Possible Involvement of miR-98 in the Regulation of PGRMC1 During Decidualization

Round 1

Reviewer 1 Report

Tsuru et al. demonstrate that PGRMC1 abundance levels are higher in proliferative phase versus secretory phase endometrial patient biopsies (fig.1). In primary endometrial stem cell (ESC) culture they demonstrate that chemical induction (by db-cAMP/P4) of pseudo-decidualization in culture is accompanied by decreased PGRMC1 protein levels (fig.2). In primary ESCs they argue that reduction of PGRMC1 levels by siRNA, or inhibition of PGRMC1 function by AG-205, both lead to increased abundance of decidualization markers IGFBP1 and PRL upon db-cAMP/P4-induced decidualization (Fig.3). There is some concern about the specificity of the AG-205 inhibitor here and throughout, which weakens the deductive reasoning and is discussed below. If higher PGRMC1 levels are indeed critical for the suppression of decidualization, as is reasoned by the authors, then it should be possible to maintain PGRMC1 levels artificially to inhibit decidualization, as discussed below. There is also concern about the statistical evaluation of this result, and others of same designe, as discussed below. In Fig.4 the authors argue that decidualization markers are induced by both db-cAMP and db-cAMP/P4, and that therefore P4 is not involved in the PGRMC1 response. The experiemtnal design for this conclusion is problematic, as described below. The authors go on to show that expogenous expression of miR-98 lowers PGRMC1 abundance levels (a more apt description than the fuzzy description of “PGRMC1 downregulation”, which could be by a number of mechanisms as discussed below).

This paper pursues an important theme. Unfortunately, the quality of experimental design and results processing are somewhat problematic. The work would be worthwhile publishing in a revised format, where it should be a well cited publication. Therefore, a resubmission after major revision is recommended. Secific and detailed comments follow.

The introduction succinctly provides background relevant for the following research description.

In general, the manuscript refers often to PGRMC1 expression or downregulation. These are fuzzy terms for proteins. It is much preferable to refer to protein abundance, and changes in abundance. Such changes may occur at the translational or post-translational level. Translational level changes can be due to changed levels of transcription, to altered mRNA stability (such as that proposed here for miR-98), or to altered efficiency of translation of a steady state mRNA pool. The authors are strongly advised to change from e.g. ‘expression’ to ‘abundance’ throughout.

Additionally, the methods do not say that all results were repeated at least once, and that a representative results figure is presented. Therefore, the reviewer assumes that experiments have only been performed once. The entire set of experiments needs to be replicated to demonstrate reproducibility before it can be considered to publish these results.

Line 24. AG-205. The authors must refer to the fact that AG-205 is not specific for PGRMC1, contrary to many citations in the literature (such as would be the case here, if uncorrected). Cite PMID: 34944026, PMID: 34680104, PMID: 32924377. Sensitivity of a phenomenon to AG-205 is consistent with possible PGRMC1 involvement, but does not demonstrate PGRMC1 involvement.

It is also suggested throughout to use “AG-205” (the correct name for the reagent: https://www.sigmaaldrich.com cat#: a1487) rather than AG205 (often incorrectly used in the literature).

Line 50. “(10-12); however,” should be a full stop. Two discrete sentences “(10-12). However,”

Methods.

Appropriate human ethics approvals were obtained.

Lines 76-78. Statistical sample sizes were unfavourably small. (n= 3 or 4). The methods could describe that all subjects were ethnically Japanese/Asian (or otherwise).

Lines 88-98. Cell culture. What characterisation was performed to confirm the identity of cells as ESCs? In general, there is very little detail on these primary cultured lines. How long are they held in culture? Are they stored cryogenically? How are they re-seeded into culture?

Lines 121-133. What percentage acrylamide were the SDS PAGE gels. For what length of time and at what voltage settings was electrophoretic transfer to PVDF? In what device?

Line 126-7. Give the catalogue number of the secondary antibody.

Line 131. On what device was ECL light detected? What were the device settings? How was pixel saturation dealt with?

Line 132. Which ImageJ plug-in was used for quantification?

Lines 134-137.

Result errors should not be expressed as SEM, but as standard deviation (s.d.). SEM is a function of how many measurements were made, and does not reflect sample variability. S.d. reflects variability between replicate measurements. All results should show s.d. error bars.

Line 150. The legend to fig. 1. inappropriately ends with “2.2. Downregulation of PGRMC1 During ESC in vitro Decidualization.” This seems to be the next section heading, in which case it should follow a line break, and say: “3.2. Downregulation of PGRMC1 During ESC in vitro Decidualization.”

All subsequent section header numbers in results should be modified to accommodate the new section.

Lines 153-156, Figure 2. We are told in the methods that primary cell cultures were obtained from 3 patients (see lines 88-89). Figure 2 seems to depict just one cell culture (which is not even named), with 4 replicates of each measurement. The result must be shown for at least each ESC cell culture (each culture or patient named separately) obtained from each patient to demonstrate that the described results are representative of multiple patients, and not potentially atypical results from an atypical patient. This result must be demonstrated in a substantively convincing manner.

