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The authors submitted the revised manuscript "ZFPM2-AS1 promotes the proliferation, migration, and invasion of human non-small cell lung cancer cells involving the JAK-STAT and AKT pathways". The authors responded to all comments so that this manuscript is now acceptable.
Congratulations!
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
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The authors submitted the revised manuscript” ZFPM2-AS1 promotes the proliferation, migration, and invasion of human non-small cell lung cancer cells involving the JAK-STAT and AKT pathways”，I really appreciate the authors responded to my comments. The author uses a more accurate title. To make sure it is consistent in the whole paper, you still need to avoid these conclusive sentences line 42-45; Line 264-265; Line 278-279; line 327-328, or similar sentences in the whole paper. [Please keep in mind, “the current data is not enough to prove ZFPM2-AS1 regulate its downstream effectors (proliferation/migration/invasion, ZFPM2, PD-L1) through JAK-STAT3 and AKT pathway, although they showed similar change pattern. The only way to prove them is using pathway inhibitors to block the signaling, and then check if the inhibitors can abolish/attenuate the effect of ZFPM2-AS1 on its effectors.”.]
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I would like to thank the authors to address reviewer's comments. The revised manuscript is clearly improved and easier for follow. I recommend this manuscript to be accepted and published as is.
Thank you for your revision in your manuscript. However, some corrections still need to be made in your revised article.
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The authors submitted the revised manuscript ” ZFPM2-AS1 promotes the proliferation, migration, and invasion of human non-small cell lung cancer cells via the JAK-STAT and AKT pathways”. Thank you for a point-to-point response to my comments, the current manuscript had a great improvement in language and data presentation, which makes readers clearer about the object of the research. Here, I have several concerns that need to be addressed.
1. the authors presented excellent data to prove the ZFPM2-AS1 regulates proliferation, migration, and invasion of NSCLC cell lines and correlate with NSCLC clinical characteristics. Additionally, the readers can also make a clear conclusion from your data:
(1) ZFPM2-AS1 enhance the IFN-gamma effect on proliferation/ migration/ invasion of NSCLC cells
(2) ZFPM2-AS1 negatively regulate ZFPM2
(3) ZFPM2-AS1 positively regulate PD-L1
(4) ZFPM2-AS1 activates JAK-STAT3 and AKT pathway.
However, the current data is not enough to prove ZFPM2-AS1 regulates its downstream effector (ZFPM2, PD-L1) through JAK-STAT3 and AKT pathway. It is a good thing to prove JAK-STAT3 and AKT pathways are the factors that contribute to proliferation/ migration/ invasion as well as the change of PD-L1 and ZFPM2, but you do not have to prove them, you just need a more comprehensive and accurate title.
If you want to prove them, the most helpful experiment is the JAK inhibitors(and/or AKT inhibitors) assay (I mentioned in the last comments), I also reviewed the supplemental figure 1, you didn’t perform the assay in incorrect way. The following cohorts may be used for validating ZFPM2-AS regulate cell proliferation/ migration/ via the JAK-STAT pathway.
a. Vector
b. ZFPM2-AS
c. Vector + JAK inhibitors
d. ZFPM2-AS+JAK inhibitors
And if you want to further prove its association with PD-L1, these 4 cohorts need to be performed in the presence OR absence of IFN-gamma (Totally 8 groups).
2. There are still have some typos/grammar/tense need to be corrected, although the revised manuscript was greatly improved in language.
3. The revised Figure 3C is different from the previous version, please confirm and explain.
The revised manuscript is much better written and easier to follow. I would like to thank the authors for taking my concerns into account and sufficiently addressing them specifically in the aspect of language and grammar. These revisions make the science and the rationale provided by these authors easier to follow for a wider, more international audience. The pdf version of their manuscripts requires some minor edits as pointed out below
(1) Lines 92-94 , lines 134 and 180 : missing letters "fl" in words like influence, confluence etc. Please double check
(2) Line 135 : please change "applied" to "used"
(3) Line 150 : Remove the phrase "following that". Unnecessary and causes ambiguity.
Apart from these changes in the text, the following minor edits need to be addressed in the figures
(1) Figures 3E, 3G : please add the cell line labels to the images of the Colony formation assay
(2) Please move figure 3D to panel H and group all the siRNA data consecutively. Figure 3D is the only one that has overexpression data
(3) Figures 4E, G, I, K : please add cell line labels to the crystal violet images and magnification bars
(4) Figure 5G, H - Please add the percentages associated with each cell cycle stage consistent with the other panels in the figure.
