Review for 10.1186/s12876-022-02097-0 (2022)

Title: PADI4 promotes epithelial-mesenchymal transition(EMT) in gastric cancer via the
upregulation of interleukin 8
Version: 3 Date: 19 Oct 2021
Reviewer's report:
PADI4 (Protein Arginine Deiminase 4) catalyzes the citrullination/deimination of arginine
residues of a protein. This citrullination/deimination affects the protein’s structural stability
and its function. Gastric cancer is very deadly cancer and often diagnosed with metastatic
property. Previous studies have documented the association between PADI4 and many cancers,
including ovarian cancer, colorectal cancer, lung cancer, cervical squamous cell carcinoma,
gastric cancer and thyroid carcinoma. In this study entitled as “PADI4 promotes epithelial-
mesenchymal transition (EMT) in gastric cancer via the upregulation of interleukin 8”, Chang
et.al. showed that,  PADI4 is overexpressed in Gastric cancer tissues compared to the adjacent
mucosal tissues. Using transfection assays with PADI4 expressing plasmid, this study has
shown that PADI4 overexpression increases the migration ability of cells as well as decreases
the expression of E-cadherin and increases expression levels of Slug and vimentin in vitro.
In addition, this study also demonstrated that PADI4 overexpression facilitates the colony
forming and proliferation capabilities of the cells. The percentage of apoptosis (FITC staining
assay) was significantly decreased in cells transfected with PADI4 plasmid compared to the
control. Furthermore, this study has documented that mice injected with PADI4-overexpressing
virus transfected MGC80-3 cells have significantly increased tumor growth. Finally, using
siRNA transfection assay against IL8 this study has established that IL-8 silencing significantly
inhibited the increased migratory capacity in PADI4-overexpressing cells.
The authors have carried out in vitro, in vivo assays to demonstrate that PADI4 increased the
proliferation, migration, metastasis, clone forming ability, and tumorigenic ability, but reduced
the apoptosis ability. The overexpression of PADI4 has also been confirmed in the tumor tissues.
In addition the authors have shown that PADI4 promotes the IL8 expression which leads to the
metastasis stage in gastric cancer cells.
However there are some issues that need to be addressed:
1. Rearrangement of the methods according to the sequence of results would be better for the
reader
2. Please mention the gene name (PADI4) in Fig1 B and C
3. Indicate the percentages of cells in each quadrant in the Fig3E.
4. Please cite the authors’ previous study mentioned in the section “IL-8 knock-down reversed
the effects of PADI4 on GC cells EMT and migration” (P-12)
5. Indicating each and every p value in the result section will help the reader.
6. In vivo results could be mentioned in separate part with another heading.
7. The values of the expression of E-Cadherin (Fig 2B) need to be checked. The difference with
the control is very small.
8. Please provide the full data of cytokines measured in this study particularly in the figure 5A
and B in the supplementary. The difference in cytokine levels with the control is very little.
9. The level of the silencing (in percentage) after the siRNA transfection against IL-8 is very
marginal (about 20% as per the figure 6 B and D). At least 50% silencing will provide better
result.