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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 40  |  Issue : 1  |  Page : 34-43

MiR-375 and miR-5691 exert anti-fibroproliferative effects on hypertrophic scar fibroblasts by suppressing thrombospondin 1 expression


1 Department of Trauma Emergency Center, The Affiliated Ganzhou Hospital of Nanchang University, Nanchang, Jiangxi, China
2 Department of Rheumatic and Immunity, The Affiliated Ganzhou Hospital of Nanchang University, Nanchang, Jiangxi, China

Date of Submission02-Jul-2021
Date of Decision21-Feb-2022
Date of Acceptance01-Mar-2022
Date of Web Publication30-Mar-2022

Correspondence Address:
Dr. Jing Lv
Department of Rheumatic and Immunity, The Affiliated Ganzhou Hospital of Nanchang University, No. 17, Hongqi Avenue, Zhanggong District, Ganzhou, Jiangxi 341000, Nanchang
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ds.ds_13_22

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  Abstract 


Background: Hypertrophic scar (HS) is characterized by the hyperproliferation of fibroblasts and the excessive deposition of extracellular matrix (ECM). Thrombospondin 1 (THBS1) is a component of the ECM, which has been implicated in HS formation. Objectives: This study aimed to explore whether miR-375/miR-5691 could modulate HS formation by targeting THBS1. Methods: The expression levels of miR-375/miR-5691/THBS1 in HS and normal skin tissues were measured by quantitative reverse transcription-polymerase chain reaction. 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide and Western blot assays were performed on fibroblasts isolated from HS tissues (HSFBs) to determine cell proliferation and the expression levels of proliferating cell nuclear antigen (PCNA), apoptosis-related proteins (caspase3/9, cleaved caspase3/9, Bax, and Bcl-2), and ECM-related proteins. The binding sites between THBS1 and miR-375/miR-5691 were predicted by the TargetScan. Dual-luciferase reporter and anti-Ago2 immunoprecipitation assays were applied to confirm the interactions between THBS1 and miR-375/miR-5691. Results: The expression levels of both miR-375 and miR-5691 were downregulated in HS tissues and HSFBs, which were negatively correlated with THBS1 expression levels. The overexpression of miR-375/miR-5691 inhibited cell proliferation and ECM production, and promoted apoptosis of HSFBs, while silencing of miR-375/miR-5691 led to an opposite result. In the mechanism analysis, THBS1 was confirmed as the direct target gene of miR-375/miR-5691. Furthermore, rescue experiments showed that the suppressed growth of HSFBs and ECM production induced by silencing of THBS1 was reversed by miR-375/miR-5691 inhibitors. Conclusion: MiR-375/miR-5691 was downregulated in HS tissues, and it could suppress the hyperproliferation and ECM production of HSFBs by targeting THBS1.

Keywords: fibroblasts, hypertrophic scar, miR-375, miR-5691, thrombospondin 1


How to cite this article:
Zhou X, Ye H, Wang X, Tu J, Lv J. MiR-375 and miR-5691 exert anti-fibroproliferative effects on hypertrophic scar fibroblasts by suppressing thrombospondin 1 expression. Dermatol Sin 2022;40:34-43

How to cite this URL:
Zhou X, Ye H, Wang X, Tu J, Lv J. MiR-375 and miR-5691 exert anti-fibroproliferative effects on hypertrophic scar fibroblasts by suppressing thrombospondin 1 expression. Dermatol Sin [serial online] 2022 [cited 2022 May 16];40:34-43. Available from: https://www.dermsinica.org/text.asp?2022/40/1/34/341351




  Introduction Top


As one of the common dermal disorders caused by abnormal responses to trauma, hypertrophic scar (HS) is characterized by unrestrained dermal fibroproliferation with persistent inflammation and excessive deposition of extracellular matrix (ECM) proteins.[1] Although HS is not generally a life-threatening disease, it frequently leads to cosmetically disfigured and fundamental clinical challenges, seriously influencing patients' quality of life.[2] To date, various treatment modalities for HS have been presented, such as corticosteroid injection, laser therapy, cryotherapy, and surgical excision.[3] However, these modalities have exhibited a limited efficacy.[4] Therefore, exploration of efficient strategies to prevent HS development and progression is vital.

