The stress-induced lincRNA JUNI is a critical factor for cancer cell survival whose interactome is a prognostic signature in clear cell renal cell carcinoma

  1. Vikash Kumar
  2. Xavier Sabaté-Cadenas
  3. Isha Soni
  4. Esther Stern
  5. Carine Vias
  6. Doron Ginsberg
  7. Carlos Romá-Mateo
  8. Rafael Pulido
  9. Martin Dodel
  10. Faraz K. Mardakheh
  11. Iddo Z. Ben-Dov
  12. Alena Shkumatava
  13. Eitan Shaulian

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Abstract

Cancer cells rely on adaptive mechanisms to survive the multiple stressors they encounter, including replication stress, toxic metabolic products and exposure to genotoxic drugs. Understanding the factors involved in these stress responses is crucial for developing effective treatments. Here, we describe a previously unstudied long non-coding RNA (lncRNA), JUNI ( JUN-DT , LINC01135 ), which is regulated by MAPK and responsive to stress. JUNI positively regulates the expression of its neighboring gene JUN , a key transducer of signals that regulate multiple transcriptional outputs. Our findings reveal that silencing JUNI sensitizes cancer cells to chemotherapeutic drugs or UV radiation, and that its prolonged silencing leads to cell death regardless of stress exposure, highlighting the pro-survival importance of JUNI. We identified 57 proteins that interact with JUNI and found that the activity of one of them, the MAPK phosphatase and inhibitor DUSP14, is inhibited by JUNI . This effect results in c-Jun induction following exposure of cancer cells to UV radiation and promotes cellular survival. Although JUNI regulates c-Jun and its downstream targets, the pro-survival effects in cells not exposed to stress are only partially dependent on c-Jun regulation.

JUNI expression levels significantly correlate with patients survival across 11 different types of cancer. Interestingly, the correlation of DUSP14 expression levels with patients survival in nine of these tumors is coherently inverse, indicating contradicting effects that are relevant not only for c-Jun induction and cellular survival but also in human cancer. Notably, we observed particularly significant antagonistic correlations in clear cell renal cell carcinoma (ccRCC) (p=5.7E-05 for JUNI and p=2.9E- 05 for Dusp14). In fact, the expression levels of 76% of JUNI -interacting proteins predict the prognosis of ccRCC patients significantly. Furthermore, a combined hazard ratio calculation demonstrates that this gene combination serves as a highly specific prognostic signature for ccRCC. Overall, our findings reveal a new important factor in stress signaling and cellular survival that is involved in ccRCC.

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    Reply to the reviewers

    Reviewer #1:

    Comment: The author investigated the role of the stress sensor pathway in the mechanism of tumor cell survival
    They identified a long noncoding RNA as JUNI that regulates antagonizing MAP phosphatase and favors the JUN transcription. JUNI correlated with the survival of several cancer histotypes, particularly in RCC, as a highly specific and correlated prognosis.

    The abstract although not always required from the journal should be divided into methods used to reach the main findings and clear presentation of results

    Response: We do not know yet to which Journal the paper will be sent. The format will be adjusted to the Journal requirements.

    it is unclear whether JUNI is a positive or negative regulator of JUI (I assume the reviewer meant JUN)

    Response: The text in the abstract was changed to” JUNI positively regulates the expression of its neighboring gene JUN, a key transducer of signals that regulate multiple transcriptional outputs.”

    Hope it is clearer now

    When the author indicates that JUNI antagonizes MAP PHOSPHATASE is not correct the term antagonism is related to receptors but the authors did not show any receptor.

    Response: The term "antagonism" does not only refer to receptor drugs. In pharmacology, antagonism generally describes the interaction between a drug (or other molecule) and a receptor or biological target that results in the inhibition or blocking of the receptor's activity. However, this concept can extend beyond receptor drugs and apply to various biological interactions.

    Outside of the realm of drugs and receptors, antagonism can also refer to antagonistic relationships between different biological processes, molecules, or organisms.

