Clinical Investigation
Identification of Collagen 1 as a Post-transcriptional Target of miR-29b in Skin Fibroblasts: Therapeutic Implication for Scar Reduction

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Abstract

Background

Excessive collagen synthesis and deposit during skin wound healing results in scar formation. MicroRNAs (miRNAs) are endogenous noncoding RNA regulators that mediate diverse biological functions through repressing target genes and hold great potentials for clinical therapeutic applications. The aim of the present study was to identify miRNAs as post-transcriptional regulators of collagen 1 in skin fibroblasts.

Methods

miRNA candidates that potentially target collagen 1 were predicted by computational algorithms PicTar and TargetScan. The expression changes of collagen subunits 1α1 and 1α2 were measured by real-time reverse transcription–polymerase chain reaction and western blot after the primary skin fibroblasts were transfected with miR-29b mimics or inhibitor, respectively. A luciferase reporter assay was performed to further determine whether both collagen 1 subunits were probably direct targets of miR-29b.

Results

Computational predictions identified several miRNAs as possible regulators for collagen 1 synthesis, including miR-29b. Enforced overexpression of miR-29b resulted in remarkable decrease of collagen 1α1 and 1α2, whereas knockdown of endogenous miR-29b induced pronounced increase of collagen 1α1 and 1α2 at both the messenger RNA and the protein levels. The luciferase activities were significantly inhibited when cells were cotransfected with reporter constructs and miR-29 mimics in vitro. Moreover, miR-29b transcriptional abundance inversely related to the levels of both collagen 1 subunits in skin scar as compared with normal skin.

Conclusions

Our data indicate that miR-29b is a potent post-transcriptional repressor of collagen 1 in skin fibroblasts and its deregulation might be implicated in scar formation, suggesting that miR-29b might represent a potential therapeutic target for scar reduction.

Section snippets

Bioinformatics Prediction and Analysis

To genome-wide screen miRNA candidates targeting human collagen 1, the PicTar (4-way) and TargetScanS algorithms provided by miRGen database were initially used with default settings.10 Because of dozens of potential miRNAs estimated by each program, the overlapped targets of both programs were considered as putative candidates for further analysis to reduce possible false positives.11

Cell Cultures and Lines

Primary human dermal fibroblasts were obtained from adult skin specimens discarded from reconstructive surgery

Computational Prediction of miRNA Candidates Targeting Collagen 1

To investigate which miRNAs are potentially involved in collagen 1 biosynthesis, we first screened possible miRNA-binding sites in the 3′UTRs of col1α1 and col1α2 via a bioinformatic approach. As expected, for both collagen 1 subunits, PicTar and TargetScanS algorithms estimated dozens of miRNA candidates probably involved in collagen 1 post-transcriptional regulation. Several overlapped miRNAs, such as let-7 family, miR-196a/b, miR-29a/b/c and miR-92, were predicted as putative regulators of

DISCUSSION

In the present study, our data indicate that miR-29b is a potent post-transcriptional repressor of collagen 1 synthesis in skin fibroblasts. miR-29b modulates col1α1 and col1α2 at both mRNA and protein levels, which was probably mediated by canonical miRNA-induced post-transcriptional silencing. Moreover, decreased miR-29b abundance was found in skin scar as compared with normal skin and its transcriptional level also inversely related to levels of both collagen 1 subunits in scar tissue.

REFERENCES (21)

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Supported by National Natural Science Foundation of China (No. 81100737), Natural Science Foundation of Jiangsu Province (No. BK2011762) and Priority Academic Program Development of Jiangsu Higher Education Institution (No. 2011137).

The authors declare no conflicts of interest.

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