To our knowledge, a comprehensive understanding on the pathological mechanisms of HS is critical, to develop additional effective therapeutic strategies for HS. With the rapid development of sequencing techniques, the biological functions of noncoding RNAs (ncRNAs) in diverse human diseases have markedly attracted researchers' attention in the recent decades.[5],[6] MicroRNAs (miRNAs) are a class of well-studied ncRNAs with a length of approximately 22 nucleotides, which have been proven as crucial regulators.[7] As reported previously, miRNAs can bind to the 3'UTR region of the target gene to modulate the expression levels of the target genes at the posttranscriptional level, thereby regulating disease development and progression.[8]

A growing body of evidence has indicated that HS formation is closely associated with the dysregulation of miRNAs. Besides, miR-519d has been shown to suppress the expression levels of ECM-associated genes, reduce the proliferation and induce the apoptosis of HS fibroblasts by regulating SIRT7 expression.[9] Zhang et al.[10] identified a miR-130a/CYLD/Akt pathway involved in the development of HS based on in vivo and in vitro experiments. Li et al.[11] demonstrated that miR-3613-3p exerted a suppressive effect on HS formation through reducing ARGLU1 expression. It is widely accepted that transforming growth factor beta-1 (TGF-β1) plays an important role in wound healing and tissue repair, and it is a central molecule that contributes to skin fibrosis.[12] In the past decade, increasing evidence has supported that thrombospondin 1 (THBS1) contributes to the development of several fibrotic diseases (e.g., HS) because of its activation role in TGF-β1 signaling pathway.[13] A recent study reported that THBS1 is highly expressed in HS tissues, and it is positively correlated with the development of HS.[14] As further attention has recently been paid to the role of miRNAs in HS, we aim to assess whether there are miRNAs that can target THBS1 to inhibit the development of HS and play a therapeutic role. Recently, several scholars exploited miRNA microarray analysis to identify miRNAs that are differentially expressed in HS and normal skin (NS) tissues;[15],[16] however, only few miRNAs have undergone further functional verification. To comprehensively understand the pathological mechanisms of HS, the regulatory role of various miRNAs in HS development is worthy of further investigation.

MiR-375 and miR-5691 were identified as significantly downregulated miRNAs of HS,[15],[16] both of which have putative binding sites at the THBS1 mRNA 3'UTR as predicted by the TargetScan web server. Hence, in the present study, miR-375 and miR-5691 were selected to explore their roles and to confirm their interaction with THBS1 in HS formation.


  Materials and Methods Top


Collection of samples and isolation and culture of cells

In the present study, 18 h tissue samples and 18 matched NS tissues were collected from 18 h patients who were aged 15–52 (mean ± standard deviation, 31 ± 13) years old during scar excision surgery at our hospital. During surgery, the scar was excised along with the minimum amount of NS from patients to ensure that the scar was fully excised. Hence, the center of excised skin specimens of HS was regarded as HS tissue and the edge was taken as NS tissue into account. All the enrolled patients did not receive any medication or radiotherapy, and they signed written informed consent forms before commencing the research. The study protocol was approved by the Ethics Committee of Ganzhou Hospital of Nanchang University (approved number: TY-HKY2020-012-03).

Primary cultures of HS fibroblasts (HSFBs) and NS fibroblasts (NSFBs) were isolated from clinical tissues as previously described.[10] Briefly, the epidermis and dermis from collected tissues were separated by digesting with Dispase II (Sigma-Aldrich, St. Louis, MO, USA; #D4693) after removing excessive adipose tissues. Next, dermis tissues were washed with phosphate-buffered saline and gently shaken, followed by chopping and incubation with collagen type I (Sigma-Aldrich; #SCR103) for 3 h to obtain HSFs or NSFs. The isolated cells were cultured in a Dulbecco's Modified Eagle's medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C with 5% CO2.

