lncRNA2919 Suppresses Rabbit Dermal Papilla Cell Proliferation via trans-Regulatory Actions

Round 1

Reviewer 1 Report

The Authors organized the article in a clear presentation to describe the function of the lncRNA2919 in HF. Results are interesting but the article is missing for important information to replicate adequately the experiments. Moreover, some results have to be improved furnishing more information.

Major comments.

Methods are missing of important information that are useful to replicate experiments. If different conditions were used to obtain results displayed in different figures, please explain the conditions used for each figure in the figure caption.
- How many cells per well were plated in the transfection experiments?
- How much RNA was used for the RACE experiments?
- How much RNA was used in the retrotranscription to clone targets in the pcDNA3.1 plasmid?
- How much cDNA was used for the cloning PCRs?
- Better describe methods used for the plasmids preparation (transformation, bacteria, minipreps/midipreps/maxipreps)
- Include sequencing results of the cloned genes.
- How much RNA was retrotrascribed for qRT-PCRs?
- How much cDNA was used in the qRT-PCR experiments?
- Why the Authors used GAPDH as reference gene? Is it demonstrated to be a reference gene in the cells they are using?
- Please include the amplification efciency of the primers used in the qRT-PCR experiments.
- Which quantity of proteins was loaded in the gel to perform WB experiments?

- How many cells per well were plated to perform cell proliferation and apoptosis assays?

- How was defined the promoter region of KRTAP11-1? I do not understand if in the Table S5 are listed the primers to clone the promoter region.
- How many cells were used in the transfections for luciferase assays?

- What was the protein concentration used for the EMSA experiments? Authors say they used 2 ul of nuclear proteins but it is uninformative

- Please, indicate in the supplementary tables the portion of the primer that is able to bind the target and the portion of the primer that is used for the cloning processes, or in vitro transcription or other.

- Please indicate the concentration of biotinilated RNA and proteins used for the RNA poll down assay.

- Table S9 represents possible interactor of lncRNA and cites co-expression. Please, indicate the co-expresison value. I suppose that Authors did Pearon or other correlations.

- It would be interesting to introduce the transcription direction in the figure 1B.

- Please, include a supplementary table describing the list of 449 proteins interacting with lncRNA antisense and 440 proteins that interact with the sense RNA. This could be data used for further studies or to support other studies.

- Please Authors can explain what does Input 1% means in the figure 3E? My question is referred to 1%.

- I do not understand why after the overexpression of KRTAP11-1 the protein expression is decreased.

 

 

Author Response

Dear Reviewer:

Thank you for your letter and for the reviewer’s comments concerning our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Reviewer 1：

Major comments.

Methods are missing of important information that are useful to replicate experiments. If different conditions were used to obtain results displayed in different figures, please explain the conditions used for each figure in the figure caption.

Thanks for the reviewer’s suggestion, the related sentence has been revised according to the reviewer’s advices.
1. How many cells per well were plated in the transfection experiments?

In the transfection experiments, at approximately 1.0*10⁶ per well in 6-well or 2.0*10⁵ per well in 24-well were seeded in the plates.
2. How much RNA was used for the RACE experiments?

According to the manufacturer’s instructions, 1 µg total RNA was used for the RACE.
3. How much RNA was used in the retrotranscription to clone targets in the pcDNA3.1 plasmid?

Thanks for the reviewer’s question. According to the manufacturer’s instructions, 1 µg total RNA was used for the retrotranscription. In the manuscript, we have provided the information of kits, the experiments were strictly carried out according to the manufacturer’s instructions, therefore, considering for the article length, many details weren’t showed in the manuscript.
4. How much cDNA was used for the cloning PCRs?

Thanks for the reviewer’s question. According to the manufacturer’s instructions, 1µl cDNA used for the cloning PCRs. In the manuscript, we have provided the information of kits, the experiments were strictly carried out according to the manufacturer’s instructions, therefore, considering for the article length, many details weren’t showed in the manuscript.
5. Better describe methods used for the plasmids preparation (transformation, bacteria, minipreps/midipreps/maxipreps)

Thanks for the reviewer’s suggestion. The PCR products were purified using MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, China), the sequence were subcloned into the pcDNA3.1(+) vector (Invitrogen, USA) and transformed into E.coli DH5α Competent Cells (TaKaRa, China), then the plasmids were obtained using EndoFree Maxi Plasmid Kit (Tiangen, China). The related sentences have been revised in the manuscript.
6. Include sequencing results of the cloned genes.

