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All my comments have been addressed by authors. I think this paper can be accepted for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Sonia Oliveira, a PeerJ Section Editor covering this Section #]
Thank you for your thorough revision and for addressing the majority of the points raised in the initial review. I have reviewed your changes, and the manuscript has improved substantially.
I understand your explanation for the difficulty in acquiring the necessary antibodies for these additional experiments in the animal level. I acknowledge your adherence to the journal’s policy of including one GAPDH band per protein. This approach is acceptable and ensures consistency in your results. The addition of measurement units in your IF data is appreciated and improves the clarity of these figures. This correction addresses the initial concern and makes the results more transparent. The inclusion of NLRP3 mRNA level testing following different treatments in Figure 2B adds depth to the analysis and aligns with the original feedback. This strengthens your argument regarding the regulatory role of MALAT1 and miR-22-3p in pyroptosis and apoptosis. The testing of siMALAT1 inhibition efficiency, as presented in Figure 1A, adequately addresses the need for validation of the knockdown efficiency. This is an essential addition to support your mechanistic claims. Your effort to correct the spelling errors and formatting issues throughout the manuscript has significantly improved its readability and professionalism.
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Overall, your revisions have addressed most of the initial concerns, and the manuscript is much stronger as a result. I think it can be accepted by this journal.
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The author's revision basically solved my concerns.
1. The English wording and grammar of the manuscript need to be further refined to improve the readability and clarity of expression.
2. The abstract should clearly specify the main apoptosis and pyroptosis indicators affected by MALAT1. Additionally, at the end of the abstract, the significance of the research results and potential applications should be further discussed and prospected.
3. The introduction lacks a description of the pathogenesis and current research status of ulcerative colitis. The research background and significance of MALAT1 are insufficiently explained.
4. The Materials and Methods section should provide more experimental details, such as specific siRNA sequences, plasmid information, qPCR reaction systems and conditions, to facilitate replication by other researchers.
5. It is important to specify whether FHCs were first treated with LPS to establish the model and then subjected to MALAT1 interference or overexpression. Some of the data points in Figure 1 are not accurately described and should be revised.
6. The rationale for detecting pyroptosis in Figure 2 needs to be explained more clearly in the text. The activation of caspase-1 and cleavage of GSDMD mediated by the NLRP3 inflammasome are important indicators for assessing pyroptosis.
7. The reasons for focusing on NLRP3 should be further elaborated in the discussion based on previous literature, suggesting that MALAT1 may affect intestinal epithelial cell inflammation through this signaling pathway.
8. The description of the results in Figures 4-6 should be more precise, with more details on miRNA screening and direct binding evidence.
9. Measurement units should be labeled in all immunofluorescence results.
10. The mRNA level of NLRP3 after treatment with different reagents should be tested in Figure 2B.
11. The knockdown efficiency of si-MALAT1 should be demonstrated in the manuscript.
12. There are numerous spelling errors throughout the manuscript that should be carefully corrected during revision, such as "miR-22-3p n" in line 33 and "increase IL-10 levels [12]." in line 56.
[# PeerJ Staff Note: The review process has identified that the English language must be improved. PeerJ can provide language editing services if you wish - please contact us at [email protected] for pricing (be sure to provide your manuscript number and title). Your revision deadline is always extended while you undergo language editing. #]
In this manuscript, the authors mainly demonstrated that MALAT1 induces apoptosis and pyroptosis by sponging the miR-22-3p to enhance the expression of NLRP3. Interestingly, MALAT1 expression can be markedly increased in LPS-treated FHCs. Conversely, inhibition of MALAT1 significantly facilitated cell proliferation and decreased apoptosis that was involved in the inhibition of NLRP3, which activates Caspase 1 and cuts the IL-1β, IL-18, and GSDMD, and then induces cell pyroptosis. Additionally, the authors demonstrated that miR-22-3-p can bind with MALAT1 and NLRP3 3’-UTR and inhibit colonic epithelial cell apoptosis and pyrotosis by silencing the NLRP3 expression.
Overall, this manuscript has clear logic and solid results. However, depending on its current condition, it can’t be published in this journal and needs a future revision.
1: There are some papers, as shown below, that have reported the relationship between MALAT1 and NLRP3. The authors should explain and discuss this during the revision.
