PONE-D-24-45289LncRNA SENRC knockdown alleviates β-glycerol phosphate-mediated vascular calcification via miR-4731-5p by suppressing endoplasmic reticulum stressPLOS ONE

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

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Reviewer #1: No

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Reviewer #1: No

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5. Review Comments to the Author

Reviewer #1: In this paper, the authors investigate the mechanism underlying vascular calcification (VC) in an in vitro model consisting of VSMCs treated with β-glycerol phosphate (β-GP). Through pharmacological and molecular approaches, they found that β-GP positively regulates the expression of LncRNA SENRC in an ERS-dependent manner. They proposed that the LncRNA SENRC sponges miR-4731-5p to modulate the osteogenic differentiation of VSMCs via Runx2.

Understanding the mechanisms of vascular calcification is of paramount importance for the development of new therapies. Although ER-stress and long non-coding RNA have been involved in this process, the authors identify a novel possible SENRC/miR-4731-5p axis that has previously been shown to regulate VSMCs proliferation (https://doi.org/10.1002/jcp.29775). From this point of view the paper is sufficiently novel and relevant to the field. However, there are several points that need to be addressed.

Major points

1) The data presented are limited to an in vitro model of VC. To confirm the relevance of the SENRC/miR-4731-5p axis and propose it as a possible target for pharmacological intervention, the authors should verify the modulation of the two transcripts (SENRC/miR-4731-5p) in preclinical or clinical models (they could also analyse published data sets).

2) ENGLISH. The whole text would benefit from an English style revision. Several sentences are unclear or ambiguous. Just a few examples, lines 45-49 or 63-66, 248-250, 251-253, 253-254.

Lines 265-267 please reconsider the sentence because two opposite effects of β-GP were proposed “the study revealed two novel findings: (1) excess β-GP increased SENRC levels by activating ERS during the procalcific process, and (2) β-GP-mediated downregulation of SENRC promoted the ERS-dependent osteogenic differentiation of VSMCs by sponging miR-4731-5p”.

Lines 240-242 please reconsider the sentence. The data description refers to fig 5C and D but not to fig 5B

3) DISCUSSION. Reconsider the discussion. There is often repetition in the text. For example, lines 271-274/276/277

The first part of the discussion is almost an introduction

The Limitation and conclusion section is unclear, please reformulate

4) REFERENCES. Some sentences are not supported by the relevant literature. Just a few examples:

Lines 79-81 “Given the close association between excess β-glycerol phosphate, SENRC, ERS, and VC,”

Other examples lines 263-265/267-269/280-281/282

Lines 306-307 Accumulating evidence suggests that SENRC binds to microR-4731-5p as a sponge to inhibit downstream effects via a β-GP-induced Runx2-dependent pathway. This sentence is very relevant to the paper, but there is no relative reference

Reference 30 does not refer to LncSENRC

Although the SENRC/miR-4731-5p axis has been implicated for the first time in VC, the functional interaction between SENRC and miR-4731-5p has already been predicted by https://doi.org/10.1016/j.prp.2023.154483. Not cited in the text.

5) DATA ANALYSIS AND PRESENTATION. Often the number of experimental replicates is 3/4, please report the individual values in the bars (at least for Figures 1-3).

Please indicate in each figure legend the type of statistical analysis performed (t-test, two-way ANOVA, etc.)

Representative blots do not always correspond to their quantification, especially those involving Gpr78.

Figure 2. There is an apparent inconsistency between mRNA and protein levels for Sm22. Sm22 increases as mRNA but decreases as protein. This data should be discussed.

6) PRESENTATION OF THE FIGURES. Figures 4 and 5 are very complex. To avoid confusion, please report the letter in the upper left corner of each panel. Also in figure 4, report the title for the 4 pictures in panel 4B

Minor points

1) Please provide the complete image of the representative blots in the supplementary materials for referees. It would be preferable to present a couple of samples, rather than just one, as shown.

2) Abbreviations: Please define the acronyms on first mention within the text. Additionally, several acronyms remain unidentified (SENCR, TS1, etc.).

VC is vascular calcification, not vascular calcium (abstract row 24).

3) Please ensure that the correct nomenclature of genes and proteins is used.

4) Row 200: This is probably fig 3A.

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Reviewer #1: No

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The lncRNA SENCR knockdown alleviates vascular calcification via miR-4731-5p by suppressing endoplasmic reticulum stress

PONE-D-24-45289R1

Dear Dr. Yongpan Huang,

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Kind regards,

Maria Cristina Vinci, PharmD, PhD

Academic Editor

PLOS ONE