STM2457 Inhibits the Invasion and Metastasis of Pancreatic Cancer by Down-Regulating BRAF-Activated Noncoding RNA N6-Methyladenosine Modification
Round 1
Reviewer 1 Report
This study investigates the potential therapeutic target of BRAF-activated noncoding RNA (BANCR) in pancreatic cancer. The study focuses on the effect of STM2457 on BANCR m6A methylation and its impact on the malignant behaviors of pancreatic cancer cells. The expression of BANCR is found to be overexpressed in pancreatic cancer tissues and cells, and it is associated with poor clinical outcomes. The study suggests that targeting BANCR m6A modification with STM2457 may inhibit the invasion and metastasis of pancreatic cancer.
Key points
BANCR is overexpressed in pancreatic cancer tissues and cells.
STM2457 inhibits the invasion and metastasis of pancreatic cancer cells by down-regulating BANCR m6A modification.
The study highlights the potential therapeutic value of targeting BANCR m6A modification in preventing and treating pancreatic cancer invasion and metastasis.
Points for revision:
Introduction: The introduction section could be expanded to provide more background information on pancreatic cancer and the role of BANCR in its invasion and metastasis. This would help readers better understand the significance of the study.
Methods: The authors could provide additional information on the specific protocols and techniques used in the study, such as the MeRIP-PCR and Real-Time qRT-PCR procedures. This would enhance the reproducibility of the experiments and allow readers to understand the methodology better.
Results: The results section should include more quantitative data and statistical analysis to support the conclusions drawn. This would enhance the credibility of the study and provide a more comprehensive understanding of the results.
While the study mentions the overexpression of BANCR in pancreatic cancer tissues and cells, including more detailed data on the expression levels and statistical analysis would be beneficial. This would strengthen the findings and provide a clearer understanding of the relationship between BANCR and pancreatic cancer.
Discussion: The discussion section should discuss the implications of the findings in the context of existing literature. It would be beneficial to compare the results with previous studies and highlight any novel insights or discrepancies. The authors could expand on the potential clinical implications of their findings. This could involve discussing how targeting BANCR m6A modification with STM2457 could be translated into therapeutic strategies for pancreatic cancer patients. Providing insights into the practical applications of the research would make the article more relevant and impactful.
Limitations: It would be helpful for the authors to acknowledge any limitations or potential biases in their research. This could include factors such as sample size, selection criteria, or any other potential confounding variables. Addressing these limitations would improve the overall credibility and reliability of the study.
Conclusion: The conclusion section should summarize the study's main findings and their potential implications. It would be helpful to provide suggestions for future research directions based on the current findings.
References: The references section should be updated to include the most recent and relevant studies in the field. This would ensure that the study is grounded in the current scientific literature.
Overall, implementing these suggestions would strengthen the article by providing more detailed experimental information comprehensive results, acknowledging limitations, discussing clinical implications, and supporting the claims with additional references.
Need attention and should be improvised.
Author Response
Response to Reviewer 1 Comments
Introduction: The introduction section could be expanded to provide more background information on pancreatic cancer and the role of BANCR in its invasion and metastasis. This would help readers better understand the significance of the study.
Response： Thank you suggestion.We have modified it according to your opinion and added that literature.
This change is as follows: "Although surgery, radiotherapy and chemotherapy treatment have greatly improved over time, the prognosis of PC has not significantly improved in the past 20 years[4]. Recent advances revealed that only <2% transcripts of human genome code for proteins and the remaining 98% transcripts encode different classes of non-coding RNAs[5].Though Long non-coding RNAs (lncRNAs) are comprising more than 200 nucleotides that lack protein coding capacity, they are involved in diverse biological processes, including cell growth, differentiation and proliferation[6]. There is mounting evidence that lncRNAs play essential roles in PC cell proliferation, apoptosis, and metastasis. Accordingly, it is important to investigate the mechanisms that lncRNA contribute to metastasis in pancreatic cancer. " And "In addition, Liu et al.[12] identified that BANCR was upregulated in pancreatic cancer tissues and cell lines and promotes pancreatic cancer tumorigenesis through miR-195-5p/Wnt/β-catenin axis may serve as a potential target for diagnostics and therapeutics in pancreatic cancer. "
Methods: The authors could provide additional information on the specific protocols and techniques used in the study, such as the MeRIP-PCR and Real-Time qRT-PCR procedures. This would enhance the reproducibility of the experiments and allow readers to understand the methodology better.
