Exosomale microRNA-21 Promotes Keloid Fibroblast Proliferation and Collagen Production by inhibiting Smad7

Abstract In keloid fibroblasts, microRNA-21 (miR-21) enhances activation of the TGF-β–Smad-signaling pathway by downregulating Smad7 expression, thereby promoting keloid fibroblast proliferation and collagen production. However, it is unclear whether miR-21 performs the above-mentioned functions through exosomal transport. Here, we extracted exosomes from the culture supernatants of keloid and normal skin fibroblasts, and observed that exosomes from both cell types secreted exosomes; however, keloid fibroblasts secreted significantly more exosomal miR-21 than normal skin fibroblasts (P < 0.001). Interestingly, we also observed that exosomal miR-21 could enter target keloid fibroblasts. In addition, inhibiting exosomal miR-21 upregulated Smad7 protein expression and reduced Smad2 and Smad3 protein levels in target keloid fibroblasts. Furthermore, inhibiting exosomal miR-21 downregulated collagen I and collagen III expression in target keloid fibroblasts, increased the proportion of apoptotic cells, and reduced cell proliferation. Taken together, these results show that exosomal miR-21 promoted proliferation and collagen production in keloid fibroblasts by inhibiting Smad7. Thus, we identified regulatory roles for miR-21 in promoting keloid fibroblast proliferation and participating in keloid formation and development. These findings imply that miR-21 may serve as a novel target for controlling the development of keloids.

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Secreted Factors from Keloid Keratinocytes Modulate Collagen Deposition by Fibroblasts from Normal and Fibrotic Tissue: A Pilot Study

Biomedicines ◽

10.3390/biomedicines8070200 ◽

2020 ◽

Vol 8 (7) ◽

pp. 200

Author(s):

Mansour A. Alghamdi ◽

Laith N. AL-Eitan ◽

Andrew Stevenson ◽

Nutan Chaudhari ◽

Nicole Hortin ◽

...

Keyword(s):

Elective Surgery ◽

Normal Skin ◽

Skin Fibroblasts ◽

Collagen Production ◽

Skin Cells ◽

Secreted Factors ◽

Primary Fibroblasts ◽

Keloid Fibroblasts

Interactions between keratinocytes and fibroblasts in the skin layers are crucial in normal tissue development, wound healing, and scarring. This study has investigated the role of keloid keratinocytes in regulating collagen production by primary fibroblasts in vitro. Keloid cells were obtained from removed patients’ tissue whereas normal skin cells were discarded tissue obtained from elective surgery procedures. Fibroblasts and keratinocytes were isolated, cultured, and a transwell co-culture system were used to investigate the effect of keratinocytes on collagen production using a ‘scar-in-a-jar’ model. Keloid fibroblasts produced significantly more collagen than normal skin fibroblasts in monoculture at the RNA, secreted protein, and stable fibrillar protein level. When keloid keratinocytes were added to normal skin fibroblasts, expression of collagen was significantly upregulated in most samples, but when added to keloid fibroblasts, collagen I production was significantly reduced. Interestingly, keloid keratinocytes appear to decrease collagen production by keloid fibroblasts. This suggests that signaling in both keratinocytes and fibroblasts is disrupted in keloid pathology.

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Overexpression of miR-340-5p Inhibits Skin Fibroblast Proliferation by Targeting Kruppel-like Factor 2

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190725112304 ◽

2019 ◽

Vol 20 (13) ◽

pp. 1147-1154 ◽

Cited By ~ 2

Author(s):

Ling Chen ◽

Qian Li ◽

Xun Lu ◽

Xiaohua Dong ◽

Jingyun Li

Keyword(s):

Gene Ontology ◽

Skin Fibroblast ◽

Kegg Pathway ◽

Normal Skin ◽

Skin Fibroblasts ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Fibroblast Proliferation ◽

Pathway Analyses ◽

Normal Skin Fibroblast

Objective: MicroRNA (miR)-340-5p has been identified to play a key role in several cancers. However, the function of miR-340-5p in skin fibroblasts remains largely unknown. Methods: Gain of function experiments were performed by infecting normal skin fibroblast cells with a lentivirus carrying 22-bp miR-340-5p. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. To uncover the mechanisms, mRNA-seq was used. Differentially expressed mRNAs were further determined by Gene Ontology and KEGG pathway analyses. The protein levels were analysed by Western blotting. A dual-luciferase reporter assay was used to detect the direct binding of miR-340-5p with the 3'UTR of Kruppel-like factor 2 (KLF2). Results: MiR-340-5p lentivirus infection suppressed normal skin fibroblast proliferation. The mRNAseq data revealed that 41 mRNAs were differentially expressed, including 22 upregulated and 19 downregulated transcripts in the miR-340-5p overexpression group compared with those in the control group. Gene Ontology and KEGG pathway analyses revealed that miR-340-5p overexpression correlated with the macromolecule biosynthetic process, cellular macromolecule biosynthetic process, membrane, and MAPK signalling pathway. Bioinformatics analysis and luciferase reporter assays showed that miR-340-5p binds to the 3'UTR of KLF2. Forced expression of miR-340-5p decreased the expression of KLF2 in normal skin fibroblasts. Overexpression of KLF2 restored skin fibroblast proliferation in the miR-340-5p overexpression group. Conclusion: This study demonstrates that miR-340-5p may suppress skin fibroblast proliferation, possibly through targeting KLF2. These findings could help us understand the function of miR-340-5p in skin fibroblasts. miR-340-5p could be a therapeutic target for preventing scarring.

