The Molecular Mechanisms and Function of miR-15a/16 Dysregulation in Fibrotic Diseases

Round 1

Reviewer 1 Report

 

Many novel miRNAs are now associated with fibrosis, both organ-specific and systemic, as in the prototypical fibrotic disease systemic sclerosis. Dysregulated miRNAs are amenable to therapeutic modulation. In this paper, Da-Da Wen et al reviews the abnormal expression level and the molecular mechanism of miR-15a/16 in liver, lung, heart, kidney, and other fibrotic disease. Therefore, they suggested that miR-15a/16 could be developed as a biomarker for the diagnosis of fibrosis and also as a therapeutic target for anti-fibrosis.

This should be a brief, very relevant and interesting review article. It would be nice if the authors could give a small summary of the role of miRNAs in fibrosis (pro-fibrotic or anti-fibrotic) and the possibility of these being used as therapeutic targets and highlight the advantages of miR-15a/16. And it is recommended that authors have English speakers or professionals revise their manuscripts to make grammar and sentence trends easier for readers to navigate and understand.

Author Response

Response to Reviewer 1 Comments
Point 1: It would be nice if the authors could give a small summary of the role of miRNAs in fibrosis (pro-fibrotic or anti-fibrotic) and the possibility of these being used as therapeutic targets and highlight the advantages of miR-15a/16.

Response 1: We sincerely appreciate your careful review and these constructive suggestions!

Based on your kind suggestions, we have rewritten the Conclusions section as follows:

“In summary, miR-15a/16 is an important molecule in regulating the occurrence and development of fibrotic diseases. miR-15a/16 can inhibit the proliferation and phenotypic transformation of fibroblasts, inhibit ECM synthesis, promote ECM degradation, induce cell apoptosis, and affect the release of fibrotic mediators by targeting the fibrosis signal pathway, inflammation signal pathway, cell proliferation and apoptosis signal pathway, collagen synthesis, and other related genes, thereby weakening liver fibrosis, pulmonary fibrosis, and renal fibrosis. It can also accelerate the progression of myocardial fibrosis by activating the signal transduction of the pro-fibrotic factor TGF-β and by interfering with normal physiological processes such as cell growth and apoptosis. Because there are differences in the target genes in different cell types and the pathogenesis or immune cell response between different fibrosis diseases, the dysregulation, mechanism, and function of miR-15a/16 in the process of fibrosis in different tissues and organs are different. Therefore, further exploring the target genes of miR-15a/16 in different cell types and the relationship between miR-15a/16 and fibrotic diseases will help to clarify the pathogenesis of fibrotic diseases so as to provide new ideas for the treatment of fibrotic diseases.” (Page 11, Line 297-312)

Point 2: it is recommended that authors have English speakers or professionals revise their manuscripts to make grammar and sentence trends easier for readers to navigate and understand.

Response 2: We sincerely thank you for these comments. Based on your kind suggestion, we asked a professional native speaker of English to revise my manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript summarizes the molecular mechanisms and the role of miR-15 and miR-16 in fibrotic diseases. This review is interesting, although the information on each reference is scarce.  

·      In the summary of each reference mentioned in the manuscript, must detail more information about the experimental design and sample type. For example, when the manuscript describes references 42 and 44, it is important to indicate that the miRNAs are derived from exosomes and extracellular vesicles respectively. Therefore, please add a column in table 2 with the information on the sample type that was used in each study.

·      It is important to be precise in each reference if the study is about miR15a/16 as a cluster or as independent miRNAs. For example, line 120, 142,153, and 165 says miR15a/miR16 but the examples describe miRNA-15 or miRNA-16 as independent miRNAs.

·      Please check carefully that all the references mentioned in the manuscript are also described in table 2. For example, references 42 and 43 are not present.

·      Add a paragraph with the following recent reference

 

Dehmel S, Weiss KJ, El-Merhie N, Callegari J, Konrad B, Mutze K, Eickelberg

O, Königshoff M, Krauss-Etschmann S. microRNA Expression Profile of Purified Alveolar Epithelial Type II Cells. Genes (Basel). 2022 Aug 10;13(8):1420. DOI:10.3390/genes13081420. PMID: 36011331; PMCID: PMC9407429.

