Reviewer's report
Title:The NFKB1 polymorphism (rs4648068) is associated with the cell
proliferation and motility in gastric cancer
Version:4Date:19 November 2014
Reviewer: Kirsten Stone
Reviewer's report:
The authors attempt to answer an important and interesting question about the
mechanism of NF#B polymorphism - dependent differences in gastric cancer
incidence. Data are presented to show that expression of NF#B is correlated to
genotype; other results show increased binding of C/EBP# to the NFKB1
promoter and increased luciferase activity in the presence of the GG genotype. In
addition in vitro data are presented that tie these results to an increased
proliferation and migration rate as well as a decreased apoptosis rate. These
data would be an important contribution if they were more convincing.
Major Compulsory Revisions
1. Figure 1 shows NF#B immunohistochemistry of tissues from gastric cancer
patients. However, the actual origin and tissue type is not described in either
methods or results. Quantification is presented but it is not clear what the
denominator is. Is it all nuclei in the tissue? Were multiple sections from multiple
patients evaluated? Also, the presented tissue sections are not comparable
between genotypes. There seem to be different tissue types in each panel. Panel
c) shows mostly connective tissue and fat, b) epithelial tissue in one panel,
connective tissue in the other, and a) a mix of tissues with what appears to be
mostly lymphocytes in the right hand panel. The method section provides a
formula but does not describe how positive cells were counted. If subjective
measures were used, the intensity of staining may be variable between slides
and not necessarily indicative of quantity. How was the statistical analysis done?
A better approach to quantify the expression of NF#B in gastric cancer tissue is
using qPCR and/or western blot analysis.
2. Figure 3 includes a nice approach to show the role of NFKB polymorphisms in
transcriptional activity. However, the result section should be expanded to
provide a clear description of all panels and a summary of the results of these
experiments in both non-gastric and gastric cancer cell lines. For data presented
in table 1, it is not clear which luciferase plasmid was used. If the description in
the legend means that the data presented are the difference between luciferase
activities of GG and AA reporters, it should be clearly described in the results
section.
3. Figure 5 provides data supposed to demonstrate the physiological importance
of the described effect. However, there are several problems with the
presentation. It is not clear whether the plasmids are stably transfected into cells.
A transient transfection will usually become ineffective within 3 days, so it is not
clear whether the cells still contained the constructs after 6 days. More
importantly, it is not clear to me how transfected promoter regions regulating a
luciferase gene can change the transcription of endogenous NFKB target genes.
The authors should explain how that works and provide evidence from the
literature that this is a valid approach.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:The NFKB1 polymorphism (rs4648068) is associated with the cell
proliferation and motility in gastric cancer
Version:4Date:19 November 2014
Reviewer: Kirsten Stone
Reviewer's report:
The authors attempt to answer an important and interesting question about the
mechanism of NF#B polymorphism - dependent differences in gastric cancer
incidence. Data are presented to show that expression of NF#B is correlated to
genotype; other results show increased binding of C/EBP# to the NFKB1
promoter and increased luciferase activity in the presence of the GG genotype. In
addition in vitro data are presented that tie these results to an increased
proliferation and migration rate as well as a decreased apoptosis rate. These
data would be an important contribution if they were more convincing.
Major Compulsory Revisions
1. Figure 1 shows NF#B immunohistochemistry of tissues from gastric cancer
patients. However, the actual origin and tissue type is not described in either
methods or results. Quantification is presented but it is not clear what the
denominator is. Is it all nuclei in the tissue? Were multiple sections from multiple
patients evaluated? Also, the presented tissue sections are not comparable
between genotypes. There seem to be different tissue types in each panel. Panel
c) shows mostly connective tissue and fat, b) epithelial tissue in one panel,
connective tissue in the other, and a) a mix of tissues with what appears to be
mostly lymphocytes in the right hand panel. The method section provides a
formula but does not describe how positive cells were counted. If subjective
measures were used, the intensity of staining may be variable between slides
and not necessarily indicative of quantity. How was the statistical analysis done?
A better approach to quantify the expression of NF#B in gastric cancer tissue is
using qPCR and/or western blot analysis.
2. Figure 3 includes a nice approach to show the role of NFKB polymorphisms in
transcriptional activity. However, the result section should be expanded to
provide a clear description of all panels and a summary of the results of these
experiments in both non-gastric and gastric cancer cell lines. For data presented
in table 1, it is not clear which luciferase plasmid was used. If the description in
the legend means that the data presented are the difference between luciferase
activities of GG and AA reporters, it should be clearly described in the results
section.
3. Figure 5 provides data supposed to demonstrate the physiological importance
of the described effect. However, there are several problems with the
presentation. It is not clear whether the plasmids are stably transfected into cells.
A transient transfection will usually become ineffective within 3 days, so it is not
clear whether the cells still contained the constructs after 6 days. More
importantly, it is not clear to me how transfected promoter regions regulating a
luciferase gene can change the transcription of endogenous NFKB target genes.
The authors should explain how that works and provide evidence from the
literature that this is a valid approach.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:The NFKB1 polymorphism (rs4648068) is associated with the cell
proliferation and motility in gastric cancer
Version:4Date:19 November 2014
Reviewer: Kirsten Stone
Reviewer's report:
The authors attempt to answer an important and interesting question about the
mechanism of NF#B polymorphism - dependent differences in gastric cancer
incidence. Data are presented to show that expression of NF#B is correlated to
genotype; other results show increased binding of C/EBP# to the NFKB1
promoter and increased luciferase activity in the presence of the GG genotype. In
addition in vitro data are presented that tie these results to an increased
proliferation and migration rate as well as a decreased apoptosis rate. These
data would be an important contribution if they were more convincing.
Major Compulsory Revisions
1. Figure 1 shows NF#B immunohistochemistry of tissues from gastric cancer
patients. However, the actual origin and tissue type is not described in either
methods or results. Quantification is presented but it is not clear what the
denominator is. Is it all nuclei in the tissue? Were multiple sections from multiple
patients evaluated? Also, the presented tissue sections are not comparable
between genotypes. There seem to be different tissue types in each panel. Panel
c) shows mostly connective tissue and fat, b) epithelial tissue in one panel,
connective tissue in the other, and a) a mix of tissues with what appears to be
mostly lymphocytes in the right hand panel. The method section provides a
formula but does not describe how positive cells were counted. If subjective
measures were used, the intensity of staining may be variable between slides
and not necessarily indicative of quantity. How was the statistical analysis done?
A better approach to quantify the expression of NF#B in gastric cancer tissue is
using qPCR and/or western blot analysis.
2. Figure 3 includes a nice approach to show the role of NFKB polymorphisms in
transcriptional activity. However, the result section should be expanded to
provide a clear description of all panels and a summary of the results of these
experiments in both non-gastric and gastric cancer cell lines. For data presented
in table 1, it is not clear which luciferase plasmid was used. If the description in
the legend means that the data presented are the difference between luciferase
activities of GG and AA reporters, it should be clearly described in the results
section.
3. Figure 5 provides data supposed to demonstrate the physiological importance
of the described effect. However, there are several problems with the
presentation. It is not clear whether the plasmids are stably transfected into cells.
A transient transfection will usually become ineffective within 3 days, so it is not
clear whether the cells still contained the constructs after 6 days. More
importantly, it is not clear to me how transfected promoter regions regulating a
luciferase gene can change the transcription of endogenous NFKB target genes.
The authors should explain how that works and provide evidence from the
literature that this is a valid approach.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests.
assistant