A: Blocking the LncRNA MALAT1/miR-224-5p/NLRP3 Axis Inhibits the Hippocampal Inflammatory Response in T2DM With OS.
B: LncRNA MALAT1 sponges miR-133 to promote NLRP3 inflammasome expression in ischemia-reperfusion injured heart.
C: Expression and diagnostic value of lncRNA MALAT1 and NLRP3 in lower limb atherosclerosis in diabete.
D: Long noncoding RNA MALAT1 promotes high glucose-induced human endothelial cells pyroptosis by affecting NLRP3 expression through competitively binding miR-22.
E: LncRNA MALAT1 promoted high glucose-induced pyroptosis of renal tubular epithelial cell by sponging miR-30c targeting for NLRP3
2: In all the figures involved WB about the N-GSDMD and Clea-Caspase-1, the authors should test and show the total level of GSDMD and Claspase-1.
3: In all WB results, one GAPDH band is enough as the loading control.
4: All the IF results should be labeled with the measurement unit.
5: In Figure 2B, the authors should test the mRNA level of NLRP3 after treatment with different reagents.
6: In this manuscript, the authors should test and demonstrate the inhibited efficiency of si MALAT1.
7: This manuscript contains many spelling errors, which should be carefully corrected and modified during the revision. Such as line 33 “miR-22-3p n”, 56 “increase IL-10 levels [12].”, 264 “sis. F1000Res 2020; 9(”.
The authors present a very meaningful study to identify the therapeutic target of ulcerative colitis. They are identified that MALAT1 had correlation to the proliferation, apoptosis, and pyroptosis of LPS-treated human colon cells. I feel the manuscript in its current form effectively clarifies these important findings. However, I have one main suggestion that I hope can furthermore support the author's research.
1) While the manuscript effectively presents the research ideas and findings, the English language requires further polishing to enhance readability and ensure that the ideas are communicated more clearly and fluently. I recommend a thorough proofreading by a native English speaker or professional editing service.
2)Pyroptosis and NLRP3 effect on inflammation, ulcerative colitis should be described in Introduction section.
3)Line 183, four sets should be described.
4)Line 243, what are the similarities and differences between the effects of MALAT1 on apoptosis, pyroptosis, inflammation, and UC in this study and previous literature?
5)Similarly, what are the similarities and differences between the effects of miR-22-3p and NLRP3 on UC in this study and previous literature?
6)The overall experimental design is logical and aligns with the study's objectives. However, more clarity is needed on the rationale behind selecting specific concentrations and time points for treatments (e.g., 10 ng/mL LPS for FHCs and incubation times for proliferation assays). It would be beneficial if the authors provided more detailed justifications for these choices, referencing relevant literature or preliminary experiments that support these parameters.
7)The manuscript describes the construction of the UC cell model by treating FHCs with LPS. To ensure the scientific validity of this approach, it is recommended that the authors cite relevant literature that supports the use of this method for modeling ulcerative colitis (UC) in vitro. Providing such citations will strengthen the credibility of the model and align the study with established methodologies in the field.
8)Line 52: Specific types of drugs?
9)Line 72: “a colitis cell model was first established”. Colitis cell model should be specified in detail.
10)Please add supplier including city/country in Materials and methods section.
11)Primer sequences of miR-22-3p and NLRP3
12)CCK8 kit?
13)Please give catalogue no. for each ELISA kit?
14)Please added the catalogue no. of antibody?
15)Pairwise comparison method after one-way ANOVA analysis?
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This research investigated the role of lncRNA MALAT1 in ulcerative colitis (UC) by studying its impact on colonic epithelial cell apoptosis and pyroptosis. They found that MALAT1 expression was significantly increased in LPS-treated human colonic epithelial cells (FHCs), promoting apoptosis and inflammation. Further analysis revealed that MALAT1 knockdown suppressed these harmful effects, suggesting it plays a critical role in UC development. The study discovered that MALAT1 interacts with the microRNA miR-22-3p and the NLRP3 protein, which are key players in inflammation and pyroptosis. They found that MALAT1 binds to miR-22-3p and also regulates the expression of NLRP3. By manipulating these interactions, they demonstrated that MALAT1 plays a crucial role in regulating colonic epithelial cell fate in UC. The research concludes that targeting MALAT1 could be a potential therapeutic strategy for managing UC by reducing inflammation and cell death in the colon. Overall, this research provides valuable insights into the intricate molecular mechanisms underlying UC and offers a promising target for developing new therapies to treat this debilitating disease. However, there are still many issues that need to be improved in this study.
