Expression of microRNA-296 in rabbit hypertrophic scars and its role to human fibroblasts

Guo Bingyu; Lin Feng; Bai Zeming; Tao Kai; Wang Hongyi

doi:10.3760/cma.j.cn501120-20210420-00142

Volume 37 Issue 8

Aug. 2021

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2021 > 37(8): 725-730

Guo BY,Lin F,Bai ZM,et al.Expression of microRNA-296 in rabbit hypertrophic scars and its role to human fibroblasts[J].Chin J Burns,2021,37(8):725-730.DOI: 10.3760/cma.j.cn501120-20210420-00142.

Citation:

Guo BY,Lin F,Bai ZM,et al.Expression of microRNA-296 in rabbit hypertrophic scars and its role to human fibroblasts[J].Chin J Burns,2021,37(8):725-730.DOI: 10.3760/cma.j.cn501120-20210420-00142.

Citation:

Guo BY,Lin F,Bai ZM,et al.Expression of microRNA-296 in rabbit hypertrophic scars and its role to human fibroblasts[J].Chin J Burns,2021,37(8):725-730.DOI: 10.3760/cma.j.cn501120-20210420-00142.

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Expression of microRNA-296 in rabbit hypertrophic scars and its role to human fibroblasts

doi: 10.3760/cma.j.cn501120-20210420-00142

Department of Burns and Plastic Surgery, General Hospital of Northern Theater Command, Shenyang 110016, China

Funds:

Youth Project of National Natural Science Foundation of China 82002047

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Corresponding author: Wang Hongyi, Email: why1_0@163.com
Received Date: 2021-04-20

Abstract

Abstract

Objective To investigate the expression of microRNA-296 (miR-296) in rabbit hypertrophic scars and its role in human fibroblasts (HFbs). Methods The experimental method was used. Twelve healthy adult New Zealand long-eared rabbits regardless gender were randomly divided into normal control group and scar group, with 6 rabbits in each group. The rabbit ear hypertrophic scar model was created in scar group according to the literature, and the rabbits in normal control group did not receive any treatment. On 60 days after setting up the models in scar group, hematoxylin-eosin staining was performed to observe the growth and arrangement of fibroblasts (Fbs) in the ear scars and skin tissue of rabbits in the two groups. The mRNA expressions of miR-296 and transforming growth factor-β₁ (TGF-β₁) in ear scars and skin tissue of rabbits in the two groups were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction, and the correlation of mRNA between miR-296 and TGF-β₁ was performed with Pearson regression analysis. Two batches of HFbs were used and transfected respectively with corresponding sequences, with the 1st batch being divided into TGF-β₁ wild type+miR-296 negative control group and TGF-β₁ wild type+miR-296 mimic group and the 2nd batch being divided into TGF-β₁ mutant type+miR-296 negative control group and TGF-β₁ mutant type+miR-296 mimic group. At 48 h after transfection, luciferase reporter gene detection kit was used to detect the luciferase and renal luciferase expression of TGF-β₁ in the cells of each group, with their ratio being used to reflect the gene expression level. Two batches of HFbs were used, and each batch of cells were divided into miR-296 negative control group and miR-296 mimic group, being transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfecting the first batch of cells, the cell proliferation was detected by thiazolyl blue method. At 24 h after transfecting the second batch of cells, the expression of TGF-β₁ and collagen type Ⅰ was detected by Western blotting. The number of samples in cell experiments was 3. Data were statistically analyzed with analysis of variance for factorial design, independent sample t test. Results On 60 days after setting up the models in scar group, the Fbs of rabbit ear scar tissue in scar group proliferated and arranged disorderly, while the growth and arrangement of Fbs in rabbit ear skin tissue in normal control group were normal. The mRNA expression of miR-296 of rabbit scar tissue in scar group (0.65±0.11) was significantly lower than 1.19±0.12 of rabbit ear skin tissue in normal control group (t=5.175, P<0.01). The mRNA expression of TGF-β₁ of rabbit ear scar tissue in scar group (1.47±0.06) was significantly higher than 1.10±0.03 of rabbit ear skin tissue in normal control group (t=12.410, P<0.01). Pearson regression analysis showed that there was a negative correlation between the mRNA expression of miR-296 and TGF-β₁ in the ear scars and skin tissue of 12 rabbits (F=7.278, P<0.05). At 48 h after transfection, the gene expression of TGF-β₁ of cells in TGF-β₁ wild type+miR-296 mimic group was significantly lower than that in TGF-β₁ wild type+miR-296 negative control group (t=35.190, P<0.01), while the gene expression of TGF-β₁ of cells in the two TGF-β₁ mutant type groups were close (P>0.05). The HFbs proliferation ability in miR-296 mimic group was significantly lower than that in miR-296 negative control group at 12, 24, 36, and 48 h after transfection(t=3.275, 11.980, 10.460, 17.260, P<0.05 or P<0.01). At 24 h after transfection, the protein expressions of TGF-β₁ and type Ⅰ collagen of cells in miR-296 negative control group were significantly higher than those in miR-296 mimic group (t=3.758, 29.390, P<0.05 or P<0.01). Conclusions The miR-296 expression in rabbit hypertrophic scars is down-regulated; miR-296 can inhibit the proliferation of HFbs and the expression of type Ⅰ collagen by down regulating the expression of TGF-β₁.
- Cicatrix,
- Fibroblasts,
- Transforming growth factor beta 1,
- microRNA