Response：Thank you suggestion.We've added "RNA Isolation and Quantitative Real-Time PCR" in the method.
This change is as follows: " Total RNA was extracted from cells or tissues using AipPure TRIzol Total RNA Ex-traction Reagent (i-presci , Beijing ,China) according to the manufacturer’s instructions. For miRNA assays, TaqMan Mi-croRNA Assays (Applied Biosystems; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA)were used to quantify the expression levels of mature miRNAs. cDNA was synthesized from total RNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosys-tems; Thermo Fisher Scientific, Inc.) with assay-specific TaqMan primers, and quantitativereal-time PCR was performed using 2× TaqMan Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following assay IDs were used: BANCR (ncRNA, 100885775), METTL3 (protein coding, 56339) and GAPDH (protein coding, 2597), (all purchased from Applied Biosys-tems; Thermo Fisher Scientific, Inc.). The reactions were performed on a 7500 Real-TimePCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression levels were calculated using the 2−∆∆Ct method."
Results: The results section should include more quantitative data and statistical analysis to support the conclusions drawn. This would enhance the credibility of the study and provide a more comprehensive understanding of the results.
While the study mentions the overexpression of BANCR in pancreatic cancer tissues and cells, including more detailed data on the expression levels and statistical analysis would be beneficial. This would strengthen the findings and provide a clearer understanding of the relationship between BANCR and pancreatic cancer.
Response：Thank you suggestion. This change is as follows:”As shown in Fig.1A, the level of BANCR in 77.4% (24/31) pancreatic cancer tissues(2.45±0.18) was higher than that in adjacent normal tissues(1.31±0.12). The expression of BANCR in pancreatic cancer cell lines SW1990(11.04±1.26) and PANC-1(12.77±1.03) was higher than normal pancreatic ductal epithelium cell (HPDEC) (Fig.1B). ”
Discussion: The discussion section should discuss the implications of the findings in the context of existing literature. It would be beneficial to compare the results with previous studies and highlight any novel insights or discrepancies. The authors could expand on the potential clinical implications of their findings. This could involve discussing how targeting BANCR m6A modification with STM2457 could be translated into therapeutic strategies for pancreatic cancer patients. Providing insights into the practical applications of the research would make the article more relevant and impactful.
Response：Thank you suggestion. This change is as follows:” So we think STM2457 could be a targeted drug for pancreatic cancer treatment. Our work is the first attempt to demonstrate that STM2457 targeting BANCRm6A may be an alternative strategy for controlling pancreatic cancer progression. But the molecular mechanism by which STM2457 inhibits BANCR m6A remains to be explored, which is also the goal of our next work. In addition, STM2457 may play an anticancer role in BANCR and other types of cancers with high levels of m6A, and its efficacy and safety should be evaluated in more clinically relevant cancer models in the future.”
Limitations: It would be helpful for the authors to acknowledge any limitations or potential biases in their research. This could include factors such as sample size, selection criteria, or any other potential confounding variables. Addressing these limitations would improve the overall credibility and reliability of the study.
Response：Thank you suggestion. This change is as follows:” Despite the significant results of the current study, there are still some shortcomings to be considered. The present study was a single-center retrospective study with a small sample size, and therefore further studies with larger sample sizes should be conducted. The molecular mechanism of BANCRm6A inhibition by STM2457 needs to be further explored.The inhibitory effect of STM2457 on pancreatic cancer has not been verified in experimental animals.
Conclusion: The conclusion section should summarize the study's main findings and their potential implications. It would be helpful to provide suggestions for future research directions based on the current findings.
Response：Thank you suggestion. We have made the additions accordingly, as follows:”In the future, we want to further investigate the molecular mechanism by which STM2457 affects BANCRm6A and find the structural location of the interaction. To lay the foundation for STM2457 to be used in clinical treatment.”
References: The references section should be updated to include the most recent and relevant studies in the field. This would ensure that the study is grounded in the current scientific literature.