    Overall, while antagonism is commonly discussed in the context of receptor drugs, the concept of antagonism can apply to a broader range of interactions in biology and other fields.

    Response: The p values for the prognostic values of JUNI and DUSP14 in RCC were added to the abstract.

    Generally, Jun oncogene correlated with poor overall survival while the table indicates promote survival so good prognosis?

    Response: This manuscript describes for the first time the biological activity and cancer relevance of JUNI. It positively regulates stress induced c-Jun and can be used as prognostic marker in ccRCC.

    The significance of JUNI and its interactome in ccRCC prognosis is unequivocal, according to data analysis of cancer relevant data (TCGA) regardless to its effects on c-Jun. The concern raised by reviewer 1 and 2 is whether the cancer-relevant effects are mediated by c-Jun regulation. We suggest that despite regulating stress induced c-Jun, they are not! This suggestion is based on three points: 1. We show in the manuscript that a large portion of JUNI dependent effects on cellular survival activity is c-Jun independent. 2. We describe many interacting proteins that may, in a JUN-independent manner, affect tumorigenesis. 3. In this study we examined JUNI’s functions which are cell-autonomous. However, neither the non -autonomous effects nor effects on cells that compose the tumor environment were studied. Reports that lncRNAs may have a role in immune responses and high expression of JUNI in CD8 cells may suggest this direction for future investigation (Carpenter, S et al. science, 341(6147), pp.789-792; Mickaël, M. et al https://doi.org/10.1101/2021.12.01.470587)

    Therefore, we assume that direct correlations in every biological activity between JUNI and JUN is an over simplified consumption. Analogy for that can be found with another major regulator of c-Jun, JNK, which is stress induced, c-Jun regulator involved in stress-induced cell death, whereas c-Jun itself is contributing in many cases to drug resistance.

    The introduction contains the main information to follow the role of JUN and renal carcinoma
    However, should be improved with background on the key role of stress genes in the pro-survival pathway of tumors during progression and hypoxia condition. Too many references on long noncoding compared to the JUN complex with AP-1 and transformation

    Response: A section describing the major stress pathway in ccRCC, HIF 1 and its role in ccrCC was added. Due to the limitation of word count in most journals we cannot expend this section further

    Results In Figure 1 the authors showed expression levels of JUNI and JUN that are clearly different after UV stimuli. they demonstrate that are both regulated by UV but the amount and the time are different. the author should comment on these data if they want to study the regulative mechanism

    Response: The following comment was added at the end of the first section: Overall, these results suggested that JUNI is a stress-induced gene whose expression pattern resembles that of JUN, therefore, we investigated the potential existence of regulatory effects between the two genes, especially post exposure of cells to stress.

    Figure 1 F the cellular distribution of JUNI which is the rational of this experiment to provide that is into nucleus while normally is into the cytoplasm? What adds this experiment?

    Response: This is the first reported description of JUNI. We attempted to characterize it as much as possible. It’s localization was not described previously and we suggest that it is mainly nuclear. A novel important information that should be presented.

    In Figure 2 the authors provided that the kinase pathway is important for Jun regulation but the effect on JUNI a Luciferase assay needs to be provided

    Response: We respectfully disagree with the reviewer. We believe that examining the expression from a DNA fragment identical to the endogenous one is superior to artificial system, such as luciferase.

    In Figure 3 for Migration assay is necessary to see cells on the other side of the filter by staining not a graphical representation

    Response: The graphical representation is an accumulated result of at least 3 experiment. However, a figure representing a single experiment was added as a supplement figure s1.

    The experiment on kinase does not add any data to what is already known on jun probably should be shifted in Figure 6

    Response: We apologize, this question was not fully understood as there is no experiment on kinase in figure 3. If case the reviewer was referring to kinase inhibition in Fig 2A we do think it is needed as a positive control for the kinases activity.

