DDP-resistant ovarian cancer cells-derived exosomal microRNA-30a-5p reduces the resistance of ovarian cancer cells to DDP.

Exosomes carrying microRNAs (miRNAs) have been demonstrated to play critical roles in the regulation of development, growth and metastasis of cancer. Bioinformatic predictions identified differentially expressed SRY-box 9 (SOX9) in OC, and the regulatory miRNA miR-139-5p. Here, we aim to evaluate the function of exosomal miR-139-5p in the sensitivity of ovarian cancer (OC) cells to cis-diamminedichloroplatinum(II) (DDP). Expression pattern of miR-139-5p and SOX9 in ovarian cancer cells (SKOV3) and DDP-resistant cells (SKOV3/DDP) was identified using reverse transcription quantitative polymerase chain reaction and western blot analysis. The relationship between miR-139-5p and SOX9 was validated using a dual-luciferase reporter assay. SKOV3/DDP cell line was developed and introduced with miR-30a-5p mimic to analyse the effects of miR-30a-5p on resistance to DDP. The in vitro and in vivo effects of exosomal miR-30a-5p on resistance of SKOV3 cells to DDP were assessed in a co-culture system of exosomes and OC cells as well as in tumour-bearing nude mice. High expression of SOX9 and low expression of miR-30-5p were witnessed in OC. Furthermore, miR-30-5p, a downregulated miRNA in SKOV3/DDP cells, increased the rate of cell apoptosis and enhanced the sensitivity of SKOV3/DDP cells to DDP by targeting SOX9. Moreover, exosomes carrying miR-30a-5p were identified to sensitize SKOV3/DDP cells to DDP both in vitro and in vivo. These data together supported an important conclusion that DDP-resistant OC cell-derived exosomal miR-30a-5p enhanced cellular sensitivity to DDP, highlighting a potential strategy to overcome drug resistance.

cancer. The authors should create an siRNA that specifically targets Sox9 or use an overexpression vector to show that overexpression of Sox9 is sufficient to cause resistance.
2. In figure 2, the binding site shown is labeled as being for miR 23a-5p, not miR 30a-5p. Is this an error? If not, please explain how the two molecules are related. Exosomes were labelled with pkh67, a dye that may form nanoparticles similar to exosomes in size. Ultracentrifugation used to isolate exosomes would not separate PKH67 nanoparticles from exosomes. Sucrose gradient separation is a feasible way to remove dye aggregates.
3. In Figure 5, the microscopy was performed using PKH26 while exosomes were stained with PKH67. While biochemically similar, these two dyes have different fluorescent characteristics with PKH67 visualized in the GFP channel and PKH26 in the red.
4. I also recommend substantial editing as there are many typos and grammar issues, examples: -Fig 5b Y-axis label numbei instead of number -Sentence beginning with "And". " On the contrary, the expression of miR-30a-5p prominently decreased in exosomes derived from SKOV3 cells that had been transfected with miR30a-5p inhibitor. And the expression of . . . " -Fig7 description scar bar vs scale bar.

Review form: Reviewer 2
Recommendation Accept with minor revision (please list in comments)

Do you have any ethical concerns with this paper? No
Comments to the Author Liu et al. have presented a significant body of work describing the relationship between miR-30a-59/Sox9 and cisplatin resistance in ovarian cancer. Through the use of a miR-mimic, the authors demonstrate that miR-30a-5p targets Sox9 mRNA, which in turn decreases Sox9 protein levels. Increased Sox9 was observed in cisplatin resistant cells and decreasing Sox9 returned sensitivity to drug. Finally, the authors report that exosomes containing miR-30a-5p reduced both in vitro cell line proliferation and in vivo tumor growth. Overall the report is well written and the data is presented well. To strengthen the relationship between Sox9 and cisplatin sensitivity I would suggest silencing Sox9 by RNAi and/or overexpressing (Sox9) is OC cells. This will confirm whether sensitivity is due to regulation of Sox9 or other miR-30a-5p target genes. Minor: 1. Define DDP in the Abstract and manuscript body. Likewise, minimize switching between DDP and cisplatin (e.g. Abstract vs Introduction). 2. Define DEGs in the first results section. 3. The first paragraph of the introduction (references 3-6) could be condensed as it currently repetitive. 4. Figure 7A and B. Order the tumor images the same as the adjacent graph key (Fig. 7B. Decision letter (RSOB-19-0173.R0)