Response：Thank you suggestion. We have added reference 4,5,12,29, 30, 31 (PMID: 25626938, 22955620, 31367258, 35574332, 36898427, 36932427).
All changes in the text are marked in red.
Reviewer 2 Report
The manuscript of Hao et al. reports an overexpression of a long non-coding RNA BANCR in pancreatic cancer tissues, its effects on the cell invasion, proliferation and migration, m6A modification of BANCR and its correlation with METTL3, a key player in the methylation. Finally, the authors investigate a potential treatment with an STM2457, a small molecule, and the effects of the treatment on the cell invasion, migration and proliferation.
Altogether, the authors identified BANCR and pathways related to it as a potential an interesting therapeutic target, much needed given that several small molecules proved not to be effective in pancreatic cancer.
The manuscript is well-written, the introduction explanatory and the discussion highlights the benefits of the study. Figures are of good quality and materials and methods clearly described.
Comments:
1) Title please spell out PC
2) Abstract: Please write it more clearly, e.g. explain what is STM2457, METTL3. Also, some parts of the abstract are in bold.
3) Table 1 and a supplementary table are mentioned, but I cannot find them. In the supplementary materials, I found only WB
4) Lines 168-172 should be included in the methods
5) Please include the authors' contributions and the number of ethical approval
Author Response
Response to Reviewer 2 Comments
Point1. Title please spell out PC
Response1：Thank you for the comments. We have spelled out it, as follows:”STM2457 inhibits the invasion and metastasis of pancreatic cancer by down-regulating BANCR m6A modification”
Point2. Abstract: Please write it more clearly, e.g. explain what is STM2457, METTL3. Also, some parts of the abstract are in bold.
Response2：Thank you suggestion . We have defined “a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457)”,” methyltransferase-like 3 (METTL3), the key enzymeinvolved in m6A methylation.”
Point3. Table 1 and a supplementary table are mentioned, but I cannot find them. In the supplementary materials, I found only WB
Response3: Thank you for the comments. We have uploaded it.
Point4. Lines 168-172 should be included in the methods
Response 4: Thank you suggestion. This change is as follows: The expression of BANCR in pancreatic cancer cell lines(SW1990, PANC-1) was higher than normal pancreatic ductal epithelium cell (HPDEC). And added "RNA Isolation and Quantitative Real-Time PCR" in the method.” Total RNA was extracted from cells or tissues using AipPure TRIzol Total RNA Ex-traction Reagent (i-presci , Beijing ,China) according to the manufacturer’s instructions. For miRNA assays, TaqMan Mi-croRNA Assays (Applied Biosystems; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA)were used to quantify the expression levels of mature miRNAs. cDNA was synthesizedfrom total RNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosys-tems; Thermo Fisher Scientific, Inc.) with assay-specific TaqMan primers, and quantitativereal-time PCR was performed using 2× TaqMan Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following assay IDs were used: BANCR (ncRNA, 100885775), METTL3 (protein coding, 56339) and GAPDH (protein coding, 2597), (all purchased from Applied Biosys-tems; Thermo Fisher Scientific, Inc.). The reactions were performed on a 7500 Real-TimePCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression levels were calculated using the 2−∆∆Ct method.”
Point5. Please include the authors' contributions and the number of ethical approval
Response 5: Thank you suggestion. We have made the additions accordingly, as follows:”Author Contributions: Conceptualization, Wei Han and Shaolong Hao ; validation, Shaolong Hao, Hao Sun, Bo Zhang and Kailun Jiï¼› formal analysis, Shaolong Hao; investigation, Wei Han.; writing—original draft preparation, Shaolong Hao, Haitao Sun; visualization, Peng Liu，Fang Nie; supervision, Wei Han and Haitao Sun; funding acquisition, Wei Han and Shaolong Hao . All authors have read and agreed to the published version of the manuscript.”
“Ethics approval and consent to participate: The institution's Ethics and Research Committees of Beijing Luhe hospital Capital Medical University approved the study. All patients signed informed consent for participation in the study, which was conducted according to the Declaration of Helsinki.Institutional Review Board Statement: This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Research Ethics Committee on Genetic Analysis at Beijing Luhe hospital Capital Medical University (approval no. 2022-LHKY-030-02).”
All changes in the text are marked in red.