Quantitative reverse transcription-polymerase chain reaction

Reverse transcription-polymerase chain reaction (RT-qPCR) was exploited to detect the expressions of miR-375, miR-5691, and THBS1 at the transcription level. The total RNA of tissues or cells was extracted by the RNAiso Plus kit (Takara, Shiga, Japan; #740406.50). The miScript II RT kit (Qiagen, Hilden, Germany; #218160) and the PrimeScript™ RT Reagent kit (Takara; #RR037B) were, respectively, utilized to synthesize the cDNA of miRNAs and mRNA. Next, the cDNA was amplified with a SYBR® Green Quantitative RT-qPCR kit (Sigma-Aldrich; #QR0100) to perform RT-qPCR on a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). In addition, U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the endogenous control to normalize the expressions of the studied miRNAs and mRNA. The relative expression of RNA was calculated based on the 2−ΔΔCt method.[17] The primer sequences of miR-375, miR-5691, THBS1, U6, and GAPDH are listed in [Supplementary Table 1].



Immunohistochemistry

Paraffin-embedded HS and NS tissues were cut into 5-μm sections. After being blocked with 5% bovine serum albumin (BSA), the sections were incubated with primary antibody against THBS1 (Abcam, Cambridge, UK; cat. no., #ab1823; 1/200 dilution) for 1.5 h, followed by incubation with a biotinylated secondary antibody for 30 min. Primary antibody binding sites in sections were visualized using 3,3′-diaminobenzidine (DAB) solution. Finally, sections were counter-stained with hematoxylin and observed under a light microscope.

Immunofluorescence staining

HSFBs and NSFBs were fixed with 4% paraformaldehyde, followed by being permeabilized with 0.2% Triton X-100 and blocked with 5% BSA. Then, cells were incubated with primary antibody against alpha-smooth muscle actin (α-SMA) (Abcam; cat. no., #ab7817; 1/3000 dilution) overnight at 4°C, followed by incubation with FITC-conjugated secondary antibody. Cells were counterstained with 4′,6-diamidino-2-phenylindole. Fluorescence microscopy was carried out under a fluorescence microscope (Leica, Wetzlar, Germany).

Cell transfection

The miRNA mimics, inhibitors, and the corresponding negative control (NC) for miR-375 and miR-5691, small interference RNA-targeted THBS1 (siTHBS1), and siRNA- NC (si-NC) were synthesized by GenePharma Co., Ltd.(Shanghai, China). Cell transfection was performed using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. At 48 h post-transfection, transfection efficiency was assessed by RT-qPCR.

Western blot assay

Transfected HSFBs were harvested and lysed with RIPA Lysis and Extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. #89900) on ice to extract total protein. After quantifying the total protein by a BCA Protein Assay kit (Millipore, MO, USA; cat. no. #71285-M), the equal amount of protein from each group was subjected to separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transferring onto polyvinylidene difluoride membranes. Next, membranes were blocked with skimmed milk for 1.5 h and subsequently incubated with primary antibodies overnight before incubation with secondary antibodies for 2 h. The protein bands were visualized with an ECL Western Blotting Substrate kit (Abcam; cat. no. #ab65623). Both primary and secondary antibodies used in this study were purchased from Abcam and were listed below: caspase-3 (cat. no. #ab32351; 1/5000 dilution), cleaved-caspase-3 (C-caspase-3) (cat. no. #ab32042; 1/500 dilution), caspase-9 (cat. no. #ab32539; 1/3000 dilution), C-caspase-9 (cat. no. #ab2324; 1/500 dilution), Bax (cat. no. #ab32503; 1/5000 dilution), Bcl2 (cat. no. #ab32124; 1/1000 dilution), proliferating cell nuclear antigen (PCNA) (cat. no. #ab29; 1/2000 dilution), TGF-β1 (cat. no. #ab92486; 1/1000 dilution), α-SMA (cat. no. #ab7817; 1/3000 dilution), Fibronectin (#ab2413; 1/1000 dilution), collagen type I (cat. no. #ab34710; 1/3000 dilution), collagen type III (cat. no. #ab7778; 1/3000 dilution), GAPDH (cat. no. #ab8245; 1/8000 dilution), and goat anti-rabbit IgG H and L (HRP) (cat. no. #ab6721; 1/3000 dilution).