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References(30)

References

[1]	KirkpatrickLD,ShuppJW,SmithRD,et al.Galectin-1 production is elevated in hypertrophic scar[J].Wound Repair Regen,2021,29(1):117-128.DOI: 10.1111/wrr.12869.
[2]	LeX,WuWW.The therapeutic effect of interleukin-18 on hypertrophic scar through inducing Fas ligand expression[J].Burns,2021,47(2):430-438.DOI: 10.1016/j.burns.2020.07.008.
[3]	TanJ,ZhouJ,HuangL,et al.Hypertrophic scar improvement by early intervention with ablative fractional carbon dioxide laser treatment[J].Lasers Surg Med,2021,53(4):450-457.DOI: 10.1002/lsm.23301.
[4]	LiQ,ChenX,ChenL,et al.LINC00173 promotes the apoptosis of hypertrophic scar fibroblasts through increasing β-catenin expression[J].Mol Cell Biochem,2021,476(2):1005-1014.DOI: 10.1007/s11010-020-03966-6.
[5]	LabergeA,MerjanehM,ArifS,et al.Shedding of proangiogenic microvesicles from hypertrophic scar myofibroblasts[J].Exp Dermatol,2021,30(1):112-120.DOI: 10.1111/exd.14178.
[6]	ZhangT,WangXF,WangZC,et al.Current potential therapeutic strategies targeting the TGF-β/Smad signaling pathway to attenuate keloid and hypertrophic scar formation[J].Biomed Pharmacother,2020,129:110287.DOI: 10.1016/j.biopha.2020.110287.
[7]	SongJ,LiX,LiJ.Emerging evidence for the roles of peptide in hypertrophic scar[J].Life Sci,2020,241:117174.DOI: 10.1016/j.lfs.2019.117174.
[8]	NongX,RajbanshiG,ChenL,et al.Effect of artesunate and relation with TGF-β1 and SMAD3 signaling on experimental hypertrophic scar model in rabbit ear[J].Arch Dermatol Res,2019,311(10):761-772.DOI: 10.1007/s00403-019-01960-7.
[9]	WeiG,XuQ,LiuL,et al.LY2109761 reduces TGF-β1-induced collagen production and contraction in hypertrophic scar fibroblasts[J].Arch Dermatol Res,2018,310(8):615-623.DOI: 10.1007/s00403-018-1849-1.
[10]	HuangY,WangY,WangX,et al.The effects of the transforming growth factor-β1 (TGF-β1) signaling pathway on cell proliferation and cell migration are mediated by ubiquitin specific protease 4 (USP4) in hypertrophic scar tissue and primary fibroblast cultures[J].Med Sci Monit,2020,26:e920736.DOI: 10.12659/MSM.920736.
[11]	LiYH,YangJ,ZhengZ,et al.Botulinum toxin type A attenuates hypertrophic scar formation via the inhibition of TGF-β1/Smad and ERK pathways[J].J Cosmet Dermatol,2021,20(5):1374-1380.DOI: 10.1111/jocd.13842.
[12]	LiL,HanW,ChenY,et al.MiR-3613-3p inhibits hypertrophic scar formation by down-regulating arginine and glutamate-rich 1[J].Mol Cell Biochem,2021,476(2):1025-1036.DOI: 10.1007/s11010-020-03968-4.
[13]	ZhangQ,GuoB,HuiQ,et al.miR-137 inhibits proliferation and metastasis of hypertrophic scar fibroblasts via targeting pleiotrophin[J].Cell Physiol Biochem,2018,49(3):985-995.DOI: 10.1159/000493236.
[14]	LiuB,GuoZ,GaoW.miR-181b-5p promotes proliferation and inhibits apoptosis of hypertrophic scar fibroblasts through regulating the MEK/ERK/p21 pathway[J].Exp Ther Med,2019,17(3):1537-1544.DOI: 10.3892/etm.2019.7159.
[15]	FangL,EllimsAH,MooreXL,et al.Circulating microRNAs as biomarkers for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy[J].J Transl Med,2015,13:314.DOI: 10.1186/s12967-015-0672-0.