We have provided the cloning sequence of lncRNA2919, KRTAP11-1 and STAT1 in the Supplementary file 2.
7. How much RNA was retrotrascribed for qRT-PCRs?

According to the manufacturer’s instructions, 1μg total RNA was retrotrascribed for qRT-PCRs.
8. How much cDNA was used in the qRT-PCR experiment

According to the manufacturer’s instructions, 1µl cDNA was used in the qRT-PCR experiment.
9. Why the Authors used GAPDH as reference gene? Is it demonstrated to be a reference gene in the cells they are using?

Thanks for the reviewer’s question. GAPDH gene has highly conserved sequence, and highly expressed in almost tissues. The mRNA and protein expression of GAPDH were generally constant in the cells and tissues. Many studies has been demonstrated that GAPDH acted as the reference gene in dermal papilla cells, such as: https://doi.org/10.1016/0923-1811(94)90038-8, https://doi.org/10.1016/j.bbrc.2018.04.067, https://doi.org/10.1016/j.gendis.2022.04.015, https://doi.org/10.1016/j.ijbiomac.2022.02.177.

10. Please include the amplification efciency of the primers used in the qRT-PCR experiments.

Thanks for the reviewer’s suggestion, the Ct value of qRT-PCR is approximately 15 to 30 in the study. I don’t know how to show the amplification efficiency of the primers, because the mRNA relative expression is significantly difference in the different samples, but we could ensure the results of qRT-PCR were accurate and repeatable. If necessary, the reviewer may contact to us to get the raw files of qRT-PCR.
11. Which quantity of proteins was loaded in the gel to perform WB experiments?

Protein Simple Wes system was used in the study, according to the manufacturer’s instructions, 5µl protein samples was used for the Wes analysis.

12. How many cells per well were plated to perform cell proliferation and apoptosis assays?

In the study, 1*10⁴ cells per wells in 96-wells were plated to perform cell proliferation, and 2.0*10⁵ cells per wells in 24-wells were plated to perform apoptosis assays.

13. How was defined the promoter region of KRTAP11-1? I do not understand if in the Table S5 are listed the primers to clone the promoter region.

In order to verified the promoter activities of KRTAP11-1, the luciferase activities on -2818 ~ 0 loci upstream of ATG of the KRTAP11-1 were investigated (Figure 5A), Table S5 was the Primers used for constructing the luciferase reporter vector of KRTAP11-1 promoter segments.

14. How many cells were used in the transfections for luciferase assays?

In the study, 2.0*10⁵ cells per wells in 24-wells were used in the transfections for luciferase assays.

15. What was the protein concentration used for the EMSA experiments? Authors say they used 2 ul of nuclear proteins but it is uninformative

According to the manufacturer’s instructions, 2 μg protein (1μg/μl) used for the EMSA experiments.

16. Please, indicate in the supplementary tables the portion of the primer that is able to bind the target and the portion of the primer that is used for the cloning processes, or in vitro transcription or other.

Thanks for the reviewer’s suggestion, the legend of supplementary tables has been indicated the purposes of primers, all the primers could be identified by the primer designer software, such as NCBI Primer-blast, Primer Premier 5 and so on.

17. Please indicate the concentration of biotinilated RNA and proteins used for the RNA poll down assay.

Thanks for reviewer’s suggestion, 50 pmol biotinylated RNA and 200 μg proteins used for the RNA pull down assay.

18. Table S9 represents possible interactor of lncRNA and cites co-expression. Please, indicate the co-expresison value. I suppose that Authors did Pearon or other correlations.

Thanks for reviewer’s suggestion, we have revised the Table S9 according the reviewer’s advice.

19. It would be interesting to introduce the transcription direction in the figure 1B.

Thanks for the reviewer’s suggestion, we have revised the related content.

20. Please, include a supplementary table describing the list of 449 proteins interacting with lncRNA antisense and 440 proteins that interact with the sense RNA. This could be data used for further studies or to support other studies.

Thanks for the reviewer’s suggestion, we have showed the data in the Table S11.