Cell proliferation assay

Transfected HSFBs were seeded into 96-well plates and incubated for 24, 48, and 72 h, respectively. Afterward, 3-(4,5)-dimethylthiahiazo (-z-y1)- 2,5-di-phenytetrazoliumromide (MTT) reagent was added into each well at designed time points. After additional incubation for 4 h, purple formazan crystals conversed from MTT were dissolved by dimethyl sulfoxide. Finally, the absorbance values at 570 nm of each well were measured using a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Prediction of target genes

TargetScan (http://www.targetscan.org/vert_72/)[18] was applied to predict the potential binding sites between THBS1 and miR-375/miR-5691.

Dual-luciferase reporter assay

To construct the luciferase reporter vector, the wild-type or mutated (MUT) 3'UTR of the THBS1 cDNA fragments incorporated with the miR-375 or miR-5691 targeting site was amplified to insert into the upstream of the reporter gene in the pmirGlo dual-luciferase vector (Addgene, Waltham, MA, USA). To perform dual-luciferase reporter assay, HSFBs cultured in a 24-well plate were co-transfected with the constructed vectors (THBS1 3'UTR WT/MUT) and miR-375 or miR-5691 mimics. After 48 h of transfection, the Firefly fluorescence was normalized to the Renilla fluorescence to calculate the luciferase activity.

Anti-Ago2 RNA immunoprecipitation assay

Anti-Ago2 RNA immunoprecipitation (RIP) assay was performed using a Magna RIP kit (Millipore; cat. no. #17-700). Cells were lysed in RIP lysis buffer and then diluted with RIP immunoprecipitation buffer. Afterward, the cell suspension was immunoprecipitated with magnetic beads conjugated with antibody against Ago2 (Abcam; cat. no. #ab32381). After overnight incubation at 4°C, the coprecipitated magnetic beads were harvested and subjected to protein digestion through proteinase K. Finally, immunoprecipitated RNA was analyzed through RT-qPCR. IgG served as a NC.

Statistical analysis

The statistical analysis was carried out using GraphPad Prism 8.0.1 software (GraphPad Software Inc., San Diego, CA, USA). The Students' t-test or one-way analysis of variance with post hoc test (Bonferroni) was employed to analyze the differences among the groups. P < 0.05 was considered statistically significant. All experiments were performed at least three times.


  Results Top


MiR-375 and miR-5691 were lowly expressed in hypertrophic scar tissues and hypertrophic scar fibroblasts

To delineate the potential roles of miR-375 and miR-5691 during HS development, the expression levels of miR-375 and miR-5691 in HS tissues and HSFBs were detected by RT-qPCR. The expression levels of both miR-375 and miR-5691 were significantly downregulated in HS tissues compared with those in NS tissues [Figure 1]a. To characterize fibroblasts derived from NS and HS tissues, the immunofluorescence staining of α-SMA on NSFBs and HSFBs was performed, which showed a higher positive staining of α-SMA in HSFBs than that in NSFBs [Figure 1]b. Then, RT-qPCR analysis of NSFBs and HSFBs revealed that the expression levels of miR-375 and miR-5691 in HSFBs were significantly lower than those in NSFBs [Figure 1]c, which was similar to those in HS and NS tissues. These findings suggested that the downregulation of miR-375 and miR-5691 might be associated with the formation of HS.
Figure 1: Downregulation of miR-375/miR-5691 is observed in hypertrophic scar tissues and hypertrophic scar fibroblasts. (a) The expression profiles of (left) miR-375 and (right) miR-5691 in hypertrophic scar and normal skin tissues. (b) Immunofluorescence analysis of alpha-smooth muscle actin in hypertrophic scar fibroblasts and normal skin fibroblasts. (c) The expression profiles of (left) miR-375 and (right) miR-5691 in hypertrophic scar fibroblasts and normal skin fibroblasts. Note: *P < 0.05.