    Table 1 is cited two times once in the context of Figure 3 and then in Figure 6 indicating that the authors go forward and back on their experimental design

    Response: Table 1 is indeed referred to in two places. It is first mentioned when we investigated the potential relevance of JUNI for human cancer, given its regulatory impact on the neighboring JUN gene and its influence on motility. Later, the types of cancers described in figure 1 were further processed in order to examine relations between JUNI and DUSP14 in human cancer. We do not see it as a flaw in experimental design but rather as further evolution of the story based on data discovered in earlier stages.

    in figure 4 the apoptotic cells are not clearly visible a specific staining marker is necessary to provide the phenomenon

    Response: Two corrections were made to demonstrate apoptosis clearly. The pictures in Figure 4 panel A were replaced with a better-quality image with addition of DNA staining to demonstrate the cell death clearer, appearance of cell blebbing and nuclear fragmentation. Panel B demonstrating increase in cleaved caspase 3 in JUNI silenced cells after all treatment was added.

    Additionally XTT assay should be reported as the percentage of survival cells not staining incorporated compared to untreated cells over time

    Response: We do apologize for the legend omission, but XTT assays, colonies formation and soft agar colonies formation are presented in Figure 4 H-J and Figure S3 for all cell lines

    The data on prognosis and correlation of gene expression are not clearly presented and discussed

    Response: Figure S4 was replaced by table S3 to demonstrate clearer the differences in Medians survival caused by JUNI of DUSP 14. Text was changed in the last section of results.

    The western blot need to be quantified

    Response: All blots were quantified

    Reviewer #2:

    1. While the experimental data showed JUNI, like c-JUN, is pro-survival of cancer cells, the clinical sample analyses correlated it positively with patients' survival. This discrepancy casts doubts in significance of the findings. The authors need to re-evaluate their data and conclusion

    Response: This manuscript describes for the first time the biological activity and cancer relevance of JUNI. It positively regulates stress induced c-Jun and can be used as prognostic marker in ccRCC.

    The significance of JUNI and its interactome in ccRCC prognosis is unequivocal, according to data analysis of cancer relevant data (TCGA) regardless to its effects on c-Jun. The concern raised by reviewer 1 and 2 is whether the cancer-relevant effects are mediated by c-Jun regulation. We suggest that despite regulating stress induced c-Jun, they are not! This suggestion is based on three points: 1. We show in the manuscript that a large portion of JUNI dependent effects on cellular survival activity is c-Jun independent. 2. We describe many interacting proteins that may, in a JUN-independent manner, affect tumorigenesis. 3. In this study we examined JUNI’s functions which are cell-autonomous. However, neither the non -autonomous effects nor effects on cells that compose the tumor environment were studied. Reports that lncRNAs may have a role in immune responses and high expression of JUNI in CD8 cells may suggest this direction for future investigation (Carpenter, S et al. science, 341(6147), pp.789-792; Mickaël, M. et al https://doi.org/10.1101/2021.12.01.470587)

    Therefore, we assume that direct correlations in every biological activity between JUNI and JUN is an over simplified consumption. Analogy for that can be found with another major regulator of c-Jun, JNK, which is stress induced, c-Jun regulator involved in stress-induced cell death, whereas c-Jun itself is contributing in many cases to drug resistance.

    Response: The Western blotting data need at least triplicate biological experiments and quantification. This is particularly important for trivial differences, such as shown in Fig. 6.

    Response: All westerns X=3. Representative experiments are depicted. Quantification was added.

    The identification and gene structure of LINC01135 and its relevance to c-Jun need better clarity

    Response: First result section. “According to ENCODE data, JUNI contains five main exons and has multiple isoforms. Twenty-seven different transcript isoforms were described according to LNCipedia ranging from 213 to 6213 bases {Volders, 2019 #****2907}. The relevance to c-Jun was referred to in discussion: Both the effects of JUNI on c-Jun induction and cellular survival were demonstrated using under-expression conditions by targeting, the common, first, exon of JUNI. Nevertheless, this exon was also sufficient for c-Jun induction upon stress exposure, under conditions of overexpression.