All comments have been addressed.
All comments have been addressed.
I would like to thank the authors for their effort in addressing all the concerns raised. The experiments designed and conducted add credence to their data. The written language better supports the data presented .
Dear Dr. Li,
Thank you for your submission to PeerJ.
It is my opinion as the Academic Editor for your article - ZFPM2-AS1 promotes the proliferation, migration and invasion in human non-small cell lung cancer cells by targeting JAK/AKT/STAT pathway - that it requires a number of Major Revisions.
My suggested changes and reviewer comments are shown below and on your article 'Overview' screen.
Please address these changes and resubmit. Although not a hard deadline please try to submit your revision within the next 55 days.
With kind regards,
Zekiye Altun
Academic Editor, PeerJ
The manuscript needs careful editing by English native speaker OR professional language editing service.
Most experiments perform with rigorous design. only one experiment may need more evidence.
The conclusion needs to be re-written.
Wang et. al. submitted the manuscript ” ZFPM2-AS1 promotes the proliferation, migration and invasion in human non-small cell lung cancer cells by targeting JAK/AKT/STAT pathway” The authors provide clinical evidence to show the correlation between ZFPM2-AS1 and NSCLC and use multiple methods (MTT, colony forming, wound healing, and Transwell assay and Flow cytometry,) to confirm that ZFPM2-AS1 regulates the proliferation/colony formation, migration, and invasion of lung cancer cell lines. In addition, they employed ZFPM2-AS1-siRNA and western blot to show ZFPM2-AS1 might regulate via JAK-STAT and AKT pathway and PD-L1. The data is enough to support the ZFPM2-AS1 is involved in NSCLC. The ZFPM2-AS1 might serve as a potential target of lung cancer treatment/immunotherapy in the future. However, the whole manuscript was poorly written, and results/figures are not well-organized, and it still needs more evidence to prove ZFPM2-AS1 involved in the JAK-STAT and AKT pathway. The manuscript requires substantial revision before acceptance. Here I have some suggestions and comments that may be helpful to improve the manuscript.
Major point:
A. Your most important issue is scientific paper writing.
your manuscript needs careful editing by English native speaker OR professional language editing service. Please pay attention to sentence structure, verb tense, and clause construction. Additionally, the authors should write the paper in logistic order, especially the result section (Figure 7).
B. The next most important item is Figure 6.
It still needs more convincing evidence to support ZFPM2-AS1 involved in JAK-STAT and AKT pathway. JAK-STAT and AKT pathway is the new and most important finding of ZFPM2-AS function in your study.
C. All the figures need to be carefully edited.
All details are listed below:
1. The author should use “targeting” carefully in the title. According to the results you presented, you did not figure out the real target (gene/miRNAs) of LncRNA ZFPM2-AS1. In addition, it is generally believed that JAK-STAT and AKT are two distinct signaling pathways, and the AKT pathway is not the direct upstream of STAT, although these signaling pathways can be integrated in some cases.
2. Line 29. Please make sure if you use “RT-PCR” rather than “qRT-PCR” to examine the transcription level of ZFPM2-AS1.
3. Line 41, the author mentioned the “PD-1” in abstract, but there is no description concerning PD-1 in the result section.
4. Line 64” warranted” in here is very confused.
5. There are many sentences with sentence structure problems, i.e. Line 51-53, line 62-64… please addressed these issues in the whole manuscript.
6. The introduction was not well-written, your need to make a comprehensive review on the LncRNA of your interest (ZFPM2-AS1)[there are more than 10 papers in PubMed (https://pubmed.ncbi.nlm.nih.gov/?term=ZFPM2-AS1)], and don’t pay too much attention to other unrelated lncRNAs in JAK-STAT and AKT pathway(line 71-85). And it needs some background information about IFN-γ to make your audience clear about your study.
7. Line 97 “different ”
8. Line 110 &131, ”CO2” (“2” here should be subscript)
9. Line 125, 4x103; (“3” here should be superscript), please carefully check the same issues in otherwhere, line 135, line 143…etc.
10. Line 165, please indicated the manufacture information of “IP lysis”, check if “IP lysis” is the correct name of this reagent.
