Title: miR-541 is associated with the prognosis of liver cirrhosis and directly targets JAG2 to
inhibit the activation of hepatic stellate cells
Version: 0 Date: 21 Oct 2023
Reviewer's report:
Comment
Focusing on JAG2 as a new mechanism of liver fibrosis, the findings regarding the progression
of fibrosis through the Notch signal pathway and the application of therapeutic drugs are new
and interesting. The utility of miR-541 as a prognostic marker for liver disease is also of interest.
What is the possible source of serum miR-541 and the mechanism by which expression of
miR-541 decreases? Also, is the expression of miR-541 in liver tissue and serum miR-541
correlated?
Compared to compensated cirrhosis, decompensated liver cirrhosis exhibits changes such as
increased blood ammonia, increased bilirubin, decreased albumin, and decreased renal function.
Basically, miRNA is degraded by RNase in the blood, so it is unlikely that the nucleic acid exists
alone, and it is highly likely that it coexists with a carrier protein. It is highly likely that the
amount of carrier protein in the blood is decreased in the state of decompensated liver cirrhosis,
so is it not possible that it simply reflects the amount of carrier protein?
I have believed that caution should be taken when interpreting the results of experiments
conducted using LX-2.
While the human hepatic stellate cell line LX-2 has the advantage of being relatively easy to
grow and use in experiments, it does not store Vit A so it is difficult to regard quiescent stellate
cells even before activation with TGF#.
Moreover, it is also known that repetitive passase of LX-2 cells  develops myofibroblast
characteristics. Therefore, when evaluating hepatic stellate cells, it is better to confirm
reproducibility by experimenting with another cell line. For example, it is preferable to use
immature cells such as Li90 or commercially available primary cultured cells.
At what stage of chronic liver injury does JAG2 induce liver fibrosis?
In liver fibrosis, hypoxic conditions are induced by inflammation, and capillary formation
of hepatic trunk cells occurs. Capillary formation further exacerbates hypoxia, and hepatic
stellate cells are activated by induction of inflammatory cytokines such as TNF#. At that
time, extracellular matrix is produced, the network between hepatocytes deteriorates, and liver
function declines.
If liver fibrosis is treated using the miR-541/JAG2 axis system, which part of the liver fibrosis
process is most likely to be improved? If this point is clarified, it is expected that the feasibility
of drug discovery as a new drug for the treatment of liver fibrosis will also become clear.
Since miR-541 has many target genes, it may control various pathways involved in liver fibrosis
and, as a result, may control liver fibrosis.
For example, it is known that Wnt/#catenin signal and EMT are closely related to liver fibrosis,
so even if the expression of molecules included in these pathways is controlled with siRNA,
there is a high possibility to conduct similar result in vitro experiment. Is it possible that miR-541
regulates other fibrosis-related factors? What role does miR-541/JAG2 axis play in the multiple
pathways of liver fibrosis progression (how important is it? ), I would like to ask for comments
on this point.
Regarding clinical specimens, were you able to track changes in miR-541 expression during the
course of treatment in the same patient?  If we want to claim the clinical usefulness of miR-541,
I think it would be better to show changes in miR-541 expression associated with treatment.
Title: miR-541 is associated with the prognosis of liver cirrhosis and directly targets JAG2 to
inhibit the activation of hepatic stellate cells
Version: 0 Date: 21 Oct 2023
Reviewer's report:
Comment
Focusing on JAG2 as a new mechanism of liver fibrosis, the findings regarding the progression
of fibrosis through the Notch signal pathway and the application of therapeutic drugs are new
and interesting. The utility of miR-541 as a prognostic marker for liver disease is also of interest.
What is the possible source of serum miR-541 and the mechanism by which expression of
miR-541 decreases? Also, is the expression of miR-541 in liver tissue and serum miR-541
correlated?
Compared to compensated cirrhosis, decompensated liver cirrhosis exhibits changes such as
increased blood ammonia, increased bilirubin, decreased albumin, and decreased renal function.
Basically, miRNA is degraded by RNase in the blood, so it is unlikely that the nucleic acid exists
alone, and it is highly likely that it coexists with a carrier protein. It is highly likely that the
amount of carrier protein in the blood is decreased in the state of decompensated liver cirrhosis,
so is it not possible that it simply reflects the amount of carrier protein?
I have believed that caution should be taken when interpreting the results of experiments
conducted using LX-2.
While the human hepatic stellate cell line LX-2 has the advantage of being relatively easy to
grow and use in experiments, it does not store Vit A so it is difficult to regard quiescent stellate
cells even before activation with TGF#.
Moreover, it is also known that repetitive passase of LX-2 cells  develops myofibroblast
characteristics. Therefore, when evaluating hepatic stellate cells, it is better to confirm
reproducibility by experimenting with another cell line. For example, it is preferable to use
immature cells such as Li90 or commercially available primary cultured cells.