    Page 9-10, Line 198-199, there are no results in Fig. 1 showing that JUNI induction was dependent to serum stimulation of starved cells

    Response: “ Similar to JUN, the induction was dose dependent (Fig 1C), and the rapid response to stress (Fig 1D) as well as to serum stimulation of starved cells, identified by others (36), qualifies it as an “immediate early” lncRNA.”

    Serum stimulation is described in reference 36

    What is the Y-axis in figures 2B, 4E-G

    Response: Legend was added to Y-axis of Figures 2B and 4 E-G

    In Fig. 3B, actin image is missing

    Response: Actin was hidden in the graphic process. Corrected.

    In Fig. 4. brightfield images are inaccurate for distinguishing apoptosis and necrosis. Additional molecular markers need to be used, such as caspase-3 cleavage and LDH release

    Response: Two corrections were made to demonstrate apoptosis clearly. The pictures in Figure 4 panel A were replaced with a better-quality image with addition of DNA staining to demonstrate the cell death clearer, appearance of cell blebbing and nuclear fragmentation. Panel B demonstrating increase in cleaved caspase 3 in JUNI silenced cells after all treatment was added.

    The inconsistency of using four cell types in each assay. For example, in Fig. 4A, B, E-G and Suppl Fig. 1, HMCB, MDA-MB-231 and CHL1 cells were used to test the short-term effect of JUNI knockdown on cell survival, whereas Hela, MDA-MB-231 and CHL1 cells were chosen to determine the long-term effect of JUNI knockdown. Similar case in other figures.

    Response: Effects on Jun regulation and the effects on long term survival were tested in all four cell lines both by XTT and clonogenic assays whereas effects on short term survival were tested in three out of the four cell lines. It is practically impossible to perform a study of this magnitude were all assays were tested in all cell lines. Using four cell lines was applied to prove the major points.

    In Fig. 5D, no difference of c-Jun expression between NS and siJUN groups

    Response: Correct, the western in 5D was replaced by a more representative one

    Cell survival in Fig. 5 lacked statistical analyses

    Response: Error bars were mistakably omitted. The figure was corrected.

    In Suppl Fig. 2C, there is no figure to show the reduced colonies formation in soft agar in MDA-MB-231 cells, contradicting to that stated in the manuscript

    Response: Indeed Figure 4 J and S3 C presented colonies formation in HMCB and HeLa cells. The text was corrected.

    Reviewer #3: "linc01135" - this is a human gene, should be capitalized

    Response: linc01135 was capitalized

    Please indicate primers in Fig1A and mention this in relevant part of Results

    Response: The following section was added: “Importantly, ENCODE predicts that the first exon is shared by all, therefore, all primers to analyze JUNI’s expression as well as siRNAs to silence it, were targeted for this exon.”

    Fig1C-F - please add a legend to explain the colors

    Response: Legend was added into the Figure as well

    Copy number: It is important to establish the approximate copy number of JUNI RNAs in the cell lines tested. FISH would be one appropriate method. This could also be referenced back to the RNA-seq TPM values. Are we talking about <1 copy /cell, or many? Quick inspection of ENCODE RNA-seq in the UCSC browser suggest an intermediate value that varies between cell lines. This value is very important when interpreting mechanistic experiments later on

    Response: The copy number in HMCB and MDA-MB-231 was calculated by comparison of CT values obtained from RNAs from a known number of cells relative to calibration curve of known concentrations of JUNI. The following section was added to the first paragraph of the results: “quantitation of JUNI’s copy number in untreated HMCB and MBA-MD-231 cells revealed the presence of minimal amount of about 8 copies per cell”

    Fig3 - again, no figure legends, difficult for reader

    Response: Legend was added to Fig. 3A

    In general, the figures could be much more clearly annotated and presented with more care. They do not do justice to the quality of the work itself. For example, Fig4E-G why not label each panel with the time course, the cell line tested etc etc to save us the work of digging through the Legends?