 

 

And as minor corrections:

 

·      Review all the text, because it has many mistakes in the spaces between words. Also, correct some typographic mistakes, for example, on page 4, line 127 “fibroblasts.rly?” or line 139,” b-FGF”?.

·      Regarding table 2. Remove the plural form in the subtitles (Disease type and Expression)

·       In the column of “biological processes affected” edit the information because it is overlapped

Author Response

Response to Reviewer 2 Comments

Point 1: In the summary of each reference mentioned in the manuscript, must detail more information about the experimental design and sample type. For example, when the manuscript describes references 42 and 44, it is important to indicate that the miRNAs are derived from exosomes and extracellular vesicles respectively. Therefore, please add a column in table 2 with the information on the sample type that was used in each study.

Response 1: We sincerely appreciate your careful review and these constructive suggestions! Based on your kind suggestion, we have added the information on the sample type that was used in each study in the table 2 as follows:

miRNA	Disease type	Expression	Target gene	Biological processes affected	Study model/cell	reference
miR-16	Liver fibrosis	Upregulation	HGF and  Smad7	Affects the TGF-β/Smad signaling pathway and promotes liver fibrosis	QSG-7701 cells and Huh-7.5.1 cells	[19]
miR-16	Liver fibrosis	Downregulation	Smad2/3	Inhibits the TGF-β/Smad pathway	HSCs	[21]
miR-16	Liver fibrosis	Downregulation	Bcl-2	Affects the mitochondrial apoptosis pathway and induces apoptosis	HSCs	[37,38,40]
miR-16	Liver fibrosis	Downregulation	LOXL1	Inhibits Smad2/3 phosphorylation and inhibits the TGF-β/Smad pathway	LX2 cells	[39]
miR-16	Liver fibrosis	Downregulation	Gα12	Affects its binding to ATG12-5 and induces autophagy	HSCs	[41]
miR-15a	Pulmonary fibrosis	Downregulation	YAP1/Twist	Modulates the lncRNA PFAR/ YAP1/Twist axis to inhibit cell activation and ECM deposition	Primary lung fibroblasts	[16,27]
miR-16	Pulmonary fibrosis	Downregulation	Rictor	Inhibits the mTORC2 signaling pathway, downregulates SPARC expression and reduces ECM synthesis	Human fetal lung fibroblast (HFL-1) cells	[28]
miR-16	Pulmonary fibrosis	Downregulation	TLR4	Inhibits the TLR4/NF-kB pathway and the NLRP3 inflammasome signaling pathway	A549 cells and 293 T cells	[34]
miR-16	Pulmonary fibrosis	Downregulation	ND	ND	Serum exosomes of IPF patients	[42]
miR-16	Pulmonary fibrosis	Downregulation	Bcl2, NF-kB, IL-1, JNK, Smad3/7, COL-I, and TGFβ-R	Affects apoptosis, Wnt signaling, TLR signaling, and the TGF-β/Smad pathway	Bleomycin mouse model of IPF	[43]
miR-16	Pulmonary fibrosis	Downregulation	Wnt ligands(including Wnt10b and Wnt3a)	Attenuates the Wnt signaling pathway, inhibits fibroblast transformation, and induces epithelial senescence	HBEC EVs and mouse models of BLM-induced lung fibrosis	[44]
miR -16	Pulmonary fibrosis	Downregulation	ATG5/7	Affects mitochondrial dysfunction and cellar senescence	MRC-5 cells and endothelial cells	[45]
miR -16	Pulmonary fibrosis	Downregulation	MAP2K1, MAP2K4, JUN and BCL2	Inhibits the TGF-β/Smad pathway	A549 cells and primary murine ATII cells	[46]
miR -15a	Cardiac fibrosis	Upregulation	ND	Promotes the MAPK and TGF-β/Smad pathways	Serum of diffuse myocardial fibrosis patients	[47]
miR - 15a, miR - 16	Cardiac fibrosis	Upregulation	ND	Represses cardiomyocyte proliferation	C57BL/6 mice	[48]
miR - 15a	Cardiac fibrosis	Upregulation	SMAD7	Promotes the TGF-β/Smad pathway	Lipopolysaccharide (LPS) stimulated H9C2 cells	[49]
miR-16	Hypertensive heart disease	Upregulation	ADRA1A	Promotes cardiomyocyte apoptosis	Primary myocardial cells and H9C2 cells	[50]
miR - 15a	Cardiac fibrosis	Downregulation	ND	Affects the TGF-β/Smad and p53 pathways	Human primary cardiac fibroblasts and Type 2 diabetic mice	[51]
miR-16	Renal fibrosis	Downregulation	MMP-9 and IL-6	Affects the fibrotic/inflammatory genes in the kidney tissue	Urinary exosomes (UEs) of diabetic rats	[53]
miR-16	Renal fibrosis	Downregulation	DEC2	Inhibits the TLR4 signaling pathway	SV40 MES-13 cells and Fcgamma receptor II-b-deficient (Fcgr2b−/−) mouse	[54]
miR - 15a	Renal fibrosis	Downregulation	ND	Affects the TGF-β/Smad signaling pathway	Remnant kidney model and NRK-52E cells	[55]
miR-16	Skin fibrosis	Downregulation	Smad3	Inhibits the TGF-β/Smad pathway	Exosomes from HaCaT cells and HDFs	[22]
miR-16	Systemic sclerosis	Downregulation	Notch2	Inhibits Notch signaling and reduces the synthesis of collagen and other ECM components	HDFs and HSFs	[26]
miR-16	Systemic sclerosis	Upregulation	ND	Induces apoptosis	Serum of systemic sclerosis patients	[56]
miR-16	Systemic sclerosis	Downregulation	Survivin and p53	Inhibits cell proliferation and promotes cell apoptosis	HSFs	[57]