21. Please Authors can explain what does Input 1% means in the figure 3E? My question is referred to 1%.

Input 1% is the control group, which used for the measurement of RIP-qPCR. We have revised the related sentences and figure in the manuscript.

22. I do not understand why after the overexpression of KRTAP11-1 the protein expression is decreased.

Thanks for reviewers’ suggestion. Sorry for our negligence, and we have revised the related sentence.

Reviewer 2 Report

The story of this manuscript is relatively simple; lncRNA2919 may prohibit DPC growth and suppress some HF growth related genes, and STAT1-KSTAP11-1 element regulate this lncRNA. On the contrary, there are too many figure data. Pease consider staying essential data in the Figures and the rest should be moved into the supplementary files.

 

The title should change since all the data were in vitro cell culture data and gene expression data, there was no direct evidence to prove lncRNA2919 suppresses hair follicle growth and development at all.

 

Following are additional concerns for labels and legends for Figures.

1.       Please specify DPC cells used for the experiments in detail, such as origin, sex, age, race, and the location, healthy or alopecia patient, passages, single resource, or multiple individuals etc.

2.       Figure Legends are too simple so that some figure data were difficult to understand. For example, what does “U6” means in Fig1 E and “Input(1%)” means in Figure 3.E.

3.       Figure legend for Figure 2 was obviously mixed up and I cannot understand what the authors want to say by Figure 2. Figure2 E should be apoptosis data, but legend does not state this. Also, the statement in the manuscript seems to be mixed up Fig2C and E.

4.       Legend for Figure 2C and Figure 4C was also too simple, need words at least such as “overexpression and knockdown”.  

Author Response

Dear Reviewer:

Thank you for your letter and for the reviewer’s comments concerning our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Reviewer 2：

The story of this manuscript is relatively simple; lncRNA2919 may prohibit DPC growth and suppress some HF growth related genes, and STAT1-KSTAP11-1 element regulate this lncRNA. On the contrary, there are too many figure data. Pease consider staying essential data in the Figures and the rest should be moved into the supplementary files.

Thanks for reviewers’ suggestion, considering to show the integrity of our work, we added some supplementary materials according to another reviewers, but we didn’t move some figures in the supplementary files because we think the figures was necessary to display out works.

The title should change since all the data were in vitro cell culture data and gene expression data, there was no direct evidence to prove lncRNA2919 suppresses hair follicle growth and development at all.

Thanks for reviewers’ suggestion, we have revised the title in the manuscript.

Following are additional concerns for labels and legends for Figures.

1. Please specify DPC cells used for the experiments in detail, such as origin, sex, age, race, and the location, healthy or alopecia patient, passages, single resource, or multiple individuals etc.

Thanks for reviewers’ suggestion. In the study, DPCs procured from our research group, which were separated from the 6-month-old male Angora rabbit dorsal HF.

2. Figure Legends are too simple so that some figure data were difficult to understand. For example, what does “U6” means in Fig1 E and “Input(1%)” means in Figure 3.E.

Thanks for reviewers’ suggestion. In the study, U6 RNA served as a positive control for nuclear gene expression. U6 snRNA always was utilized for the housekeeping gene, which was highly expressed in the nuclear. Input 1% is the control group, which used for the measurement of RIP-qPCR. We have revised the related sentences and figure in the manuscript.

3. Figure legend for Figure 2 was obviously mixed up and I cannot understand what the authors want to say by Figure 2. Figure2 E should be apoptosis data, but legend does not state this. Also, the statement in the manuscript seems to be mixed up Fig2C and E.

Thanks for reviewers’ suggestion. Sorry for our negligence, and we have revised the figure legend.

4. Legend for Figure 2C and Figure 4C was also too simple, need words at least such as “overexpression and knockdown”.  

According to reviewers’ suggestion, we have revised the figure legend.

Round 2

Reviewer 1 Report

The authors have answered most of my concerns, but some questions remain open, which I have highlighted below.

1. I asked how much cDNA was used to perform cloning experiments and the Authors responded to me 1 ul. What does it mean? It can be concentrated 10 nl/ul or 10 ug/ul. Please specify the concentration. The same problem is for the specification of the quantity of cDNA used in qRT-PCR.

