that are not convincing, especially those from which the study stems. These are detailed below:

    Authors say that LINC00176 is expressed at lower level in tumor vs normal tissues in LUAD, LUSC and COAD (box plot supply fig 1b); however, plots from GEPIA presented in Suppl Fig 1A show exactly the opposite result. Moreover, box plots do not have asterisk, which is provided by GEPIA when results are significant, indicating that the presented differences in expression are not significant. I suggest to delete these results and revise the analysis which led to inconclusive contrasting results.

    Figure 1D: Please explain in the text why HCT116 p53-/- cells show highly expressed D40p53 to allow readers to easily follow the experiments.

    Suppl. Fig.1C-E-G: Authors say that in normal tissues, LINC00176 was positively correlated with p53 levels (line 212). Again, here only in LUAD the positive correlation is significant. They also say that "in tumor tissues they were negatively correlated" (line 213). This is discordant with data presented in Suppl. Fig.1D-F-H, where no negative correlation is present (negative corr. is indicated by minus sign preceding the correlation coefficient (R) in Pearson's / Spearman's analysis).

    "This observation suggests that LINC00176 may be positively regulated by WT p53 in normal conditions and negatively regulated by mutant p53 in tumor conditions" (line 214-215): this could be easily assessed as TP53 mutational status is publicly available in the TCGA datasets.

    Figure 1. It is not indicated whether the significance derives from three independent biological replicates. This should be addressed for all the experiments presented in the study.

    Figure 2I: Please indicate on the graph which comparison the asterisks refer to. Moreover, explain better in the text that induction caused by ER stress is not obtained in absence of D40p53.

    Figure 2K-J: Quality of the WB is poor. Moreover, as the b-actin seems down regulated by si-D40/Thaps the normalization over b-act is not so informative (numbers at the bottom of the panels).

    Figure 3: Enrichments of D40p53 in ChIP experiments are really small. Usually enrichments <2folds are not very reliable.

    Figure 4F-G: Please explain better in the text the pull-down results, describing which comparisons have been made to evaluate the results.

    Figure 5G-H: Please include significance for the viability assays

    Figure 5E: The finding that o/e of LINC00176 induces D40p53 is very interesting and it's worth reinforcing this by analyzing 3 biological replicates followed by quantification of WB results and statistical analysis. I strongly suggest to evaluate D40p53 protein in cells silenced for LINC00176 as well. Interaction D40p53/LINC00176 could be stabilizing on both sides. Evaluation of cells with modulated LINC00176 in presence/absence of cycloheximide would definitely prove this. Inclusion of evaluation of this aspect with regard also to p53 is encouraged.

    For all the presented experiments number of biological replicates evaluated should be indicated.

    Minor comments:

    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately? Yes
    • Are the text and figures clear and accurate? Yes, unless indicated in the suggestions above
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? I have already included this above

    Significance

    The study highlights that D40p53 protein, besides regulating microRNAs (as previously reported) is also involved in the control of lncRNAs. The findings provide an advancement in the understanding of D40p53 function in cancer cells.

    The study might be quite relevant for the scientific community (cancer/tumor suppressors) if evaluation of cancer samples will be included.

    My expertise falls in the p53/mutant-p53 and non-coding RNA fields so I think it is appropriate to evaluate this study.

  12. Version published to 10.1101/2022.04.28.489891v2 on bioRxiv
  13. Version published to 10.1101/2022.04.28.489891v1 on bioRxiv