Were primary cultures used directly, or were they stored cryogenically? If the latter, the freezing and resuscitation conditions are critical for ESC-like properties.

Line 158/Fig. 2. The western blot (also Fig.3) result must show the entire molecular weight range (e.g. in supplemental data), including MW markers. PGRMC1 is known to be Sumoylated or ubiquitinylated and then degraded by the proteasome pathway, which lead to higher molecular weight species (PMID: 31067491). The latter could well contribute to alterations in PGRMC1 abundance cited by this study. It is important that the involvement of the ubiquitin pathway contribution be either quantified or discarded in well controlled experiments to justify the title of the paper.

Nine PGRMC1 lysines are known to be ubiquitinated (https://www.phosphosite.org/proteinAction.action?id=5744&showAllSites=true). Individual mutation of these residues could stabilise PGRMC1 levels to reduce decidualization if miR-98 is only partially responsible for reducing PGRMC1 levels. In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization. It is important to determine whether these mechanisms are both operating. At the very least this mechanism merits detailed discussion in that section. PGRMC1 protein instability could be the dominant effect involved, with miRNA transcript regulation playing only a minor part.

Line 163. Change 3.2. to 3.3.

Lines 164-172. This cell culture experiment also seems to be based upon just one primary cell culture, split into replicates. I am uncertain because the methods do not explain how the experiment was performed. Cell culture methods must be much more clearly explained so that the reader can reconstruct each exact experiment.

Lines 178-9. If PGRMC1 down-regulation is critical, it should be possible to perturb decidualization by expression of an exogenous PGRMC1 protein. This could be accomplished by making an expression plasmid encoding a codon-altered PGRMC1 gene, and cotransfecting this in a controlled matrix design with siRNA. If siRNA-resistant PGRMC1 levels are able to inhibit decidualization it would provide convincing evidence in favour of the hypothesis that lowered PGRMC1 is required for decidualization, and is perhaps even a trigger. A codon-altered PGRMC1 expression vector would cost just several hundred dollars from a provider such as Genscript, so the experiment is eminently achievable.

PGRMC1 levels are also controlled by proteolysis, mediated by the ubiquitin proteasome system as described above.

Fig.3. vs. Fig.4.

Notably, Fig. 4. lacks the western blot equivalent to Fig.3A, showing that siRNA indeed depleted PGRMC1 levels.

Critically, these figures compare the effects of two variables each, and therefor one way ANOVA is not the appropriate test. Assuming binomial distribution, a two-way ANOVA is required for these experiments.

Line 189-96. The paragraph describes a result showing that the results are independent of P4. However, the control structure of comparisons is incorrect. The simplest direct comparison would be db-cAMP/P4 vs. db-cAMP to permit the argument the authors are presenting. However, the effects of P4 in the absence of db-cAMP would also require control in cells without db-cAMP, +/- P4. The statistical significance of any result would be evaluated using multivariate analysis.

Line 204. Change 3.3. to 3.4.

Line 207: “The miRNA target prediction database, miRDB (http://www.mirdb.org), was screened to identify miRNAs with potential to interact with PGRMC1.” miR-98 is already known to regulate PGRMC1. The results of PMID: 22492871 and PMID: 21109987 MUST be cited here. This is not a novel finding.

Discussion.

The discussion must qualify the results of Salsano et al. who used AG-205 to inhibit PGRMC1. There was no control in PGRMC1 genomic/CRISPR KO cells (not si/miRNA KO) to demonstrate that effects induced by AG-205 were due to PGRMC1.

Line 261. mechanisms HAVE not (plural mechanisms)

Lines 261-262. “In the present study, we showed that microRNA-mediated PGRMC1 regulation during the process of decidualization.” This is confusingly worded. Suggest change to “In the present study, we showed that microRNA-mediated the regulation of PGRMC1 abundance during the process of decidualization.” (if this is what is meant)

Lines 271-76. This is very long and burdens the reader. Always make new sentences when possible. Suggest change to;

“Further study is required to determine the relationship between miR-98-mediatd PGRMC1 regulation and pregnancy. Comparison of the miR-98/PGRMC1 abundance in proliferative and secretory phases of endometrial biopsy may contribute to the diagnostic prediction of uterine receptivity for embryo implantation. Furthermore, controlling their expression may support the improvement of IVF (NOT IFV) outcome.

Define IVF at first use.

Lines 281-5. PGRMC1 (Hpr6.6) increased the rate of cell death (in a non-apoptotic mechanism) in MCF-7 cancer cells in response to H2O2 (PMID: 14523988). That could be discussed here in terms of mechanism.

Lines 286-287. Enunciate what kind of role(s) PGRMC1 may be involved in (up/down regulation, when, how?). Suggest observations that may be able to discriminate between different possibilities.