    Response: We thank the reviewer for this remark. All figures were corrected, legends and proteins quantification was added.

    Rescue experiments: The rescue experiments in Fig5D are nicely done and the results are interesting. However, I would request the authors to perform similar experiments with JUNI rescue. Specifically, to knock down JUNI with siRNA, and then reintroduce it from an 'immune' expression plasmid, where the siRNA site is mutated. This will further strengthen the claim that JUNI siRNA is acting through the intended target to cause observed effects on cell viability

    Response: As the effects on survival are strongest in the longer term, 14 days after silencing, rescue experiments were performed to test the rescue in the survival of HMCB and HeLa cells using clonogenic assays. Results are presented in figure 4 L

    IncPrint data: was Jun protein found to be an interactor? This might be mentioned in the text, whether it is yes or no

    Response: c-Jun was screened and did not interact with JUNI. The text was changed as following” Interestingly, c-Jun itself does not interact with JUNI (Table S2, Normalized luciferase intensity MS2, RLU =0.44). By contrast, the dual specificity protein phosphatase 14….”

    Expression: A key issue is the expression of JUNI in healthy and diseased cells and organs. Is JUNI ubiquitous (and essential to both healthy and tumor cells), or is it specific to tumor cells? Which tumor types? This would be straightforward to find out from public data. I would suggest a main figure panel. Also, is JUNI upregulated across tumors? Could find this out from GEPIA2 or other databases.

    Response: Figure 7E describing the levels of JUNI in variety of normal and tumor samples was added.

    Non-tumor cells: Like many studies, this one focusses on effect of LOF in transformed cells. However, therapeutic relevance is tied to specific effect in transformed cells. Therefore I believe the paper would be vastly strengthened, if knockdowns+viability assays were also performed in some non-transformed cells. Eg HEK293, immortalised fibroblasts, RPE1 etc

    Response: Indeed discrimination between Normal and cancer cells is an essential point for further research and translation. We examined the affects of silencing on spontaneously immortalized keratinocytes, HaCat cells, and the results are depicted in Figure 4 K.

    Alternative reagents: The siRNA experiments are well performed with two independent sequences. An important additional experiment would be to replicate these experiments with antisense oligonucleotides. This would both further strengthen the confidence in experiments, and open more lines of potential therapies. This experiment I would consider optional

    Response: Stable CRISPR can not be formed. We are currently constructing inducible CRISPR but the construction consumes longer time than the scope of this revision.

    Advanced models: All the present experiments are performed in monolayer cell lines. The authors will no doubt be aware that the paper would be substantially strenghtened if functional experiments could be replicated in more advanced models: spheroids, PDX, explants, mice...

    Response: We examined the protective role of JUNI in Doxorubicin treated spheroids of HMCB and CHL1 cells. The results are depicted in figure 4 D and E.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Kumar and colleagues present an apparently novel, cancer-promoting lncRNA 'JUNI' and perform a rather thorough and careful analysis of its in vitro functions and molecular mechanisms.JUNI is located adjacent to the Jun protein coding gene, although intriguingly the two appear to be rather indepent of each other at the level of gene products. JUNI appears to be necessary for cancer cell line growth and survival (monolayer) in multiple contexts. Particularly interesting is the demonstration that JUNI appears to function in trans. Overall this is an excellent paper - work is solidly and carefully done, hypotheses are well formulated and thoroughly tested. The JUNI lncRNA is well supported in public annotations, seems to be highly expressed, and it is surprising that virtually no work has been carried out on it so far. Furthermore, the apparently essentiality of JUNI to cancer cells has potentially important therapeutic and mechanistic ramifications.

    These are suggestions for improvement of the work.

    "linc01135" - this is a human gene, should be capitalised.