[16]	MotawiTK,ShakerOG,El-MaraghySA,et al.Serum microRNAs as potential biomarkers for early diagnosis of hepatitis C virus-related hepatocellular carcinoma in Egyptian patients[J].PLoS One,2015,10(9):e0137706.DOI: 10.1371/journal.pone.0137706.
[17]	CantaluppiV,GattiS,MedicaD,et al.Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells[J].Kidney Int,2012,82(4):412-427.DOI: 10.1038/ki.2012.105.
[18]	ZhangT,ShenZ,ZhengJ,et al.Effect of UVA1 on hypertrophic scarring in the rabbit ear model[J].Biosci Rep,2020,40(1):BSR20190007. DOI: 10.1042/BSR20190007.
[19]	de BakkerE,van der PuttenM,HeymansMW,et al.Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity[J].Exp Dermatol,2021,30(1):169-178.DOI: 10.1111/exd.14139.
[20]	YangJH,YoonJY,MoonJ,et al.Expression of inflammatory and fibrogenetic markers in acne hypertrophic scar formation: focusing on role of TGF-β and IGF-1R[J].Arch Dermatol Res,2018,310(8):665-673.DOI: 10.1007/s00403-018-1856-2.
[21]	LiuJ,ZhaoB,ZhuH,et al.Wnt4 negatively regulates the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition via targeting Smad3 and ERK[J].Cell Tissue Res,2020,379(3):537-548.DOI: 10.1007/s00441-019-03110-x.
[22]	DobaczewskiM,ChenW,FrangogiannisNG.Transforming growth factor (TGF)-β signaling in cardiac remodeling[J].J Mol Cell Cardiol,2011,51(4):600-606.DOI: 10.1016/j.yjmcc.2010.10.033.
[23]	ChenH,XuY,YangG,et al.Mast cell chymase promotes hypertrophic scar fibroblast proliferation and collagen synthesis by activating TGF-β1/Smads signaling pathway[J].Exp Ther Med,2017,14(5):4438-4442.DOI: 10.3892/etm.2017.5082.
[24]	ShiDM,LiLX,BianXY,et al.miR-296-5p suppresses EMT of hepatocellular carcinoma via attenuating NRG1/ERBB2/ERBB3 signaling[J].J Exp Clin Cancer Res,2018,37(1):294.DOI: 10.1186/s13046-018-0957-2.
[25]	FuQ,SongX,LiuZ,et al.miRomics and proteomics reveal a miR-296-3p/PRKCA/FAK/Ras/c-Myc feedback loop modulated by HDGF/DDX5/β-catenin complex in lung adenocarcinoma[J].Clin Cancer Res,2017,23(20):6336-6350.DOI: 10.1158/1078-0432.CCR-16-2813.
[26]	ZhuL,DengH,HuJ,et al.The promising role of miR-296 in human cancer[J].Pathol Res Pract,2018,214(12):1915-1922.DOI: 10.1016/j.prp.2018.09.026.
[27]	ZhangXW,WangL,DingH.Long noncoding RNA AK089579 inhibits epithelial-to-mesenchymal transition of peritoneal mesothelial cells by competitively binding to microRNA-296-3p via DOK2 in peritoneal fibrosis[J].FASEB J,2019,33(4):5112-5125.DOI: 10.1096/fj.201801111RR.
[28]	ChenY,GaoH,LiY.Inhibition of LncRNA FOXD3-AS1 suppresses the aggressive biological behaviors of thyroid cancer via elevating miR-296-5p and inactivating TGF-β1/Smads signaling pathway[J].Mol Cell Endocrinol,2020,500:110634.DOI: 10.1016/j.mce.2019.110634.
[29]	ChenM,ChenC,LuoH,et al.MicroRNA-296-5p inhibits cell metastasis and invasion in nasopharyngeal carcinoma by reversing transforming growth factor-β-induced epithelial-mesenchymal transition[J].Cell Mol Biol Lett,2020,25(1):49.DOI: 10.1186/s11658-020-00240-x.
[30]	CaoZ,LiuW,QuX,et al.miR-296-5p inhibits IL-1β-induced apoptosis and cartilage degradation in human chondrocytes by directly targeting TGF-β1/CTGF/p38MAPK pathway[J].Cell Cycle,2020,19(12):1443-1453.DOI: 10.1080/15384101.2020.1750813.