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Overexpression of miR-375 or miR-5691 suppressed the proliferation, induced the apoptosis of hypertrophic scar fibroblasts, and reduced the expression levels of extracellular matrix-related proteins in hypertrophic scar fibroblasts

To investigate the functional role of miR-375 or miR-5691 in HSFBs, we transfected miR-375 or miR-5691 mimics into HSFBs. RT-qPCR confirmed that miR-375 or miR-5691 mimics were successfully transfected into HSFBs [Figure 2]a. The results of MTT assay revealed that the absorbance was markedly reduced in HSFBs that had been transfected with miR-375 or miR-5691 mimics compared with the negative control (NC mimics), indicating that the overexpression of miR-375 and miR-5691 could suppress the proliferation of HSFBs [Figure 2]b. Besides, the expression levels of apoptotic proteins were evaluated to investigate the effects of miR-375 and miR-5691 on the apoptosis of HSFBs. Total protein levels of caspase-3 and caspase-9 in HSFBs exhibited no change after overexpression of miR-375 or miR-5691 [Figure 2]c. Of note, the transfection of miR-375 or miR-5691 mimics significantly increased the expression levels of pro-apoptotic proteins and caspases (Bax, C-caspase-3, and C-caspase-9) while decreased the expression level of anti-apoptotic protein (Bcl-2) in HSFBs [Figure 2]c. To further investigate whether miR-375 and miR-5691 could exert anti-fibroproliferative effects on HSFBs, we also detected the expression levels of proliferation-related and ECM-related proteins by Western blot assay. As shown in [Figure 2]d, the expression levels of PCNA in HSFBs were noticeably reduced by miR-375 or miR-5691 mimics compared with NC mimics. These findings indicated that both miR-375 and miR-5691 might play a suppressive role in the proliferation of HSFBs, which is consistent with the results of the MTT assay. Moreover, the overexpression of miR-375 or miR-5691 mimics also significantly weakened the expression levels of TGF-β1, α-SMA, fibronectin, collagen type I, and collagen type III in HSFBs compared with those in the NC mimics [Figure 2]d, demonstrating that miR-375 and miR-5691 were involved in the formation of HS by regulating the expression levels of ECM-related proteins. Collectively, the above-mentioned results indicated that miR-375 and miR-5691 suppressed HS formation by inhibiting cell proliferation and ECM production and promoting apoptosis of HSFBs.
Figure 2: Overexpression of miR-375 or miR-5691 suppresses the hypertrophic scar phenotypes in hypertrophic scar fibroblasts. (a) Transfection efficiency of (left) miR-375 mimic and (right) miR-5691 mimic in hypertrophic scar fibroblasts was analyzed by reverse transcription-polymerase chain reaction. (b) 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide assay examined the proliferation of hypertrophic scar fibroblasts transfected with (left) miR-375 mimics or (right) miR-5691. (c) The expression levels of caspase-3, caspase-9, C-caspase-3, C-caspase-9, Bax, and Bcl-2 in hypertrophic scar fibroblasts with diverse transfections were detected by western blotting. (d) The expression levels of proliferating cell nuclear antigen, transforming growth factor beta-1, alpha-smooth muscle actin, fibronectin, collagen type I, and collagen type III in hypertrophic scar fibroblasts with diverse transfections were measured by Western blotting. Note: *P < 0.05.

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Downregulation of miR-375 or miR-5691 promoted cell proliferation and extracellular matrix production and inhibited apoptosis of hypertrophic scar fibroblasts