    Please indicate primers and ASOs in Fig1A and mention this in relevant part of Results.

    Fig1C-F - please add a legend to explain the colors.

    Copy number: It is important to establish the approximate copy number of JUNI RNAs in the cell lines tested. FISH would be one appropriate method. This could also be referenced back to the RNA-seq TPM values. Are we talking about <1 copy /cell, or many? Quick inspection of ENCODE RNA-seq in the UCSC browser suggest an intermediate value that varies between cell lines. This value is very important when interpreting mechanistic experiments later on.

    Fig3 - again, no figure legends, difficult for reader.

    In general, the figures could be much more clearly annotated and presented with more care. They do not do justice to the quality of the work itself. For example, Fig4E-G why not label each panel with the time course, the cell line tested etc etc to save us the work of digging through the Legends?

    Rescue experiments: The rescue experiments in Fig5D are nicely done and the results are interesting. However, I would request the authors to perform similar experiments with JUNI rescue. Specifically, to knock down JUNI with siRNA, and then reintroduce it from an 'immune' expression plasmid, where the siRNA site is mutated. This will further strengthen the claim that JUNI siRNA is acting through the intended target to cause observed effects on cell viability.

    IncPrint data: was Jun protein found to be an interactor? This might be mentioned in the text, whether it is yes or no.

    Expression: A key issue is the expression of JUNI in healthy and diseased cells and organs. Is JUNI ubiquitous (and essential to both healthy and tumor cells), or is it specific to tumor cells? Which tumor types? This would be straightforward to find out from public data. I would suggest a main figure panel. Also, is JUNI upregulated across tumors? Could find this out from GEPIA2 or other databases.

    Non-tumor cells: Like many studies, this one focusses on effect of LOF in transformed cells. However, therapeutic relevance is tied to specific effect in transformed cells. Therefore I believe the paper would be vastly strengthened, if knockdowns+viability assays were also performed in some non-transformed cells. Eg HEK293, immortalised fibroblasts, RPE1 etc.

    Alternative reagents: The siRNA experiments are well performed with two independent sequences. An important additional experiment would be to replicate these experiments with antisense oligonucleotides. This would both further strengthen the confidence in experiments, and open more lines of potential therapies. This experiment I would consider optional.

    Advanced models: All the present experiments are performed in monolayer cell lines. The authors will no doubt be aware that the paper would be substantially strenghtened if functional experiments could be replicated in more advanced models: spheroids, PDX, explants, mice...

    Significance

    This is an important advance in the cancer field. It reveals a potential new lncRNA oncogene, JUNI, which appears to be necessary for cancer cell survival in multiple contexts through mechanisms defined by the authors. Future work will be required to understand the degree to which JUNI's activity is cancer specific, and its functional effects will have to be replicated in more faithful cancer models.

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    Referee #2

    Evidence, reproducibility and clarity

    This work identified a lncRNA JUNI located near c-JUN and investigated its relationships with c-JUN and stress response, survival, and cancer prognosis. Experiments are logically designed, and the research topic is novel. The main concern is weaknesses in data interpretation and significance. Additionally, the paper needs improvement in experimental rigor with statistical assessment of multiple data sets; data description and conclusion need better clarity.

    Overall comments:

    1. While the experimental data showed JUNI, like c-JUN, is pro-survival of cancer cells, the clinical sample analyses correlated it positively with patients' survival. This discrepancy casts doubts in significance of the findings. The authors need to re-evaluate their data and conclusion.
    2. The Western blotting data need at least triplicate biological experiments and quantification. This is particularly important for trivial differences, such as shown in Fig. 6.