17-Sep-2019
Dear Dr Wang, We are writing to inform you that the Editor has reached a decision on your manuscript RSOB-19-0173 entitled "Exosomal transfer of cisplatin-resistant ovarian cancer cells-derived microRNA-30a-5p reduces resistance of ovarian cells to cisplatin", submitted to Open Biology.
As you will see from the reviewers' comments below, there are a number of criticisms that prevent us from accepting your manuscript at this stage. The reviewers suggest, however, that a revised version could be acceptable, if you are able to address their concerns. If you think that you can deal satisfactorily with the reviewer's suggestions, we would be pleased to consider a revised manuscript.
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Sincerely, The Open Biology Team mailto: openbiology@royalsociety.org Reviewer(s)' Comments to Author(s): Referee: 1 Comments to the Author(s) This manuscript presents some original and interesting findings and will likely be of interest to many people in the field. Besides the scientific comments listed below, I'd like to point out that the manuscript contains a large number of typos and grammatical errors. Both new experiments and substantial editorial revisions are needed to improve the quality of the manuscript.
1. While the authors showed a correlation between Sox9/miR 30a-5p and susceptibility to drug treatment, they did not definitively show the involvement of sox9 in the pathway. They acknowledged that this miRNA is known to affect expression of other proteins in various types of cancer. The authors should create an siRNA that specifically targets Sox9 or use an overexpression vector to show that overexpression of Sox9 is sufficient to cause resistance.
2. In figure 2, the binding site shown is labeled as being for miR 23a-5p, not miR 30a-5p. Is this an error? If not, please explain how the two molecules are related. Exosomes were labelled with pkh67, a dye that may form nanoparticles similar to exosomes in size. Ultracentrifugation used to isolate exosomes would not separate PKH67 nanoparticles from exosomes. Sucrose gradient separation is a feasible way to remove dye aggregates.
3. In Figure 5, the microscopy was performed using PKH26 while exosomes were stained with PKH67. While biochemically similar, these two dyes have different fluorescent characteristics with PKH67 visualized in the GFP channel and PKH26 in the red. Referee: 2 Comments to the Author(s) Liu et al. have presented a significant body of work describing the relationship between miR-30a-59/Sox9 and cisplatin resistance in ovarian cancer. Through the use of a miR-mimic, the authors demonstrate that miR-30a-5p targets Sox9 mRNA, which in turn decreases Sox9 protein levels. Increased Sox9 was observed in cisplatin resistant cells and decreasing Sox9 returned sensitivity to drug. Finally, the authors report that exosomes containing miR-30a-5p reduced both in vitro cell line proliferation and in vivo tumor growth. Overall the report is well written and the data is presented well. To strengthen the relationship between Sox9 and cisplatin sensitivity I would suggest silencing Sox9 by RNAi and/or overexpressing (Sox9) is OC cells. This will confirm whether sensitivity is due to regulation of Sox9 or other miR-30a-5p target genes. Minor: 1. Define DDP in the Abstract and manuscript body. Likewise, minimize switching between DDP and cisplatin (e.g. Abstract vs Introduction). 2. Define DEGs in the first results section. 3. The first paragraph of the introduction (references 3-6) could be condensed as it currently repetitive. 4. Figure 7A and B. Order the tumor images the same as the adjacent graph key (Fig. 7B

Response to Reviewers
Dear Reviewers, Thank you very much for your letter and the comments from the referees about our paper entitled "DDP-resistant ovarian cancer cells-derived exosomal microRNA-30a-5p reduces resistance of ovarian cancer cells to DDP" submitted to "Open Biology". We have checked the manuscript and revised it according to the comments. We submit here the revised manuscript as well as a list of changes. If you have any question about this paper, please don't hesitate to let me know.

Comments to the Author(s)
This manuscript presents some original and interesting findings and will likely be of interest to many people in the field. Besides the scientific comments listed below, I'd like to point out that the manuscript contains a large number of typos and grammatical errors. Both new experiments and substantial editorial revisions are needed to improve the quality of the manuscript.
1. While the authors showed a correlation between Sox9/miR 30a-5p and susceptibility to drug treatment, they did not definitively show the involvement of sox9 in the pathway. They acknowledged that this miRNA is known to affect expression of other proteins in various types of cancer. The authors should create an siRNA that specifically targets Sox9 or use an overexpression vector to show that overexpression of Sox9 is sufficient to cause resistance.
Response: Following your suggestion, we have supplemented "In order to directly prove the role

of SOX9 in this regulation, we delivered si-SOX9 into SKOV3/DDP cells and found that si-SOX9
significantly decreased DDP IC 50 , blocked cell cycle in G1 phase, and enhanced the apoptosis rate significantly (Figure 4f). In addition, we noted that the inhibitory effects of miR-30a-5p Appendix A inhibitor on DDP sensitivity could be reversed by the inhibition of SOX9 (Figure 4g), while

si-SOX9 counteracted the inhibitory effect of miR-30a-5p mimic on OC resistance to DDP (Figure 4h)."
2. In figure 2, the binding site shown is labeled as being for miR 23a-5p, not miR 30a-5p. Is this an error? If not, please explain how the two molecules are related. Exosomes were labelled with pkh67, a dye that may form nanoparticles similar to exosomes in size. Ultracentrifugation used to isolate exosomes would not separate PKH67 nanoparticles from exosomes. Sucrose gradient separation is a feasible way to remove dye aggregates.