To corroborate the above-mentioned finding, HSFBs were transfected with miR-375 or miR-5691 inhibitors, and the same functional analyses were subsequently performed. The transfection efficiency of miRNA inhibitors detected by RT-qPCR is depicted in [Figure 3]a, indicating that miR-375 and miR-5691 inhibitors significantly prohibited the expression levels of miR-375 and miR-5691 in HSFBs, respectively. MTT assay and PCNA detection indicated that silencing of miR-375 or miR-5691 could remarkably promote the proliferation of HSFBs [Figure 3]b and [Figure 3]d. Simultaneously, after silencing of miR-375 or miR-5691, the expression levels of Bax, C-caspase-3, and C-caspase-9 were significantly reduced, while the expression level of Bcl-2 was noticeably elevated [Figure 3]c, indicating that miR-375 and miR-5691 exerted a pro-apoptotic effect on HSFBs. Furthermore, miR-375 or miR-5691 inhibitors also significantly enhanced the expression levels of ECM-related proteins, suggesting a suppressive role of miR-375 and miR-5691 in ECM production in HSFBs [Figure 3]d. Combined with the functional analyses of miR-375 and miR-5691 mimics in HSFBs, these data further verified that miR-375 and miR-5691 were involved in HS formation by regulating the cell proliferation, apoptosis, and ECM production of HSFBs.
Figure 3: Silencing of miR-375 or miR-5691 suppresses the hypertrophic scar phenotypes in hypertrophic scar fibroblasts. (a) Transfection efficiency of (left) miR-375 inhibitors and (right) miR-5691 inhibitors in hypertrophic scar fibroblasts was analyzed by reverse transcription-polymerase chain reaction. (b) 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide assay examined the proliferation of hypertrophic scar fibroblasts transfected with (left) miR-375 inhibitors and (right) miR-5691 inhibitors. (c) The expression levels of caspase-3, caspase-9, C-caspase-3, C-caspase-9, Bax, and Bcl-2 in hypertrophic scar fibroblasts with diverse transfections were detected by western blot assay. (d) The expression levels of proliferating cell nuclear antigen, transforming growth factor beta-1, alpha-smooth muscle actin, fibronectin, collagen type I, and collagen type III in hypertrophic scar fibroblasts with diverse transfections were determined by Western blot assay. Note: *P < 0.05 and **P < 0.01.

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THBS1 was a direct target of both miR-375 and miR-5691

Based on the outputs of a bioinformatics tool (TargetScan), it was found that THBS1 could be a common potential target of miR-375 and miR-5691. It has been reported that THBS1 is highly expressed in HS tissues and is positively correlated with the development of HS.[14] Hence, we speculated that THBS1 was a target of both miR-375 and miR-5691 in HS. RT-qPCR analysis showed that THBS1 was highly expressed in HS tissues compared with NS tissues [Figure 4]a, which was consistent with the results of a previous study.[14] Similarly, immunohistochemistry (IHC) showed that the expression level of THBS1 in HS tissues was higher than that in NS tissues [Figure 4]b. In addition, we analyzed the correlation between THBS1 and miR-375 or miR-5691, and it was revealed that the expression levels of miR-375 and miR-5691 were negatively correlated with THBS1 expression levels in HS [Figure 4]c. Besides, it was found that the THBS1 expression level in HSFBs was remarkably downregulated by miR-375 and miR-5691 mimics, which suggested that THBS1 was a potential target of miR-375 and miR-5691 mimics [Figure 4]d. The potential binding sites between THBS1 and miR-375/miR-5691 were predicted by the TargetScan [Figure 4]e. Dual-luciferase reporter assay confirmed direct interactions between THBS1 and miR-375/miR-5691 [Figure 4]f. Anti-Ago2 RIP assay further revealed that THBS1 could spatially interact with miR-375/miR-5691 through Ago2 protein [Figure 4]g. Taken together, these results confirmed our hypothesis that THBS1 was a direct target of both miR-375 and miR-5691.
Figure 4: Thrombospondin 1 is a direct target of both miR-375 and miR-5691. (a) The expression profile of thrombospondin 1 in hypertrophic scar and normal skin tissues. (b) thrombospondin 1 expression level was measured by IHC in hypertrophic scar and normal skin tissue sections. (c) The correlations between thrombospondin 1 and (left) miR-375 and (right) miR-5691 were analyzed in hypertrophic scar tissues. (d) The mRNA expression of thrombospondin 1 in hypertrophic scar fibroblasts transfected with miR-375 or miR-5691 mimics. (e) The potential binding sites between thrombospondin 1 and (top) miR-375 and (bottom) miR-5691 were predicted by the TargetScan. (f) The interactions between thrombospondin 1 and (left) miR-375 and (right) miR-5691 were confirmed by dual luciferase reporter assay. (g) Thrombospondin 1 was enriched in the Ago2 immunoprecipitates after the transfection of miR-375 or miR-5691 mimics. Note: *P < 0.05 and **P < 0.01.

