Title: METTL3 regulates N6-methyladenosine modification of ANGPTL3 mRNA and
potentiates malignant progression of stomach adenocarcinoma
Version: 1 Date: 27 Mar 2023
Reviewer's report:
This manuscript by Zhang et al. aims to see if high expression of the m6A methyltransferase
METTL3 can drive malignant phenotypes in stomach adenocarcinoma (STAD) cell lines.
They also identify ANGPTL3 mRNA as a potential target of m6A in STAD lines and show
methylation of ANGPTL3 mature mRNA decreases its half-life. They conclude that low
ANGPTL3 expression could be used a biomarker for STAD.
The identification of ANGPTL3 as a target for METTL3 does elucidate a potential new
mechanism of STAD development and the studies are convincing that high METTL3 expression
does drive oncogenic STAD phenotypes. However, some of the experimental quality is low
or poorly designed and some of the conclusions are poorly worded. I suggest the following
revisions before acceptance.
1. Vague words (mediated, altered, regulated…) are used almost everywhere to describe how
m6A methylation affects ANGPTL3 mRNA levels.  As m6A methylation often promotes mRNA
decay, but can also promote its stability, decreases and/or increases should be used instead so
one can better understand the actual model. Also, some discussion in the introduction on how
m6A methylation can promote either mRNA decay or stability, and which is happening here,
would be helpful.
2. Western blots are needed to confirm knockdown in figures 2A and 4A, and the western blots
in 1C, 3D, and 3G are very poor quality.  They are out of focus in the TIFs and highly digitalized
in the web version.
3. Immunofluorescence images from supplemental figure 2 showing co-localization of METTL3
and ANGPLT3 proteins should be removed. They are of very poor quality and hard to interpret,
but from what I can see, there is no co-localization. Also, as METTL3 methylates ANGPTL3
mRNA, it is not clear why the proteins would be expected to co-localize.
4. All the experiments in the section “The tumor suppressor effect of METTL3 silencing can
be relieved to some extent by ANGPTL3 inhibition” also do not make sense. The whole paper
is about METTL3 overexpression as an oncogene and how it results in lower expression of
ANGPTL3 (here apparently working as a tumor suppressor). If this is the model, the more
relevant experiments would be to overexpress ANGPTL3 in the STAD cell lines and see if that
relieves oncogenic phenotypes.
Minor comments:
1. In the first paragraph of the results section it is written “to confirm whether METTL3 was
a pathogenic factor in STAD…” and it goes on to say that because it is highly expressed it is
pathogenic.  Because something is highly expressed does not necessarily make it pathogenic.
This should be rewritten “to confirm high METTL3 expression in STAD…”
2. In the second section of the results section it is written “…the proliferative capacity of AGS
and HGC27 cells showed the same trend.” This gives the impression no statistical significance
was found, though the figure indicates this is statistically significant data. This sentence should
be reworded to indicate there is statistical significance. Also colony formation assays don’t
measure proliferative capacity.
3. In the third section of the results section it is written “Determining the threshold as cor > 0.2
and P < 0.05, ANGPTL3 revealed strong correlation with METTL3.” Again this wording is too
vague.  It should be written “strong negative correlation” for clarity.
4. The third paragraph of the Discussion “METTL3 can activate cell proliferation by installing
m6A on key genes that are critical for cell growth” again is incorrect if they are focusing on
methylation by METTL3 promoting mRNA degradation. It should read “…on key genes that
are critical for blocking cell growth.”
Title: METTL3 regulates N6-methyladenosine modification of ANGPTL3 mRNA and
potentiates malignant progression of stomach adenocarcinoma
Version: 1 Date: 27 Mar 2023
Reviewer's report:
This manuscript by Zhang et al. aims to see if high expression of the m6A methyltransferase
METTL3 can drive malignant phenotypes in stomach adenocarcinoma (STAD) cell lines.
They also identify ANGPTL3 mRNA as a potential target of m6A in STAD lines and show
methylation of ANGPTL3 mature mRNA decreases its half-life. They conclude that low
ANGPTL3 expression could be used a biomarker for STAD.
The identification of ANGPTL3 as a target for METTL3 does elucidate a potential new
mechanism of STAD development and the studies are convincing that high METTL3 expression
does drive oncogenic STAD phenotypes. However, some of the experimental quality is low
or poorly designed and some of the conclusions are poorly worded. I suggest the following
revisions before acceptance.
1. Vague words (mediated, altered, regulated…) are used almost everywhere to describe how
m6A methylation affects ANGPTL3 mRNA levels.  As m6A methylation often promotes mRNA
decay, but can also promote its stability, decreases and/or increases should be used instead so
one can better understand the actual model. Also, some discussion in the introduction on how
m6A methylation can promote either mRNA decay or stability, and which is happening here,
would be helpful.
2. Western blots are needed to confirm knockdown in figures 2A and 4A, and the western blots
in 1C, 3D, and 3G are very poor quality.  They are out of focus in the TIFs and highly digitalized
in the web version.
3. Immunofluorescence images from supplemental figure 2 showing co-localization of METTL3
and ANGPLT3 proteins should be removed. They are of very poor quality and hard to interpret,
but from what I can see, there is no co-localization. Also, as METTL3 methylates ANGPTL3
mRNA, it is not clear why the proteins would be expected to co-localize.