11. line 198, “one lung normal epithelial cell line, four NSCLC cell lines, and one tool cell line ”, avoid this kind of usage in a scientific paper, please state the specific name of cell lines.
12. Line 202, “including H1299 and H292”, should be “including H1299 cells and H292 cells”, please carefully proofread the similar issues in otherwhere, line 218, line 219, line 229, line 232, line233…...
13. Line 206, please indicate the abbreviation” TNM” information.
14. Line 216, please add a short description of why you use IFN-γ for your study, and there is no description in introduction and otherwhere (result and discussion), which will make the readers very confused.
15. Another confusing point: The whole paper focused on the ZFPM2-AS1 rather than ZFPM2 (although ZFPM2 is a potential target of ZFPM2-AS1 lncRNA). The authors present lots of data to prove the role of ZFPM2-AS1 in NSCLC, but at the end of the results (Result-3.8), the authors tried to explain the role of ZFPM2 in tumor immune infiltration. Please explain it OR re-organize the results to make readers clear about the object of your research.
16. Line 253 “Meanwhile, the expression of JAK2, p-STAT3 and p-AKT was decreased, compared with the control group. However, there was no significant differential expression in STAT3 and AKT”. Typically, phosphorylated JAK2 (pJAK2), rather than JAK2, represents “active status” in the signaling study, in my opinion, you should check the pJAK2 (the ratio of pJAK2/total JAK2).
17. Result 3.7, The most important finding in this article is ZFPM2-AS1 involved with JAK-STAT and AKT pathway. I guess the authors try to use the IFN-γ to activate the JAK-STAT pathway (not stated in the manuscript), however, it still cannot exclude that ZFPM2-AS1 regulate NSCLC by other pathways(like p53 pathways), in my opinion, you should use JAK inhibitors to block the JAK-STAT pathway, and then check if the inhibitors can abolish (at least attenuate) the effect of ZFPM2-AS1 on proliferation/migration/invasion of lung cancer cells. You may present at least one piece of data in this section.
18. Line 287-299. Please don’t overstate the JAK-STAT pathway that is well-known signaling in cancer, this paper focused on ZFPM2-AS1, you may discuss how ZFPM2-AS1 regulates JAK-STAT pathway and whether other pathways (for example, p53 pathway that you mentioned in the introduction) involved in ZFPM2-AS1 regulatory network.
19. Conclusion. Usually, the conclusion just briefly summarizes your finding, which differs from the abstract and result.
20. Please provide a brief title for each figure and avoid unnecessary descriptions in the text. According to the Journal requirement, use ”Figure x” rather than all capital ” FIGURE x”.
21. Figure 3, panel E is already in Figure 3, please remove figure 3E on Page 31.
22. Figure 4, there are 5 panels (A.B.C.D.E) in Figure 4, which located on different pages. The authors should re-organize the layout and combine all panels in one page OR divide them into two figures.
23. Figure 5. Combine all panels in one page. Figure 5B, please use “IFN-γ” instead of “γ”.
24. Figure 6. Combine all panels in one page. Figure 6B, I cannot see a significant difference(as the author wrote, line 259-260) of pSTAT3 level in groups, please confirm. In addition, the author should add a bar graph to each protein in Figure 6. For the phosphorylated protein, a phospho/total ratio (i.e., p-STAT3/STAT3…) need to be presented.
25. Figure 7. Combine all panels in one page.
a. The overall language used is acceptable however minor grammatical and typological errors need to be fixed. For example, in lines #59, #67, #81– words like mounting, frustrate and sponging are out of context and provide ambiguous meaning. I would recommend that the authors restructure these sentences to make it clear.
b. It is not clearly stated that the authors consider both lung adenocarcinomas and lung squamous cell carcinomas for their work presented here. It is to be assumed based on their results. However, I would recommend adding a clear statement to their methods section to remove any ambiguity.
a. The authors choose to focus specifically on signaling driven by phosphorylation of Akt on Ser47 vs Thr308. However studies have shown activation at these sites have different phenotypic effects and consequences in cancer cells such as https://doi.org/10.1038/bjc.2011.132 . The authors should address this by looking at Akt phosphorylation at this site via western blotting. (If possible).