Author Response

Responses to Reviewer 1

We appreciate your review of our manuscript and constructive comments and suggestions. Our responses to your comments are as follows.

1) In general, the manuscript refers often to PGRMC1 expression or downregulation. These are fuzzy terms for proteins. It is much preferable to refer to protein abundance, and changes in abundance. Such changes may occur at the translational or post-translational level. Translational level changes can be due to changed levels of transcription, to altered mRNA stability (such as that proposed here for miR-98), or to altered efficiency of translation of a steady state mRNA pool. The authors are strongly advised to change from e.g. ‘expression’ to ‘abundance’ throughout.

>> We agree with your comments. The term ‘expression’ has, therefore, been changed to ‘abundance’ or ‘level’ throughout the manuscript.

2) Additionally, the methods do not say that all results were repeated at least once, and that a representative results figure is presented. Therefore, the reviewer assumes that experiments have only been performed once. The entire set of experiments needs to be replicated to demonstrate reproducibility before it can be considered to publish these results.

>>All experiments were repeated at least two times. We have failed the description regarding the reproducible results in the previously submitted manuscript, and so revised the section of the methods in the revised manuscript (Page 4, lins143-143).

3) Line 24. AG-205. The authors must refer to the fact that AG-205 is not specific for PGRMC1, contrary to many citations in the literature (such as would be the case here, if uncorrected). Cite PMID: 34944026, PMID: 34680104, PMID: 32924377. Sensitivity of a phenomenon to AG-205 is consistent with possible PGRMC1 involvement, but does not demonstrate PGRMC1 involvement.

>> We agree that AG-205 is not a specific inhibitor of PGRMC1. Therefore, we examined the effects of siRNA-mediated PGRMC1 knockdown on decidualization. As you mentioned, this issue was not appropriately described in the previously submitted manuscript. The citations have been inserted in the revised manuscript (page 8, lines 258-263).

4) It is also suggested throughout to use “AG-205” (the correct name for the reagent: https://www.sigmaaldrich.com cat#: a1487) rather than AG205 (often incorrectly used in the literature).

>> The name of the reagent has been corrected to AG-205 consistently throughout the manuscript.

5) Line 50. “(10-12); however,” should be a full stop. Two discrete sentences “(10-12). However,”

>> The sentence has been separated according to your suggestion.

6) Lines 76-78. Statistical sample sizes were unfavourably small. The methods could describe that all subjects were ethnically Japanese/Asian (or otherwise).

>>The description has been modified as followed (page 2, line 69). ‘Endometrial samples were obtained from Japanese patients with a normal menstrual cycle, ….’.

7) Lines 88-98. Cell culture. What characterization was performed to confirm the identity of cells as ESCs? In general, there is very little detail on these primary cultured lines. How long are they held in culture? Are they stored cryogenically? How are they re-seeded into culture?

>> The primary cultured ESCs we used were confirmed immunocytochemically to be positive for vimentin, a marker for stromal cells, and negative for cytokeratin, a marker for epithelial cells (please see above figure). ESCs at early passages (between 2 and 6 passages) were used for the experiment. In this study, the passaged cells were used without freezing. However, we have confirmed that primary ESCs can be cryopreserved in CELLBANKER, and retain characteristics such as responsiveness to decidual stimuli. These descriptions have been added to the revised manuscript (page 3, lines 96-99).

Figure. Immunocytochemistry (ICC) of vimentin and cytokeratin in isolated human ESCs.

8) Lines 121-133. What percentage acrylamide were the SDS PAGE gels. For what length of time and at what voltage settings was electrophoretic transfer to PVDF? In what device?

>> The protein samples were separated by SDS-PAGE (5-20% gradient gel) and transferred electrophoretically to polyvinylidene difluoride membranes (8 cm x 8.5 cm) for 60 min at constant current of 128 mA using a semi-dry transfer system (ATTO, Tokyo Japan). These conditions have been added to the Materials and Methods section (Pages 3 and 4, lines 127-130).

9) Line 126-7. Give the catalogue number of the secondary antibody.

>>The catalogue number of the antibodies (PI-1000, PI-2000) were inserted (Page 4, line 134).

10) Line 131. On what device was ECL light detected? What were the device settings? How was pixel saturation dealt with?

>>The immunoreactive bands were visualized using an enhanced chemiluminescence (Western Lightning, PerkinElmer, Inc., Waltham, MA, USA) and analyzed with ImageQuant LAS 500 (semi-auto mode; GE Healthcare Japan, Tokyo Japan) (Page 4, lines 136-137).

11) Line 132. Which ImageJ plug-in was used for quantification?

>>The densitometry analysis was carried out using the “Gel Plot” plug-in of the Image J software. This plug-in has been described in the Materials and Methods (Page 4, line 139)

12) Lines 134-137.

Result errors should not be expressed as SEM, but as standard deviation (s.d.). All results should show s.d. error bars.

