MicroRNAs Regulate Ca²⁺ Homeostasis in Murine Embryonic Stem Cells

Round 1

Reviewer 1 Report

This is a review of manuscript cells-2515614 “microRNAs regulate Ca²⁺ homeostasis in murine embryonic stem cells” by Reid et al. The authors exhibited that miRNAs play an important role of maintaining the intracellular Ca²⁺ homeostasis through the indirect regulation of Itpr2 expression level in ESCs. miRNAs might affect the Rbl2, Lats2, and Cdk1a whose miRNA is important in the differentiation and cell cycle regulation of ESCs.

This reviewer found this study is interesting, however, this reviewer has following major concerns.

1.       Do these effects of loss of Dicer really represent loss of miRNAs? The authors showed only indirect evidence that miRNAs regulate Ca²⁺ homeostasis.

2.       In Fig.3, the authors investigated apoptosis using a reduced concentration of thapsigargin at 200 nM. However, the authors did not examine that the effect of 200 nM thapsigargin on Ca²⁺ response. This should be confirmed.

3.       In Fig.2A and 5A, the Ca²⁺ response to thapsigargin treatment in Dicer^-/- ESCs appears to be faster than in Dicer^fl/fl ESCs. The authors should mention about this.

4.       The authors claim that thapsigargin-induced cytoplasmic Ca²⁺ increase from the ER. But mitochondria can also be a candidate for intracellular Ca²⁺ resource. Thapsigargin inhibits only Ca²⁺ uptake into the ER, does not promote to release Ca²⁺ from the ER. Why did not the authors consider the mitochondria or other resources?

Minor comments:

1.         In the abstract, please add the explanation about Dicer.

2.         In the introduction, it is necessary to describe in more detail the miRNAs that the authors focused on.

3.         In L67-L69, please insert the reference.

4.         In Fig.1A-C, did the authors perform the statistical analysis?

5.         In the result section 3.2., the authors should more clearly mention that thapsigargin increased cytoplasmic Ca²⁺. This makes it easier for the reader to understand.

6.         In Fig.5, the authors should mention how the extracellular Ca²⁺ was removed.

Minor editing of English language required.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Overall, the paper contributes to our understanding of the regulatory role of miRNAs in Ca2+ homeostasis in murine embryonic stem cells. The findings shed light on the specific effects of miRNA deletion on Ca2+ response, apoptosis, and stress response pathways. The identification of Itpr2 as a target of miRNA regulation provides valuable insights into the mechanisms underlying Ca2+ signaling regulation in pluripotent stem cells.

Comments:

Figures: Please include the N numbers of repeated experiments in all figure legends.

Figure 1A: The legends were mislabeled. It should be "Left and right" instead of "above and below."

Figure 1E: Please explain why miR-19 is highlighted in yellow.

Section 3.4: This part of the results is not quite related to the work presented before or after. The authors need to provide further explanation regarding the source of the enhanced Ca2+ level upon adding ATP, whether it is from the extracellular space or the ER, and how this relates to Dicer depletion.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Please add a supplemental figure that the effect of 200 nM thapsigargin on Ca²⁺ response.

Author Response

We have added the figure as Figure S1, and we have modified the text in the results section with a sentence explaining the addition of the figure.