b. The data shown in figures 3C and 3D do not seem very convincing. There is significant inconsistency in the data presented with some experiments performed in only H460 and some only in A549. Specifically, in figure 3C and 3D, the difference in proliferation between the control and sample (either siZFPM-AS1 or overexpression of AFPKM-AS1) seems too small to be significant. The raw data provided show much variation in their values. I would recommend that the authors verify their statistical tests keeping the variation between replicated in mind. The authors should also address why only one cell line was chosen for certain experiments vs others or provide data for both to make their study more robust.
c. There is a lack of explanation on the relevance of the JAK-STAT pathway and the use of IFN-gamma induction of this pathway in NSCLC specifically. The authors do try to expand on this in the discussion but some further expansion on this may be helpful. The authors can refer to reviews like 10.1080/21623996.2014.999503 and https://doi.org/10.3390/cancers6020708 to further supplement the introduction and discussion.
d. In their TCGA data analysis, the authors do not mention what studies were included in their 535 lung cancer cohort. What criteria was used to be included in their study compared to normal. If this analysis was based off their previous publication that should be mentioned here.
e. The analysis/ data used for the calculation of the ROC in Figure 1B is not explained. If this is part of the TCGA provided analysis it needs to be clearly stated. Furthermore, the ROC curve is depicted in an unusual manner with the AUC shaded and the random guess line is not shown. Can the authors please comment as to why they chose to depict the results in such a manner over the conventional representation?
f. In their RNA,qRT-PCR methods section, the authors fail to mention how the relative gene expression was calculated and which housekeeping gene was used. (Lines 120-122)
g. The authors need to mention if the colonies were counted manually or under a microscope for their colony formation assay. (Line 133)
h. Lines 147-148. This is ambiguous. Please provide information on the magnification and the specifications of the microscope.
i. In figures 4D and 4E the bottom panel of the quantification of results should be H460, not A549 again. Please correct this typographical error in your figures.
j. In Figure 7A the color legend is missing to indicate what the red, blue and purple mean.
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The article ZFPM2-AS1 promotes the proliferation, migration and invasion in human non-small cell lung cancer cells by targeting JAK/AKT/STAT pathway by Wang et al is a generally well-put together article aimed at elucidating the molecular mechanism of lncRNA ZFPKM-AS1 on the tumorigenic characteristics of NSCLC. I commend the authors on testing the effects of both loss and overexpression of ZFPKM-AS1 on multiple characteristics of tumor cells. I was specifically impressed by their assessment of 50 clinical samples in this context. I further appreciate the efforts of the authors in sharing the raw data for almost figure represented in the paper to aid transparency and provide confidence in their results. However, there are some concerns with the paper in its current version as detailed above.
1. The most important issue is the English language used throughout the manuscript. I suggest the authors carefully proof-read and further improve the quality. Many sentences were grammatically incorrect. Some examples include line 60(…lncRNAs might role…), line 61 (…differential expression lncRNAs…), line 237(…in the nuclear...), etc. Some other phrases, albeit grammatically correct, were scientifically confusing. Examples include line 63 (...biological behavior of NSCLC cells...), line 86(...proliferation, migration, and invasion capacity of ZFPM2-AS1 in NSCLC cells), line 215(...the decreased efficiency...), line 261 (...distinctly inhibited...), etc.
2. Please define and spell out acronyms where they appeared for the first time. Some examples include ZFPM2-AS1, MIF, NC, etc.
3. I think it would be helpful to briefly summarize/conclude at the end of each Results subsection. The last sentence of section 3.4 was a good example.
4. Some figure labels are barely visible.
5. Line 283 needs reference
1. It would be helpful to explain the rationales of different analyses done in Figures 1B and 1C, either in the legends or text.
2. Could the authors explain why they used IFN-γ in the proliferation experiments. Because this was used extensively throughout the manuscript, it might be worth mentioning in the introduction. Along the same line, could the authors comment on why some differences were only significant in the presence of IFN-γ, e.g. Figure 3C?
I appreciate that this manuscript had extensive amount of results, most of which are well controlled. My biggest concern, however, is that the differential expression of the lncRNA ZFPM2-AS1 in various NSCLC samples and cell lines. In fact, as the authors reported, ZFPM2-AS1 was significantly downregulated in two out of four cell lines experimented. This should not be overlooked and should be commented on, given that the authors proposed this to be a promising prognostic marker and even a therapeutic target.
Please include quantification of the western blotting results in Figures 6A and 6B, normalized to internal controls.
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