>> As you indicated, all data are expressed consistently as means and standard deviation (Figs 1 to 4). In our previously submitted manuscript, we had described carelessly the data of as for figure 5B and C with s.d.

13) Line 150. The legend to fig. 1. inappropriately ends with “2.2. Downregulation of PGRMC1 During ESC in vitro Decidualization.” This seems to be the next section heading, in which case it should follow a line break, and say: “3.2. Downregulation of PGRMC1 During ESC in vitro Decidualization.”

All subsequent section header numbers in results should be modified to accommodate the new section.

>> Thank you for pointing this out. The mistakes have been corrected (Pages 4,5 and 7, lines 160, 174 and 217).

14) Lines 153-156, Figure 2. The result must be shown for at least each ESC cell culture (each culture or patient named separately) obtained from each patient to demonstrate that the described results are representative of multiple patients, and not potentially atypical results from an atypical patient. This result must be demonstrated in a substantively convincing manner.

>> Based upon your suggestions, additional experiments have been performed and added individual Western blotting data from there different ESCs (named #1-3) in Figure 2.

15) Line 158/Fig. 2. The western blot (also Fig.3) result must show the entire molecular weight range (e.g. in supplemental data), including MW markers. PGRMC1 is known to be Sumoylated or ubiquitinylated and then degraded by the proteasome pathway, which lead to higher molecular weight species (PMID: 31067491). The latter could well contribute to alterations in PGRMC1 abundance cited by this study. It is important that the involvement of the ubiquitin pathway contribution be either quantified or discarded in well controlled experiments to justify the title of the paper.

>> Western blotting for PGRMC1 in figures 2, 3, and 4 have been replaced in the data with the whole molecular weight range. As you pointed out, PGRMC1 is known to be modified by sumoylation or ubiquitinylation which results in high molecular weight. The right panel shows the western blotting of PGRMC1. There are some higher molecular bands (white arrowhead) in lysates of ESCs, however, the band’s intensity was not changed by PGRMC1 siRNA treatment. Therefore, we believe that these high molecular bands detected in our experiment were non-specific bands.

16) Nine PGRMC1 lysines are known to be ubiquitinated (https://www.phosphosite.org/proteinAction.action?id=5744&showAllSites=true).

Individual mutation of these residues could stabilise PGRMC1 levels to reduce decidualization if miR-98 is only partially responsible for reducing PGRMC1 levels. In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization. It is important to determine whether these mechanisms are both operating. At the very least this mechanism merits detailed discussion in that section. PGRMC1 protein instability could be the dominant effect involved, with miRNA transcript regulation playing only a minor part.

>> As you pointed out, the possible involvement of posttranslational modification of endometrial PGRMC1 such as sumoylation and ubiquitinylation is interesting. Discussion regarding this possibility has been inserted in the revised manuscript (Page 9, lines 300-307). We will further examine whether posttranslational modification of PGRMC1 could be associated with decidualization in the next study.

17) Line 163. Change 3.2. to 3.3.

>> The mistake has been corrected.

18) Lines 164-172. This cell culture experiment also seems to be based upon just one primary cell culture, split into replicates. I am uncertain because the methods do not explain how the experiment was performed. Cell culture methods must be much more clearly explained so that the reader can reconstruct each exact experiment.

>> All data of cell culture represent the mean ± SD of three independent experiments with at least repeated two times each experiment. The explanation of the experiments has been inserted the method (page 4, lines142-143).

19) Lines 178-9. If PGRMC1 down-regulation is critical, it should be possible to perturb decidualization by expression of an exogenous PGRMC1 protein. This could be accomplished by making an expression plasmid encoding a codon-altered PGRMC1 gene, and cotransfecting this in a controlled matrix design with siRNA. If siRNA-resistant PGRMC1 levels are able to inhibit decidualization it would provide convincing evidence in favour of the hypothesis that lowered PGRMC1 is required for decidualization, and is perhaps even a trigger. A codon-altered PGRMC1 expression vector would cost just several hundred dollars from a provider such as Genscript, so the experiment is eminently achievable.

>>Thank you for your valuable advice to prove the significance of PGRMC1 downregulation in decidualization. Salsano et al.(19), reported that overexpression of PGRMC1 in ESCs abrogated the decidual markers expression (page 8. Lines 253-254). We will try to examine the effects of enforced expression of codon-altered PGRMC1 which is resistant for siRNA on decidualization in next study.

20) Notably, Fig. 4. lacks the western blot equivalent to Fig.3A, showing that siRNA indeed depleted PGRMC1 levels.

>> Based upon your suggestions, we have added the data of PGRMC1 knockdown in figure 4. We confirmed efficient siRNA-mediate PGRMC1 knockdown by western blotting.

21) Critically, these figures compare the effects of two variables each, and therefor one way ANOVA is not the appropriate test. Assuming binomial distribution, a two-way ANOVA is required for these experiments.