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MiR-375 or miR-5691 inhibitor could restore the effect of si-THBS1 on hypertrophic scar fibroblasts

To investigate whether miR-375/miR-5691 is involved in HS formation through regulating THBS1, rescue experiments were further performed. We silenced the THBS1 expression level in HSFBs by transfecting with si-THBS1-1 or si-THBS1-2. The results revealed that the silencing efficiency of si-THBS1-1 in HSFBs was slightly higher than that in si-THBS1-2 [Figure 5]a. Hence, the si-THBS1-1 was selected to perform subsequent experiments. The MTT assay showed that the knockdown of THBS1 (si-THBS1-1) significantly reduced the proliferation of HSFBs compared with the control group (si-NC) [Figure 5]b, as confirmed by PCNA detection [Figure 5]d. These results suggested that THBS1 promoted the proliferation of HSFBs. However, the suppressive role of si-THBS1-1 in the proliferation of HSFBs was partly reversed by co-transfecting with miR-375 or miR-5691 inhibitors. Besides, silencing of THBS1 could also increase the expression levels of Bax, C-caspase-3, and C-caspase-9, and decrease Bcl-2 expression levels in HSFBs, which indicated that THBS1 was involved in the apoptosis of HSFBs [Figure 5]c. After co-transfecting with miR-375 or miR-5691 inhibitors, the pro-apoptosis effect on HSFBs induced by si-THBS1-1 was partly countered [Figure 5]c. Furthermore, it was found that the expression levels of TGF-β1, α-SMA, fibronectin, collagen type I, and collagen type III in HSFBs were positively correlated with THBS1 expression levels, as si-THBS1-1 significantly reduced the expression levels of these ECM-related proteins [Figure 5]d. As expected, miR-375 or miR-5691 inhibitors noticeably reversed si-THBS1-1-mediated suppression effects on the expression levels of ECM-related proteins in HSFBs [Figure 5]d. Overall, THBS1 promoted HS formation by enhancing the proliferation and ECM production, and suppressing apoptosis of HSFBs, which could be regulated by miR-375/miR-5691.
Figure 5: MiR-375 and miR-5691 regulate hypertrophic scar formation through targeting thrombospondin 1. (a) The transfection efficiency of si-thrombospondin 1 in hypertrophic scar fibroblasthypertrophic scar fibroblasts was analyzed by reverse transcription-polymerase chain reaction. hypertrophic scar fibroblasts with the transfection of si-thrombospondin-1 alone or combined with miR-375/miR-5691 inhibitors for 48 h before subsequent experiments. (b) 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide assay examined the proliferation of hypertrophic scar fibroblasts with diverse transfections. (c) The expression levels of caspase-3, caspase-9, C-caspase-3, C-caspase-9, Bax, and Bcl-2 in hypertrophic scar fibroblasts with diverse transfections were detected by western blotting. (d) The expression levels of proliferating cell nuclear antigen, transforming growth factor beta-1, alpha-smooth muscle actin, fibronectin, collagen type I, and collagen type III in hypertrophic scar fibroblasts with diverse transfections were measured by Western blotting. Note: *P < 0.05 and **P < 0.01.

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  Discussion Top


Recently, multiple studies indicated that miRNAs play a crucial role in the formation of HS.[19],[20] Some miRNAs have been reported to regulate the proliferation, apoptosis, and ECM production of HSFBs, thereby participating in HS formation.[10],[11],[21] However, the regulatory roles of miRNAs in HS formation remain to be investigated. Besides, miR-375 and miR-5691 were identified as significantly downregulated miRNAs in HS patients by previous studies.[15],[16] Consistent with previous studies, our study showed that both miR-375 and miR-5691 were significantly downregulated in HS tissues compared with those in NS tissues. To our knowledge, the major histological feature of HS is the hyperproliferation of fibroblasts with the excessive deposition of ECM proteins. Fibroblasts are widely considered as the key cells in HS formation,[22],[23] of which transcriptomic profile is intimately involved in the scar formation in the wound.[24] Therefore, we isolated fibroblasts from HS and NS tissues for further analysis. The differentiation of fibroblasts from myofibroblasts is closely associated with wound contraction and the remodeling of the ECM during scar formation, which is reflected by α-SMA expression. To characterize the isolation of HSFBs and NSFBs, the α-SMA expression level in HSFBs and NSFBs was identified using immunofluorescence staining. Our data showed that the α-SMA positive rate in HSFBs was noticeably higher than that in NSFBs.

