1. Author Response:

    Reviewer #1 (Public Review):

    We thank the Reviewer #1 for their valuable comments. We agree with the Reviewer that our current results are not sufficient to confirm the therapeutic effects. The statement related to therapy is removed.

    The study by Song and colleagues explores the role of circRNAs in fibrosis of the endometrium. Endometrial cells for patients with and without fibrosis were subjected to expression profiling analysis, and circPTPN12 and miR-21-5p were strongly separate in fibrosis in endometrial, with circPTPN12 acting as an inhibitory factor for miR-21-5p. Through the use of various molecular approaches, the authors further that miR-21-5p inhibition results in upregulation of ΔNp63α, and transcription factor that induces EMT. The role of circPTPN12 was also confirmed in vivo using a mouse model of mechanically induced endometrial fibrosis. The authors concluded that targeting the path circPTPN12/miR-21-5p/∆Np63α may be a therapeutic strategy for endometrial fibrosis.

    The authors clearly and convincingly show the involvement of the circPTPN12/miR-21-5p/∆Np63α in EMT and its potential involvement in endometrial fibrosis. Whether or not this can be a therapeutic target is too preliminary at this point. First because the in vivo experiments confirm the link between circPTPN12/miR-21-5p/∆Np63α at the RNA level only (p63) and it would be more convincing to see protein data as well.

    We did try to detect the protein of ΔNp63α in mouse with immunochemistry and immunofluorescence, using three antibodies (CST, cat# 67825 and 39692; Abcam, ab124762). Unfortunately, we did not obtain positive results. However, ΔNp63α mRNA was significantly changed.

    The involvement of p63 in the process remains a little elusive in this paper.

    We have reported that ΔNp63α is ectopically expressed in endometrial epithelial cells in IUA patients (Cao et al., 2018), and showed that ΔNp63α promotes the expression of SNAI1 by DUSP4/GSK3B pathway and induces EECs-EMT and fibrosis (Zhao et al., 2020). We've put this description of ΔNp63α in the discussion section (2nd paragraph).

    In addition, if the authors believe this pathway can be a real future target to treat endometrial fibrosis, they could better contextualise such a statement, specifically describe what kinds of therapeutic intervention they think of, like regression or prevention of fibrosis. These should be tested in vitro and in vivo.

    Our results showed that replenishing miR-21-5p can reverse EMT and remit endometrial fibrosis in vivo and in vitro. However, the therapeutic intervention of miR-21-5p in clinic needs more research on other animal models such as rats, pigs, and non-human primates. Thus, we removed therapeutic statement (page 1, Line 1-2; and page 2, Line 37-40; and page 4, Line 74-76; page 13, Line 273).

    More evidence of the involvement of circPTPN12/miR-21-5p/∆Np63α and the correlation between the three players using clinical material is also necessary.

    The involvement of ∆Np63α in endometrial fibrosis has been proved in our published paper and results are quoted in this paper (Zhao et al., 2020). The correlation between circPTPN12 and miR-21-5p using clinical material was listed in Figure 2J. In vivo and ex vivo experiments had confirmed that overexpression of circPTPN12 downregulates miR-21-5p and upregulates ∆Np63α (Figure 3H/Figure 4J/ Figure 5B/ Figure 5E). In addition, ex vivo experiments suggested that the decrease of ∆Np63α is secondary to the increase of miR-21-5p (Figure 4C-E).

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  2. Reviewer #2 (Public Review):

    As the most common reason for infertility, the underlying mechanism of endometrial fibrosis remains largely unknown. Although some progress has been made about the pathogenesis of endometrial fibrosis, the role of cirRNAs during this process remains elusive. In this investigation, Song et al. propose a novel mechanism that increased epithelial circPTPN12 reduces miR-21-5p, which contributes to upregulation of ΔNp63α to induce the epithelial mesenchymal transition (EMT) of EECs (EEC-EMT). There are several interesting findings in this manuscript including 1) there are hundreds of miRNAs are differentially expressed between control and endometrial fibrosis; 2) miR21-5p is mainly located in epithelial cells in normal endometrium 3) There are also some circRNAs are significantly changed between control and IUA; 4) Moreover, functional studies reveal that circPTPN12 is a critical ceRNA for miR21-5p; 5) Different in vivo evidence from the established animal model also unravels that circPTPN12-miR21-5p participate EMT process. Although the author provides comprehensive evidence to support their hypothesis, there are still some minor concerns raised during reviewing this manuscript.

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  3. Reviewer #1 (Public Review):

    The study by Song and colleagues explores the role of circRNAs in fibrosis of the endometrium. Endometrial cells for patients with and without fibrosis were subjected to expression profiling analysis, and circPTPN12 and miR-21-5p were strongly separate in fibrosis in endometrial, with circPTPN12 acting as an inhibitory factor for miR-21-5p. Through the use of various molecular approaches, the authors further that miR-21-5p inhibition results in upregulation of ΔNp63α, and transcription factor that induces EMT. The role of circPTPN12 was also confirmed in vivo using a mouse model of mechanically induced endometrial fibrosis. The authors concluded that targeting the path circPTPN12/miR-21-5p/∆Np63α may be a therapeutic strategy for endometrial fibrosis.

    The authors clearly and convincingly show the involvement of the circPTPN12/miR-21-5p/∆Np63α in EMT and its potential involvement in endometrial fibrosis. Whether or not this can be a therapeutic target is too preliminary at this point. First because the in vivo experiments confirm the link between circPTPN12/miR-21-5p/∆Np63α at the RNA level only (p63) and it would be more convincing to see protein data as well. The involvement of p63 in the process remains a little elusive in this paper. In addition, if the authors believe this pathway can be a real future target to treat endometrial fibrosis, they could better contextualise such a statement, specifically describe what kinds of therapeutic intervention they think of, like regression or prevention of fibrosis. These should be tested in vitro and in vivo. More evidence of the involvement of circPTPN12/miR-21-5p/∆Np63α and the correlation between the three players using clinical material is also necessary.

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  4. Evaluation Summary:

    The study by Song and colleagues explores the role of circRNAs in fibrosis of the endometrium. The paper is of interest for scientists working in the field of endometrial fibrosis and most likely can have implications for other endometrial disorders characterised by fibrotic tissues. The study unravel the molecular mechanism underlying the disease and the thorough experimental part fully support the author's claim.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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