>> The statistical analysis was not appropriate in the former manuscript. The data has been re-evaluated by a two-way ANOVA followed by a Turkey-Kramer multiple comparison test (page 4, line13).

22) Line 189-96. The paragraph describes a result showing that the results are independent of P4. However, the control structure of comparisons is incorrect. The simplest direct comparison would be db-cAMP/P4 vs. db-cAMP to permit the argument the authors are presenting. However, the effects of P4 in the absence of db-cAMP would also require control in cells without db-cAMP, +/- P4. The statistical significance of any result would be evaluated using multivariate analysis.

>> As you suggested, we should have presented the data in the same graph. However, the two experiments were performed independently. Therefore, we were not able to combine the results in the graph. Because treatment with P4 alone could not induce decidualization in the experiments, we examined the effect of PGRMC1 knockdown and inhibition on db-cAMP-induced decidualization. Further study would be needed to determine whether PGRMC1 is involved in the P4 action in endometrium.

23) Line 207: “The miRNA target prediction database, miRDB (http://www.mirdb.org), was screened to identify miRNAs with potential to interact with PGRMC1.” miR-98 is already known to regulate PGRMC1. The results of PMID: 22492871 and PMID: 21109987 MUST be cited here. This is not a novel finding.

 The description has been corrected and cited the references in the section (page 7, lines 223-224).

24) The discussion must qualify the results of Salsano et al. who used AG-205 to inhibit PGRMC1. There was no control in PGRMC1 genomic/CRISPR KO cells (not si/miRNA KO) to demonstrate that effects induced by AG-205 were due to PGRMC1.

>>New discussion regarding the need for PGRMC1 knockout studies to clarify the role of PGRMC1 in decidualization has been inserted in page 9, lines 281-282.

25) Line 261. mechanisms HAVE not (plural mechanisms)

>> The error has been corrected.

26) Lines 261-262. “In the present study, we showed that microRNA-mediated PGRMC1 regulation during the process of decidualization.” This is confusingly worded. Suggest change to “In the present study, we showed that microRNA-mediated the regulation of PGRMC1 abundance during the process of decidualization.” (if this is what is meant)

>> Thank you for correcting the sentence. Your correction is what we meant to mention. We have replaced this sentence in the revised manuscript.  (page9, line 284-286)

27) Lines 271-76. This is very long and burdens the reader. Always make new sentences when possible. Suggest change to; “Further study is required to determine the relationship between miR-98-mediatd PGRMC1 regulation and pregnancy. Comparison of the miR-98/PGRMC1 abundance in proliferative and secretory phases of endometrial biopsy may contribute to the diagnostic prediction of uterine receptivity for embryo implantation. Furthermore, controlling their expression may support the improvement of IVF (NOT IFV) outcome. Define IVF at first use.

>> As noted, the sentence has been separated and corrected in accordance with your suggestion (page 9, line 295-299).

28) Lines 281-5. PGRMC1 (Hpr6.6) increased the rate of cell death (in a non-apoptotic mechanism) in MCF-7 cancer cells in response to H2O2 (PMID: 14523988). That could be discussed here in terms of mechanism.

>> The report that PGRMC1 mediates H2O2-induced cell death in MCF7 cells has been inserted in discussion (page 10, lines 314-317)

29) Lines 286-287. Enunciate what kind of role(s) PGRMC1 may be involved in (up/down regulation, when, how?). Suggest observations that may be able to discriminate between different possibilities.

>> We have suggested the possibility that PGRMC1 downregulation may be involved in ESCs senescence during the decidualization (page 10, lines 319-321).

Reviewer 2 Report

The manuscript explored the mechanisms of PGRMC1 regulation during the decidualization of human ESCs. The research has a certain degree of scientific significance. However there are some issues.

1. Some of the tissue samples are from patients with endometriosis, then according to eutopic endometrium determinism, whether such selection will cause the research results to be inaccurate？
2. The sample size is small. And please provide basic information of the patient.
3. The conclusion has been verified by other 2-3 articles, and the design of this study is not rigorous enough. It is suggested to add cell lines or normal endometrial cells for verification. If further mechanism exploration is carried out, the research will be more complete and persuasive.

Author Response

Responses to Reviewer 2

We appreciate your review of our manuscript and constructive comments and suggestions. Our responses to your comments are as follows.

1) Some of the tissue samples are from patients with endometriosis, then according to eutopic endometrium determinism, whether such selection will cause the research results to be inaccurate？The sample size is small. And please provide basic information of the patient.

>>As you mentioned, our description of ESCs isolation may be inappropriate in terms of the patients with genetical differences. ESCs were isolated from histologically normal region in the patients with leiomyoma (age 42-45). It has been reported that ESCs obtained from the eutopic endometrium of the patients with endometriosis showed impaired decidualization. Therefore, we should consider the possible influence of leiomyoma on the decidual response, but we confirmed that the isolated ESCs have differentiated into decidual cells in response to cAMP analogue and progesterone, as you pointed out. The information of the patients has been inserted in the method

2) The conclusion has been verified by other 2-3 articles, and the design of this study is not rigorous enough. It is suggested to add cell lines or normal endometrial cells for verification. If further mechanism exploration is carried out, the research will be more complete and persuasive.