2. Regarding my question on WB experiments the Authors responded "Protein Simple Wes system was used in the study, according to the manufacturer’s instructions, 5µl protein samples was used for the Wes analysis." Even if the system is an automatic system it needs specific quantities of proteins expressed in ug and not in ul. How concentrated were the 5 ul used?

3. Authors did not calculate primer amplification efficiency (only for the primers used for the qRT-PCR). As an example to calculate primer amplification efficiency see https://toptipbio.com/calculate-primer-efficiencies/ but the Authors can find several other examples.

4. Please, indicate in the supplementary tables the portion of the primer that is able to bind the target and the portion of the primer that is used for the cloning processes, or in vitro transcription or other. This suggestion is to facilitate the readers that, instead of using BLAST or other tools, can understand easyer how the primers work.

5. Authors printed an arrow to describe transcription direction in the Figure 1 B. Please indicate in the caption of the figure that the arrow describes the transcription direction of both genes (I suppose).

 

Author Response

Dear Reviewer:

Thank you for your letter and for the reviewer’s comments concerning our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Reviewer 1：

The authors have answered most of my concerns, but some questions remain open, which I have highlighted below.

1. I asked how much cDNA was used to perform cloning experiments and the Authors responded to me 1 ul. What does it mean? It can be concentrated 10 nl/ul or 10 ug/ul. Please specify the concentration. The same problem is for the specification of the quantity of cDNA used in qRT-PCR.

Thanks for reviewer’s suggestion, we have revised the related sentences, 50 ng cDNA used for the cloning experiment, and 10 ng cDNA used for qPCR.

2. Regarding my question on WB experiments the Authors responded "Protein Simple Wes system was used in the study, according to the manufacturer’s instructions, 5µl protein samples was used for the Wes analysis." Even if the system is an automatic system it needs specific quantities of proteins expressed in ug and not in ul. How concentrated were the 5 ul used?

Thanks for reviewer’s suggestion, we have revised the related sentences, 1.5 ng protein samples was used for the Wes analysis.

 

3. Authors did not calculate primer amplification efficiency (only for the primers used for the qRT-PCR). As an example to calculate primer amplification efficiency see https://toptipbio.com/calculate-primer-efficiencies/ but the Authors can find several other examples.

Thanks for reviewer’s suggestion, we have calculated the amplification efficiency of primers, the details showed as below.

Gene name	Slope	R Squared	Efficiency (%)
BCL2	-2.83823	0.9610	125.08
BMP2	-3.27485	0.9989	102.00
CCND1	-2.6473	0.9379	138.64
EGF	-2.84554	0.9899	124.61
GAPDH	-3.0787	0.9930	111.26
KRTAP11-1	-3.0452	0.9863	113.00
KRT17	-2.88842	0.9805	121.93
LEF1	-2.76057	0.9626	130.27
lncRNA2919	-2.88187	0.9990	122.33
SFRP2	-3.11368	0.9983	109.49
STAT1	-2.9215	0.9987	119.93
U6	-3.26701	0.7736	102.34
WNT2	-3.1043	0.9896	109.96

 

4. Please, indicate in the supplementary tables the portion of the primer that is able to bind the target and the portion of the primer that is used for the cloning processes, or in vitro transcription or other. This suggestion is to facilitate the readers that, instead of using BLAST or other tools, can understand easyer how the primers work.

Thanks for reviewer’s suggestion. In the supplementary tables, we have revised the related content that the bold font indicates the gene-specific primers.

5. Authors printed an arrow to describe transcription direction in the Figure 1 B. Please indicate in the caption of the figure that the arrow describes the transcription direction of both genes (I suppose).

Thanks for reviewer’s suggestion, we have added the related content in the figure legend.

Reviewer 2 Report

Please add "rat" dermal papilla in the title.  That is,  "lncRNA2919 suppresses rat dermal papilla cells proliferation via 2 trans-regulatory actions".

Rest of all other revisions by the authors are fine with me.

Author Response

Dear Reviewer:

Thank you for your letter and for the reviewer’s comments concerning our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

 

Please add "rat" dermal papilla in the title.  That is, "lncRNA2919 suppresses rat dermal papilla cells proliferation via 2 trans-regulatory actions".

Thanks for reviewer’s suggestion, we have revised the tittle.