At what stage of chronic liver injury does JAG2 induce liver fibrosis?
In liver fibrosis, hypoxic conditions are induced by inflammation, and capillary formation
of hepatic trunk cells occurs. Capillary formation further exacerbates hypoxia, and hepatic
stellate cells are activated by induction of inflammatory cytokines such as TNF#. At that
time, extracellular matrix is produced, the network between hepatocytes deteriorates, and liver
function declines.
If liver fibrosis is treated using the miR-541/JAG2 axis system, which part of the liver fibrosis
process is most likely to be improved? If this point is clarified, it is expected that the feasibility
of drug discovery as a new drug for the treatment of liver fibrosis will also become clear.
Since miR-541 has many target genes, it may control various pathways involved in liver fibrosis
and, as a result, may control liver fibrosis.
For example, it is known that Wnt/#catenin signal and EMT are closely related to liver fibrosis,
so even if the expression of molecules included in these pathways is controlled with siRNA,
there is a high possibility to conduct similar result in vitro experiment. Is it possible that miR-541
regulates other fibrosis-related factors? What role does miR-541/JAG2 axis play in the multiple
pathways of liver fibrosis progression (how important is it? ), I would like to ask for comments
on this point.
Regarding clinical specimens, were you able to track changes in miR-541 expression during the
course of treatment in the same patient?  If we want to claim the clinical usefulness of miR-541,
I think it would be better to show changes in miR-541 expression associated with treatment.
Please review this paper.
user
Title: miR-541 is associated with the prognosis of liver cirrhosis and directly targets JAG2 to
inhibit the activation of hepatic stellate cells
Version: 0 Date: 21 Oct 2023
Reviewer's report:
Comment
Focusing on JAG2 as a new mechanism of liver fibrosis, the findings regarding the progression
of fibrosis through the Notch signal pathway and the application of therapeutic drugs are new
and interesting. The utility of miR-541 as a prognostic marker for liver disease is also of interest.
What is the possible source of serum miR-541 and the mechanism by which expression of
miR-541 decreases? Also, is the expression of miR-541 in liver tissue and serum miR-541
correlated?
Compared to compensated cirrhosis, decompensated liver cirrhosis exhibits changes such as
increased blood ammonia, increased bilirubin, decreased albumin, and decreased renal function.
Basically, miRNA is degraded by RNase in the blood, so it is unlikely that the nucleic acid exists
alone, and it is highly likely that it coexists with a carrier protein. It is highly likely that the
amount of carrier protein in the blood is decreased in the state of decompensated liver cirrhosis,
so is it not possible that it simply reflects the amount of carrier protein?
I have believed that caution should be taken when interpreting the results of experiments
conducted using LX-2.
While the human hepatic stellate cell line LX-2 has the advantage of being relatively easy to
grow and use in experiments, it does not store Vit A so it is difficult to regard quiescent stellate
cells even before activation with TGF#.
Moreover, it is also known that repetitive passase of LX-2 cells  develops myofibroblast
characteristics. Therefore, when evaluating hepatic stellate cells, it is better to confirm
reproducibility by experimenting with another cell line. For example, it is preferable to use
immature cells such as Li90 or commercially available primary cultured cells.
At what stage of chronic liver injury does JAG2 induce liver fibrosis?
In liver fibrosis, hypoxic conditions are induced by inflammation, and capillary formation
of hepatic trunk cells occurs. Capillary formation further exacerbates hypoxia, and hepatic
stellate cells are activated by induction of inflammatory cytokines such as TNF#. At that
time, extracellular matrix is produced, the network between hepatocytes deteriorates, and liver
function declines.
If liver fibrosis is treated using the miR-541/JAG2 axis system, which part of the liver fibrosis
process is most likely to be improved? If this point is clarified, it is expected that the feasibility
of drug discovery as a new drug for the treatment of liver fibrosis will also become clear.
Since miR-541 has many target genes, it may control various pathways involved in liver fibrosis
and, as a result, may control liver fibrosis.
For example, it is known that Wnt/#catenin signal and EMT are closely related to liver fibrosis,
so even if the expression of molecules included in these pathways is controlled with siRNA,
there is a high possibility to conduct similar result in vitro experiment. Is it possible that miR-541
regulates other fibrosis-related factors? What role does miR-541/JAG2 axis play in the multiple
pathways of liver fibrosis progression (how important is it? ), I would like to ask for comments
on this point.
Regarding clinical specimens, were you able to track changes in miR-541 expression during the
course of treatment in the same patient?  If we want to claim the clinical usefulness of miR-541,
I think it would be better to show changes in miR-541 expression associated with treatment.
assistant