 

Point 2: It is important to be precise in each reference if the study is about miR15a/16 as a cluster or as independent miRNAs. For example, line 120, 142,153, and 165 says miR15a/miR16 but the examples describe miRNA-15 or miRNA-16 as independent miRNAs.

Response 2: We sincerely thank you for these comments. We have revised the sentence in the original Line 120, 142, 153, and 165 as follows:

“In addition, miR-15a and miR-16 can also affect other signaling pathways related to ECM formation.” (Page 4, Line 120)

“On the one hand, miR-16 can directly inhibit the release of inflammatory cytokines and other pro-fibrotic mediators.” (Page 4, Line 142)

“On the other hand, miR-16 can regulate the expression of fibrosis mediators such as IL-1β and IL-18 by affecting the NOD-like receptor protein 3 (NLRsP3) inflammasome pathway.” (Page 4, Line 153)

“miR-15a and miR-16 are abnormally upregulated or downregulated in fibrotic dis-eases and promote or inhibit various fibrotic diseases by targeting different genes.” (Page 5, Line 165)

 

Point 3: Please check carefully that all the references mentioned in the manuscript are also described in table 2. For example, references 42 and 43 are not present.

Response 3: We sincerely thank you for these comments. Based on your suggestions, we have supplemented and refined the references mentioned in this article in Table 2.

 

Point 4: Add a paragraph with the following recent reference.

Response 4: We sincerely thank you for this comment. We have cited your recommended references in the pulmonary fibrosis section as follows:

“In addition, Dehmel et al. [46] performed a miRNA analysis via flow cytometry sorting and purifying alveolar epithelial cell type II (ATII) and found that miR-16 was expressed abundantly in normal alveolar cells, but the expression of miR-16 was inhibited after TGF-β stimulation, and the expression of potential targets such as MAP2K1, MAP2K4, JUN, and BCL2 was upregulated. Therefore, miR-16 can regulate cell activation pathways to maintain alveolar cell homeostasis and prevent the occurrence and development of lung injury such as fibrotic lung diseases.” (Page 8, Line 218-225)

 

Point 5: And as minor corrections:

1、Review all the text, because it has many mistakes in the spaces between words. Also, correct some typographic mistakes, for example, on page 4, line 127 “fibroblasts.rly?” or line 139,” b-FGF”?.

2、Regarding table 2. Remove the plural form in the subtitles (Disease type and Expression).

3、In the column of “biological processes affected” edit the information because it is overlapped

Response 5: We sincerely appreciate your careful review and these constructive suggestions!  Based on your kind suggestions, we have corrected some typographic mistakes and removed the plural form in the subtitles (Disease type and Expression).

For the column of “biological processes affected” section, we have re-edited the information.

Author Response File: Author Response.pdf