>> Because of the ethical difficulties of isolating ESCs from disease-free healthy women, we routinely used ESCs isolated from the patients in surgical cases. ESC lines are commercially available (T-HESC, ATCC) and maybe useful tool to investigate the role of PGRMC1 in decidualization, as you pointed out. However, the cell lines are also established from the endometrium of leiomyoma and immortalized by transfection with hTERT. Therefore, we believe that ESCs used in this study are no different than the above cells and cell lines in examining the role of PGRMC1.In the future, we plan to use several ESCs and their cell lines to clarify the precise mechanisms of PGRMC1 downregulation and decidualization.

Round 2

Reviewer 1 Report

The authors have addressed most of my concerns. However, I requested a major revision including the use of miRNA-resistant exogenous PGRMC1 expression (altered codon usage) to demonstrate that lack of PGRMC1 attenuation will prevent decidualization. That new experimental result is what elevated the required changes to a major revision. The revised manuscript as presently resubmitted is a minor revision. This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualisation. This requires stronger evidence than that currently presented. Furthermore, the required experiment is technically not challenging. The authors could consider whether an inducible miRNA-resistant PGRMC1 promoter could be designed. The fact that PGRMC1 levels fall does not mean that low PGRMC1 levels are required. This may be only correlation. Before such a claim is published in the scientific literature, it must be supported by convincing evidence. Aspiration to clarify this in future papers does not merit publication of the present paper. Some minor comments follow.

Lines 142-3.

“Data of cell culture are expressed as the mean ± SD from three independent experiments with at least repeated two times.”

Change to:

Data of cell culture are expressed as the mean ± SD from three independent experiments. All reported results were observed in at least two independent experiments.  

Line 259. PGRMC1-independe (add t)

Lines 263-267: “Because, the effect of AG-205 and PGRMC1 knock-down on decidualization was common in the present study, these findings further support for a role for PGRMC1 downregulation in promoting ESC decidualization during the secretory phase.”

The reviewer does not understand the sentence. What is being said?

Lines 281-282. There are two natural sentences here.

However, as described above AG-205 is not a specific inhibitor of PGRMC1. [full stop, new sentence] Further study of PGRMC1 knockout on decidualization is needed.

Lines 300-307.

The salient point concerning the ubiquitination events is that they are correlated for other proteins with proteasome-mediated degradation. Sabbir’s results (ref 31) imply that this also holds for PGRMC1. The new text does not conceptually link PTMs with PGRMC1 protein stability, which is the only reason they should be mentioned. To be clear, protein levels can be mediated by altered rates of transcription, translation, or degradation. This new text should be discussing the possibility of the latter also contributing to the observed effects, as well as the effects of mRNA level that the paper pursues.

Author Response

We appreciate your review of our revised manuscript, constructive comments, and suggestions, which have helped us to improve the manuscript. Our responses to your comments are as follows.

1) I requested a major revision including the use of miRNA-resistant exogenous PGRMC1 expression (altered codon usage) to demonstrate that lack of PGRMC1 attenuation will prevent decidualization. That new experimental result is what elevated the required changes to a major revision. The revised manuscript as presently resubmitted is a minor revision. This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualization. This requires stronger evidence than that currently presented. Furthermore, the required experiment is technically not challenging. The authors could consider whether an inducible miRNA-resistant PGRMC1 promoter could be designed. The fact that PGRMC1 levels fall does not mean that low PGRMC1 levels are required. This may be only correlation. Before such a claim is published in the scientific literature, it must be supported by convincing evidence. Aspiration to clarify this in future papers does not merit publication of the present paper.

>> As you pointed out, we infer that downregulation of PGRMC1 may be necessary for decidualization to proceed. To clarify this point, we further attempted to overexpress PGRMC1 by introducing expression vectors into primary ESCs under various conditions using lipofection and calcium phosphate transfection methods, as you suggested. Unfortunately, for reasons unknown, we were unable to establish ESCs overexpressing PGRMC1. In the present study, we found that not only knockdown of PGRMC1 but also functional inhibition with inhibitors promotes ESC decidualization in vitro. Furthermore, in a previous report, overexpression of PGRMC1 suppressed ESCs differentiation (Ref. 19). These results indicate that decreased PGRMC1 in the endometrium may be associated with accelerated dedifferentiation. As you suggested, we have changed the title significantly because further evidence was not available. We plan to establish a model of miR-resistant PGRMC1 overexpression using several ESCs cell lines to investigate the role of PGRMC1 in decidualization, and if possible, we would be happy to make this our next research topic.

2) Lines 142-3.“Data of cell culture are expressed as the mean ± SD from three independent experiments with at least repeated two times.” Change to:　Data of cell culture are expressed as the mean ± SD from three independent experiments. All reported results were observed in at least two independent experiments.