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Is leukotriene B4 one of the keloid marker? a fibroblast keloid study

Dermatology Reports ◽

10.4081/dr.2019.8045 ◽

2019 ◽

Author(s):

Yuli Kurniawati ◽

Oki Suwarsa ◽

A. Achadiyani ◽

Sudigdo Adi

Keyword(s):

Normal Skin ◽

Leukotriene B4 ◽

Normal Fibroblast ◽

Fibroblast Proliferation ◽

Elisa Method ◽

Inflammatory Phase ◽

Keloid Fibroblast ◽

Normal Skin Fibroblast ◽

Tgf Β1 ◽

Chemical Mediators

Keloid pathogenesis occurs due to the duration of prolonged inflammatory phase and increased production of various growth factors such as TGF-β1 which may cause increasing fibroblast proliferation and collagen synthesis. Existence of one of the chemical mediators released during inflammation, leukotriene B4 (LTB4), in keloid pathogenesis specifically in the phases of inflammation and proliferation, is still unclear. The purpose of the study is to analyze the levels of LTB4 in keloid. Methods: Fibroblast culture that was done by explanting keloid and normal skin of a keloid patient. Measurement of LTB4 on keloid and normal fibroblast was done by Elisa method. This experiment was run in triplicate. Statistical test was conducted by t test for the unpaired data and Anova test. The experiment was done at cell culture laboratory of Medical Faculty of Padjadjaran University Bandung. Levels of LTB4 in keloid fibroblast was higher than that of normal skin fibroblast (mean 23143.27 vs 18191.85 pg/ml; p<0.05). Conclusion, increased LTB4 levels in keloid fibroblast showed the existence of LTB4 role in the prolonged inflammatory process in keloid pathogenesis.

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FACTOR XIII (FXIIIa) OF BLOOD COAGULATION NORMALIZES COLLAGEN SYNTHESIS OF SCLERODERMA FIBROBLASTS

10.1055/s-0038-1644650 ◽

1987 ◽

Author(s):

M Paye ◽

T Krieg ◽

Ch M Lapière

Keyword(s):

Collagen Synthesis ◽

Normal Skin ◽

Culture Conditions ◽

Dermal Fibroblasts ◽

Skin Fibroblasts ◽

Similar Proportion ◽

Collagen Biosynthesis ◽

Active Lesion ◽

Collagen Production ◽

Collagen Lattice

Progressive systemic scleroderma (PSS) fibroblasts display, in some cases, an excessive collagen production which leads to fibrosis of the skin and internal organs. Administration of FXIIIa has been reported to be beneficial to some of the PSS patients. The effect of FXIIIa on collagen synthesis by PSS fibroblasts was studied in vitro in two different culture conditions: in a confluent monolayer on plastic and in a three-dimensional collagen lattice.Proteins and collagen synthesis was measurfed by metabolic labeling for 24 h with 3H-proline in absence or presence of FXIIIa (1 U/ml) in dermal fibroblasts from an active lesion (PSS forearm), from an uninvolved area of the skin of the same patient (control abdomen) and from the skin of a normal subject.Proteins synthesis was similar for the three strains under both culture conditions while collagen synthesis was strongly increased in PSS forearm fibroblasts as compared to the uninvolved and to the normal skin fibroblasts. The addition of FXIIIa repressed collagen synthesis of PSS forearm synthesis to the level observed in the abdominal skin fibroblasts of the patient and the normal cells in absence of FXIIIa. When cultured in a collagen lattice collagen synthesis was repressed in a similar proportion in all fibroblasts. Addition of FXIIIa further reduced collagen biosynthesis in the active PSS fibroblasts. The addition of FXIIIa in the lattice largely increased the degradation of newly synthesized collagen in all the strains of fibroblasts.The action of FXIIIa on collagen biosynthesis was also tested in monolayer in 3 other strains of PSS fibroblasts and 3 controls. In all fibroblasts, collagen biosynthesis was reduced by 75 % after addition of FXIIIa.Our results suggest that the beneficial effect of FXIIIa administration in PSS patient might be related to a reduction o1 excessive collagen production and perhaps to an increased degradation of newly synthesized molecules.