4. All the experiments in the section “The tumor suppressor effect of METTL3 silencing can
be relieved to some extent by ANGPTL3 inhibition” also do not make sense. The whole paper
is about METTL3 overexpression as an oncogene and how it results in lower expression of
ANGPTL3 (here apparently working as a tumor suppressor). If this is the model, the more
relevant experiments would be to overexpress ANGPTL3 in the STAD cell lines and see if that
relieves oncogenic phenotypes.
Minor comments:
1. In the first paragraph of the results section it is written “to confirm whether METTL3 was
a pathogenic factor in STAD…” and it goes on to say that because it is highly expressed it is
pathogenic.  Because something is highly expressed does not necessarily make it pathogenic.
This should be rewritten “to confirm high METTL3 expression in STAD…”
2. In the second section of the results section it is written “…the proliferative capacity of AGS
and HGC27 cells showed the same trend.” This gives the impression no statistical significance
was found, though the figure indicates this is statistically significant data. This sentence should
be reworded to indicate there is statistical significance. Also colony formation assays don’t
measure proliferative capacity.
3. In the third section of the results section it is written “Determining the threshold as cor > 0.2
and P < 0.05, ANGPTL3 revealed strong correlation with METTL3.” Again this wording is too
vague.  It should be written “strong negative correlation” for clarity.
4. The third paragraph of the Discussion “METTL3 can activate cell proliferation by installing
m6A on key genes that are critical for cell growth” again is incorrect if they are focusing on
methylation by METTL3 promoting mRNA degradation. It should read “…on key genes that
are critical for blocking cell growth.”
Please review this paper.
user
Title: METTL3 regulates N6-methyladenosine modification of ANGPTL3 mRNA and
potentiates malignant progression of stomach adenocarcinoma
Version: 1 Date: 27 Mar 2023
Reviewer's report:
This manuscript by Zhang et al. aims to see if high expression of the m6A methyltransferase
METTL3 can drive malignant phenotypes in stomach adenocarcinoma (STAD) cell lines.
They also identify ANGPTL3 mRNA as a potential target of m6A in STAD lines and show
methylation of ANGPTL3 mature mRNA decreases its half-life. They conclude that low
ANGPTL3 expression could be used a biomarker for STAD.
The identification of ANGPTL3 as a target for METTL3 does elucidate a potential new
mechanism of STAD development and the studies are convincing that high METTL3 expression
does drive oncogenic STAD phenotypes. However, some of the experimental quality is low
or poorly designed and some of the conclusions are poorly worded. I suggest the following
revisions before acceptance.
1. Vague words (mediated, altered, regulated…) are used almost everywhere to describe how
m6A methylation affects ANGPTL3 mRNA levels.  As m6A methylation often promotes mRNA
decay, but can also promote its stability, decreases and/or increases should be used instead so
one can better understand the actual model. Also, some discussion in the introduction on how
m6A methylation can promote either mRNA decay or stability, and which is happening here,
would be helpful.
2. Western blots are needed to confirm knockdown in figures 2A and 4A, and the western blots
in 1C, 3D, and 3G are very poor quality.  They are out of focus in the TIFs and highly digitalized
in the web version.
3. Immunofluorescence images from supplemental figure 2 showing co-localization of METTL3
and ANGPLT3 proteins should be removed. They are of very poor quality and hard to interpret,
but from what I can see, there is no co-localization. Also, as METTL3 methylates ANGPTL3
mRNA, it is not clear why the proteins would be expected to co-localize.
4. All the experiments in the section “The tumor suppressor effect of METTL3 silencing can
be relieved to some extent by ANGPTL3 inhibition” also do not make sense. The whole paper
is about METTL3 overexpression as an oncogene and how it results in lower expression of
ANGPTL3 (here apparently working as a tumor suppressor). If this is the model, the more
relevant experiments would be to overexpress ANGPTL3 in the STAD cell lines and see if that
relieves oncogenic phenotypes.
Minor comments:
1. In the first paragraph of the results section it is written “to confirm whether METTL3 was
a pathogenic factor in STAD…” and it goes on to say that because it is highly expressed it is
pathogenic.  Because something is highly expressed does not necessarily make it pathogenic.
This should be rewritten “to confirm high METTL3 expression in STAD…”
2. In the second section of the results section it is written “…the proliferative capacity of AGS
and HGC27 cells showed the same trend.” This gives the impression no statistical significance
was found, though the figure indicates this is statistically significant data. This sentence should
be reworded to indicate there is statistical significance. Also colony formation assays don’t
measure proliferative capacity.
3. In the third section of the results section it is written “Determining the threshold as cor > 0.2
and P < 0.05, ANGPTL3 revealed strong correlation with METTL3.” Again this wording is too
vague.  It should be written “strong negative correlation” for clarity.
4. The third paragraph of the Discussion “METTL3 can activate cell proliferation by installing
m6A on key genes that are critical for cell growth” again is incorrect if they are focusing on
methylation by METTL3 promoting mRNA degradation. It should read “…on key genes that
are critical for blocking cell growth.”
assistant