>> Thank you for correcting the sentence. The description has been corrected in accordance with your suggestion (page 4, lines 141-142).

3) Line 259. PGRMC1-independe (add t)

>> The error has been corrected (page 8, line 257).

4) Lines 263-267: “Because the effect of AG-205 and PGRMC1 knock-down on decidualization was common in the present study, these findings further support for a role for PGRMC1 downregulation in promoting ESC decidualization during the secretory phase.” The reviewer does not understand the sentence.

>> The description has been modified to the following (page 8, lines 261-266). “Although we cannot exclude the possible non-specific action of AG-205 on ESCs, the effect of AG-205 treatment and PGRMC1 knockdown promoted in vitro decidualization. These findings suggested that PGRMC1 downregulation may promote ESC decidualization during the secretory phase.

5) There are two natural sentences here. However, as described above AG-205 is not a specific inhibitor of PGRMC1. [full stop, new sentence] Further study of PGRMC1 knockout on decidualization is needed.

>> As noted, the sentence has been separated and corrected in accordance with your suggestion (page 9, lines 280-281).

6) The salient point concerning the ubiquitination events is that they are correlated for other proteins with proteasome-mediated degradation. Sabbir’s results (ref 31) imply that this also holds for PGRMC1. The new text does not conceptually link PTMs with PGRMC1 protein stability, which is the only reason they should be mentioned. To be clear, protein levels can be mediated by altered rates of transcription, translation, or degradation. This new text should be discussing the possibility of the latter also contributing to the observed effects, as well as the effects of mRNA level that the paper pursues.

>> As you pointed out, we did not explain the relationship between PGRMC1 stability and post-transcriptional regulation of the protein. We have added the following description (page 9, lines 304-308). ”These reports suggested that PGRMC1 protein levels can be modulated by changes in the rates of transcription, translation, and degradation. Therefore, further studies would be needed to clarify the possible involvement of the post-translational modification and degradation that may regulate PGRMC1 protein stability in ESCs during decidualization.”

Reviewer 2 Report

The author provided explanations for the questions.  The manuscript has been modified accordingly. I think the manuscript could be accepted now.

Author Response

We appreciate your review of our revised manuscript, constructive comments, and suggestions, which have helped us to improve the manuscript. We are thankful for the time and energy you expended.

Round 3

Reviewer 1 Report

I requested a major revision, involving expression of an miRNA-resistant PGRMC1 ORF to convincingly demonstrate the mechanistic involvement of PGRMC1 down-regulation in decidualization, rather than a temporal correlation. In response the authors have made minimal changes to the manuscript, effectively restricted to rewording the title, and several minor text changes. Unfortunately, the authors have been unable to demonstrate the direct involvement of PGRMC1 downregulation in decidualization. Since this is the main publishable finding, the results as they stand do not merit publication.

The authors mention technical difficulties in establishing ESCs that express miRNA-resistant PGRMC1. This may be due to stable plasmid selection conditions, which may affect the differentiation status of the ESC cells. It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1. An inducible vector may be advisable, such as tetracycline, IPTG, or other available systems. Such vectors typically include fluorescent protein reporters, enabling discrimination between infected and non-infected cells in one plate, or population separation by FACS.

In conclusion, if this published was published in present form then it would invariably be cited as demonstrating that PGRMC1 downregulation by miR-98 causes decidualization. Since that is by no means certain, the study should not be published in its present form. The opportunity should remain open for the authors to convincingly demonstrate the requirement for PGRMC1 down-regulation in decidualization by performing the requested experiments, or others of their devising. However, in the first instance the manuscript cannot be published as it stands, and should therefore be rejected now with the stated option of subsequent resubmission only if new evidence is obtained.

Author Response

Comments from the Reviewer:

In response the authors have made minimal changes to the manuscript, effectively restricted to rewording the title, and several minor text changes. Unfortunately, the authors have been unable to demonstrate the direct involvement of PGRMC1 downregulation in decidualization. Since this is the main publishable finding, the results as they stand do not merit publication. The authors mention technical difficulties in establishing ESCs that express miRNA-resistant PGRMC1. This may be due to stable plasmid selection conditions, which may affect the differentiation status of the ESC cells. It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1. An inducible vector may be advisable, such as tetracycline, IPTG, or other available systems. Such vectors typically include fluorescent protein reporters, enabling discrimination between infected and non-infected cells in one plate, or population separation by FACS.

Responses to the Reviewer's comments:

We appreciate your review of our revised manuscript, constructive comments, and suggestions for improving the manuscript. We deeply understand the significance of the experiments using miR-98-resistant PGRMC stably expressing ESCs to directly prove the implication of the miR-mediated PGRMC1 regulation in decidualization. We are transfecting the miR-resistant PGRMC1 expression vector into ESCs cell lines to explore the role of PGRMC1 in decidualization but is not going well. As you advised, lentiviral transduction of the PGRMC1 along with fluorescent protein may enable us to effectively select the overexpressing cells without losing differentiation status in response to decidual stimuli. We have acknowledged this and suggested it as a topic for further research in the “Limitation” section of the revised manuscript following editor’s comment (page 10, lines 336-348).