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MiR-4328 inhibits proliferation, metastasis and induces apoptosis in keloid fibroblasts by targeting BCL2 expression

Open Life Sciences ◽

10.1515/biol-2020-0056 ◽

2020 ◽

Vol 15 (1) ◽

pp. 638-646

Author(s):

Hongmei Tang ◽

Qi Chen ◽

Wenyuan Yu ◽

Tianlan Zhao

Keyword(s):

B Cell Lymphoma ◽

Cell Counting ◽

Nuclear Antigen ◽

Luciferase Reporter ◽

Inhibition Of Proliferation ◽

Keloid Fibroblast ◽

Promising Target ◽

Collagen Iii ◽

Bcl2 Protein ◽

Keloid Fibroblasts

AbstractKeloids are considered to be a type of benign tumor. MicroRNAs have been reported to be involved in the formation and growth of keloids. MicroRNA-4328 (miR-4328) was found to be abnormally expressed in keloids, while the role and the detailed molecular mechanism of miR-4328 in keloids remain unclear. The expression of miR-4328 and B-cell lymphoma 2 (BCL2) mRNA was detected by qRT-PCR. The proliferation, migration, invasion and apoptosis of keloid fibroblasts (KFs) was examined using Cell Counting Kit-8 assay, transwell assay or flow cytometry, respectively. Western blot was used to detect the level of proliferating cell nuclear antigen, cleaved-caspase 3, collagen I, collagen III and BCL2 protein. The interaction between miR-4328 and BCL2 was confirmed by luciferase reporter analyses. It was observed that miR-4328 was down-regulated in keloid tissues and fibroblasts, and miR-4328 restoration mediated the inhibition of proliferation, metastasis, collagen synthesis and the promotion of apoptosis in KFs. BCL2 was up-regulated in keloid tissues and fibroblasts, and BCL2 knockdown promoted the deterioration of KFs. In addition, BCL2 was confirmed to be a target of miR-4328, and the rescue experiment indicated that the inhibitory action of miR-4328 on keloid fibroblast progression was reversed by BCL2 overexpression. Thus, our results demonstrated that miR-4328 restrained the deterioration of KFs by targeting BCL2, which sheds new light on miR-4328 as a promising target for keloid development and therapeutic.

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Increased expression of the transforming growth factor β–inducible gene HIC-5 in systemic sclerosis skin and fibroblasts: a novel antifibrotic therapeutic target

Rheumatology ◽

10.1093/rheumatology/keaa200 ◽

2020 ◽

Vol 59 (10) ◽

pp. 3092-3098 ◽

Cited By ~ 1

Author(s):

Sonsoles Piera-Velazquez ◽

Jolanta Fertala ◽

Gonzalo Huaman-Vargas ◽

Natalia Louneva ◽

Sergio A Jiménez

Keyword(s):

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Normal Skin ◽

Transforming Growth Factor Β ◽

Dermal Fibroblasts ◽

Skin Fibroblasts ◽

Tissue Fibrosis ◽

Protein Levels ◽

Antifibrotic Therapy ◽

Tgf Β1

Abstract Objective SSc is a systemic fibrotic disease affecting skin, numerous internal organs and the microvasculature. The molecular pathogenesis of SSc tissue fibrosis has not been fully elucidated, although TGF-β1 plays a crucial role. The Hic-5 protein encoded by the TGF-β1-inducible HIC-5 gene participates in numerous TGF-β-mediated pathways, however, the role of Hic-5 in SSc fibrosis has not been investigated. The aim of this study was to examine HIC-5 involvement in SSc tissue fibrosis. Methods Affected skin from three patients with diffuse SSc and dermal fibroblasts cultured from affected and non-affected SSc skin were examined for HIC-5 and COL1A1 gene expression. Real-time PCR, IF microscopy, western blotting and small interfering RNA–mediated HIC-5 were performed. Results HIC-5 and COL1A1 transcripts and Hic-5, type 1 collagen (COL1) and α-smooth muscle actin (α-SMA) protein levels were increased in clinically affected SSc skin compared with normal skin and in cultured dermal fibroblasts from affected SSc skin compared with non-affected skin fibroblasts from the same patients. HIC-5 knockdown caused a marked reduction of COL1 production in SSc dermal fibroblasts. Conclusion HIC-5 expression is increased in affected SSc skin compared with skin from normal individuals. Affected SSc skin fibroblasts display increased HIC-5 and COL1A1 expression compared with non-affected skin fibroblasts from the same patients. Hic-5 protein was significantly increased in cultured SSc dermal fibroblasts. HIC-5 mRNA knockdown in SSc fibroblasts caused >50% reduction of COL1 production. Although these are preliminary results owing to the small number of skin samples studied, they indicate that Hic-5 plays a role in the profibrotic activation of SSc dermal fibroblasts and may represent a novel molecular target for antifibrotic therapy in SSc.

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Alternative Splicing Is Responsible for the Presence of Two Tissue Factor mRNA Species in LPS Stimulated Human Monocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648424 ◽

1992 ◽

Vol 67 (02) ◽

pp. 272-276 ◽

Cited By ~ 19

Author(s):

C Paul ◽

E van der Logt ◽

Pieter H Reitsma ◽

Rogier M Bertina

Keyword(s):

Alternative Splicing ◽

Tissue Factor ◽

Stop Codon ◽

Cell Types ◽

Northern Analysis ◽

Human Monocytes ◽

Mrna Species ◽

Protein Levels ◽

Intron 1 ◽

Tf Gene

SummaryAlthough normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide (LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.

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