    Specific comments:

    1. The identification and gene structure of LINC01135 and its relevance to c-Jun need better clarity.
    2. Page 9-10, Line 198-199, there are no results in Fig. 1 showing that JUNI induction was dependent to serum stimulation of starved cells.
    3. What is the Y-axis in figures 2B, 4E-G
    4. In Fig. 3B, actin image is missing.
    5. In Fig. 4. brightfield images are inaccurate for distinguishing apoptosis and necrosis. Additional molecular markers need to be used, such as caspase-3 cleavage and LDH release.
    6. The inconsistency of using four cell types in each assay. For example, in Fig. 4A, B, E-G and Suppl Fig. 1, HMCB, MDA-MB-231 and CHL1 cells were used to test the short-term effect of JUNI knockdown on cell survival, whereas Hela, MDA-MB-231 and CHL1 cells were chosen to determine the long-term effect of JUNI knockdown. Similar case in other figures.
    7. In Fig. 5D, no difference of c-Jun expression between NS and siJUN groups.
    8. Cell survival in Fig. 5 lacked statistical analyses
    9. In Suppl Fig. 2C, there is no figure to show the reduced colonies formation in soft agar in MDA-MB-231 cells, contradicting to that stated in the manuscript.

    Significance

    Experiments are logically designed, and the research topic is novel. The main concern is weaknesses in data interpretation and significance. Additionally, the paper needs improvement in experimental rigor with statistical assessment of multiple data sets; data description and conclusion need better clarity.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The author investigated the role of the stress sensor pathway in the mechanism of tumor cell survival
    They identified a long noncoding RNA as JUNI that regulates antagonizing MAP phosphatase and favors the JUN transcription. JUNI correlated with the survival of several cancer histotypes, particularly in RCC, as a highly specific and correlated prognosis.

    The abstract although not always required from the journal should be divided into methods used to reach the main findings and clear presentation of results it is unclear whether JUNI is a positive or negative regulator of JUI. When the author indicates that JUNI antagonizes MAP PHOSPHATASE is not correct the term antagonism is related to receptors but the authors did not show any receptor. Correlated with prognosis ( negative or positive ) Statistical value should be reported in the abstract. Generally, Jun oncogene correlated with poor overall survival while the table indicates promote survival so good prognosis?

    Major comments

    The introduction contains the main information to follow the role of JUN and renal carcinoma
    However, should be improved with background on the key role of stress genes in the pro-survival pathway of tumors during progression and hypoxia condition. Too many references on long noncoding compared to the JUN complex with AP-1 and transformation.
    Results In Figure 1 the authors showed expression levels of JUNI and JUN that are clearly different after UV stimuli they demonstrate that are both regulated by UV but the amount and the time are different the author should comment on these data if they want to study the regulative mechanism figure 1 F the cellular distribution of JUNI which is the rational of this experiment to provide that is into nucleus while normally is into the cytoplasm? What adds this experiment?
    In Figure 2 the authors provided that the kinase pathway is important for Jun regulation but the effect on JUNI a Luciferase assay needs to be provided
    In Figure 3 for Migration assay is necessary to see cells on the other side of the filter by staining not a graphical representation the experiment on kinase does not add any data to what is already known on jun probably should be shifted in Figure 6. Table 1 is cited two times once in the context of Figure 3 and then in Figure 6 indicating that the authors go forward and back on their experimental design
    in figure 4 the apoptotic cells are not clearly visible a specific staining marker is necessary to provide the phenomenon additionally XTT assay should be reported as the percentage of survival cells not staining incorporated compared to untreated cells over time.
    The data on prognosis and correlation of gene expression are not clearly presented and discussed

    Significance

    The authors identified a long noncoding RNA as JUNI that regulates antagonizing MAP phosphatase and favors the JUN transcription. JUNI correlated with survival of several cancer histotypes In particular in RCC as a highly specific and correlated prognosis.

    The data are not presented with a good rationale often the authors go forward and back on the experimental design. The data are not presented in the best way some data are shown as bar graph but need to be supported by cell staining of transwell staining and standard plot for survival rate The western blot need to be quantified

    In general, the experimental design does not match the rational

  5. Version published to 10.1101/2023.10.23.563579v1 on bioRxiv