“Limitations: In this study, we proposed that miR-98-mediated PGRMC1 downregulation may be involved in decidualization, but were unable to examine the effects of miR-98-resistant PGRMC1 on differentiation due to technical difficulties establishing PGRMC1-overexpressing ESCs. miR-98 may not be the only miRNA that modulates PGRMC1 expression; therefore, comprehensive analysis of transcriptional, post-transcriptional, and post-translational regulation of endometrial PGRMC1 during decidualization is required. In addition, PGRMC1 can bind to various proteins, including receptors for epidermal growth factor [46] and insulin [47], cytochrome P450 [48], and serpine mRNA-binding protein 1 (SERBP1) [19]. Further, PGRMC1 may interact with proteins associated with endomembrane trafficking/cytoskeleton and mitochondrial functions in decidualizing ESCs [49]. Thus, we plan to investigate the modulation of endometrial PGRMC1 expression and the interaction between PGRMC1 and intracellular proteins during decidualization in the future.”

Round 4

Reviewer 1 Report

The inclusion of the model of Fig. 6 in the manuscript depicts the problem. This manuscript will be cited as evidence of that inconclusive model. The reviewer is not convinced that PGRMC1 level attenuation by miR-98 mechanistically drives attenuation. Cell biology is quite complex, and it remains fully feasible that PGRMC1 levels only correlate with the decidualization process. In the lack of convincing evidence, the manuscript should be rejected.

Author Response

We appreciate your review of our revised manuscript, constructive comments, and suggestions. Our responses to your comments are as follows.

As you pointed out, we have deleted the proposed model of the study (Figure 6). We have added the “Strength and limitation of this study” and “Conclusions” in the revised manuscript in accordance with the academic editor’s comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

see word attached

Comments for author File: Comments.docx

Reviewer 2 Report

In this study the group of Yoshie and colleagues aim to study the physiological roles of PGRMC1 and its regulation during the decidualization of human ESCs. Authors conclude that “…secretory phase-specific downregulation of endometrial PGRMC1 expression during the menstrual cycle, regulated in part by miR-98, may promote decidualization for the establishment of pregnancy”. In general, the manuscript contents the small amount of novel data and, in my opinion, this work is too preliminary and requires more experiments to provide evidence to support the authors' hypothesis that miR-98 mediates downregulation of PGRMC1 in decidualization.

There are some serious difficulties with this manuscript:

1. There are concerns about the originality and novelty of the present work. The implication of PGRMC1 in the human decidualization process is well established, see for instance:

• Salsano, Stefania, et al. "Dynamic expression of PGRMC1 and SERBP1 in human endometrium: an implication in the human decidualization process." Fertility and sterility 108.5 (2017): 832-842. doi: 10.1016/j.fertnstert.2017.07.1163 – in this paper the comprehensive immunohistochemical (and qPCR) analyses confirmed variable endometrial in vivo expression of PGRMC1 throughout the menstrual cycle, with a decrease signal in the late secretory phase in stromal cells. Additionally, in vitro functional studies indicated that low PGRMC1 levels are necessary for decidualization in human ESCs, as PGRMC1 overexpression in ESCs significantly inhibited the entire process.
• Garrido-Gómez, Tamara, et al. "Deciphering the proteomic signature of human endometrial receptivity." Human reproduction 29.9 (2014): 1957-1967. doi:10.1093/humrep/deu171. – this group also demonstrated the profile of PGRMC1 expression in endometrial biopsies from women throughout the menstrual cycle, and showed a significant decrease of PGRMC1 in the mid-secretory phase.

Therefore this current manuscript is just more of the same. The only difference is that while Salsano et al. used PGRMC1 overexpression approach to inhibit decidualization, the group of Yoshie used PGRMC1-depleted  ESCs that promoted the specific markers of decidualization.

2. Continuing the above comment, the authors should focus more on the mechanism underlying PGRMC1 downregulation, which in fact is as yet unknown. The authors pointed to the miR-98-mediated downregulation of PGRMC1 in decidualization. However, in my opinion, there is not enough data linking miR-98 with PGRMC1-downregulation during decidualization. At least specific inhibitor of miR-98 should be also used in this experiment. It would be great if the authors determine whether suggested miR-98-dependent regulation of PGRMC1 takes place directly, or rather indirectly through other miR-98 targets. I think this research should go in this direction.

Other comments:

3. I think that the effect of db-cAMP/P4 and db-cAMP in the presence or absence of siPGRMC1 or AG205 should be presented in the same graph. Additionally, the effect of P4 alone in this experiment should also be included.
4. The secretion of prolactin rather than mRNA expression should be analyzed to confirm decidualization.