Moreover, the expression levels and roles of miR-375 and miR-5691 were further explored in HSFBs. As expected, the expression levels of miR-375 and miR-5691 in HSFBs were significantly reduced compared with those in NSFBs. The overexpression of miR-375/miR-5691 significantly suppressed the proliferation of HSFBs compared with that in the control. The silencing of miR-375/miR-5691 exhibited an opposite effect. Bax and Bcl-2 are known as pro-and anti-apoptotic proteins, respectively.[25] Our study showed that overexpression of miR-375/miR-5691 could increase the Bax expression level while reduce the Bcl-2 expression level in HSFBs. Besides, several caspases, including caspase-3 and caspase-9, would be changed into cleaved form to participate in cleavage of a variety of proteins during apoptosis.[26] Therefore, C-caspase-3 and C-caspase-9 were considered as reliable markers of apoptosis in the present study. The increased expression levels of C-caspase-3 and C-caspase-9 were also observed in HSFBs transfected with miR-375/miR-5691 mimics. These results indicated that miR-375/miR-5691 could play anti-proliferative and pro-apoptotic roles in HSFBs, which in turn could suppress HS formation. In addition, the overexpression of miR-375/miR-5691 noticeably decreased the expression levels of ECM-related proteins (TGF-β1, α-SMA, fibronectin, collagen type I, and collagen type III). Multiple studies demonstrated that abnormal TGF-β1 expression level plays an important role in HS formation.[27],[28] TGF-β1 induces excessive deposition of ECM and suppresses collagenase activity, thereby promoting HS formation.[29] Fibronectin is an adhesive molecule that is closely correlated with ECM production during wound healing, which can promote migration of fibroblasts in the early stage of HS formation.[30] Collagen type I and collagen type III are the most important ingredients of the ECM. As reported, collagen synthesis is seven-fold higher in HS compared with that in NS.[31] The above-mentioned results demonstrated that miR-375/miR-5691 plays a key role in HS formation by regulating fibroblast growth and ECM accumulation.

As the functions of miRNAs in diverse biological processes are mainly associated with the target genes, we further searched for the potential target genes of miR-375/miR-5691 to explore the specific mechanism underlying miR-375/miR-5691 in HS formation. THBS1 has been proven to regulate TGF-β1 expression in the biological process of pathological scars, including HS.[32] Jiang et al. recently demonstrated that THBS1 could promote HS development by inducing fibroblast growth and migration through TGF-β1 based on in vitro and in vivo experiments.[14] Some studies reported that the abnormal expression level of THBS1 in the pathological processes could be modulated by diverse miRNAs.[33],[34] Inspired by these studies, we hypothesized that miR-375/miR-5691 could regulate growth of HSFBs and ECM production through regulating THBS1 expression levels, thereby influencing HS formation. Analysis of our clinical samples revealed that there was a significantly negative correlation between the expression levels of THBS1 and miR-375/miR-5691 in HS. In addition, dual-luciferase reporter and anti-Ago2 RIP assays confirmed the interactions between the expression levels of THBS1 and miR-375/miR-5691. Rescue experiments further verified our hypothesis, indicating that the suppressed growth of HSFBs and ECM production induced by silencing of THBS1 was restored by miR-375/miR-5691 inhibitors. Collectively, the results of the present study revealed that miR-375/miR-5691 could be downregulated in HS and could suppress the hyperproliferation and ECM production of HSFBs by targeting THBS1.

In conclusion, our study revealed that the upregulation of miR-375/miR-5691 could induce the apoptosis and repress the proliferation and ECM deposition of HSFBs through THBS1, thereby inhibiting HS formation. Our findings may assist scholars to better understand the complex miRNA-mRNA interaction in HS formation and provide scientific information to develop efficient strategies for HS treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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