1. The abstract mentions "pyroptosis indicators" but doesn't explicitly list what those indicators are. It might be helpful to name a few key indicators. In addition, instead of simply stating "MALAT1 interference inhibits colonic epithelial cell apoptosis and pyroptosis to relieve ulcerative colitis via miR-22-3p/NLRP3," expand on the implications: "These findings suggest that MALAT1 represents a promising therapeutic target for the treatment of UC by modulating the miR-22-3p/NLRP3 pathway, potentially leading to novel strategies for reducing inflammation and cell death in the colon."
2. The introduction lacks a clear transition between the general information about UC and the specific focus on MALAT1. It abruptly shifts from a general overview to the specific research question without effectively connecting the two. While the introduction mentions that further exploration of MALAT1's role is needed, it doesn't clearly explain why this research is significant or what impact it could have on the field.
3. The materials and methods should provide more details about the specific materials and methods used, including product names, manufacturers, protocols, and reaction condition, such as the specific sequences of the siRNAs and the vectors used for the plasmids.
4. The materials and methods should ensure that the methods are described in enough detail that another researcher could reproduce the experiment. Such as Specify the qPCR protocol, including the reaction mixture, cycling parameters, and the number of technical replicates performed; Provide more details about the Annexin V-FITC/PI assay, including the incubation time and temperature, and the method for analyzing flow cytometry data.
5. The description doesn't clarify when each treatment was administered relative to LPS exposure. For instance, were FHCs treated with LPS first and then transfected with plasmids or siRNAs, or vice versa? This information is essential for understanding the experimental timeline. The author indicated that “Functionally, LPS treatment significantly suppressed FHC proliferation while promoted apoptosis compared to that in blank group (Fig. 1B and 1C). Proliferation was remarkably inhibited in LPS+ov-MALAT1 group while remarkably enhanced in LPS+si-MALAT1 group compared to the corresponding control group (Fig. 1B).” The author should provide a more accurate description of the results, as the cell proliferation at 24 hours does not match the description.
6. This study focuses on the role of MALAT1 in regulating colonic epithelial cell apoptosis and pyroptosis. By detecting pyroptosis, the authors can better understand how MALAT1 contributes to the inflammatory process in UC and whether it specifically promotes this particular form of cell death. In Figure 2, the author needs to clarify why it is necessary to detect pyroptosis?
7. NLRP3 is well-established in apoptosis and pyroptosis, the authors should clearly explain why they focused on NLRP3 over other potential factors influencing MALAT1's effects in Figure 3.
8. The results in Figure 4-6 lacks clarity and precision in its descriptions. Such as “The intersection of four sets of results was found the miR-22-3p (Fig. 4A)”; A detailed description of the results for the four datasets is needed, highlighting the filtering process and principle of miR-22-3p. “The potential binding sites between miR-22-3p and MALAT1/NLRP3 3'-UTR were analyzed by starbase and targetscan (Fig. 4B-Fig. 4C).” The detailed information of the results should be described.
9. To explore function of miR-22-3p on MALAT1/NLRP3, a rescue experiment was performed by transfecting miR-22-3p inhibitor along with si-MALAT1 into LTFs. There is no direct evidence in this study to verify the effect of miR-22-3p on NLRP3.
10. The discussion jumps between different topics, making it difficult to follow the central argument. It doesn't clearly connect the study's findings to the broader context of UC pathogenesis or treatment.
11. The discussion doesn't delve deeply into the implications of the findings. For example, it doesn't explain in detail how the interaction of MALAT1, miR-22-3p, and NLRP3 contributes to the observed effects on cell behavior and inflammation. The conclusion simply reiterates the findings but doesn't address the broader significance or provide specific recommendations for future research.
12. Some sentences are unnecessarily lengthy and contain awkward phrasing, making the reading experience less smooth. For example, "Currently, inflammation of intestinal epithelial cells can lead to apoptosis or pyroptosis of intestinal epithelial cells and Impairment of intestinal absorption function, which was believed to be induced the processed of UC, and may lead to the infiltration of harmful substances transfer to other tissues, causing adverse reactions caused by UC [6-8]." This sentence is overly long, convoluted, and grammatically incorrect.
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