Abstract
Keloids 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.
Highlights
Keloids, known as connective tissue hyperplasia, are the result of excessive hyperplasia of fibroblasts and collagen caused by the continuous hyperactivity of collagen anabolism during wound healing [1]
In order to evaluate the biological effects of miR-4328 in human keloid fibroblasts (KFs), the keloid cells were transfected with a miR4328 mimic, miR-negative control (NC), and an up-regulation of miR-4328 in KFs after transfection was found (Figure 2a)
Cell apoptosis was measured by flow cytometry or western blot, and results showed that the apoptosis rate was greatly elevated in KFs (Figure 2c) and the western blot data suggested an inhibition of proliferating cell nuclear antigen (PCNA) protein but enhancement of cleaved-caspase 3 protein (Figure 2d)
Summary
Known as connective tissue hyperplasia, are the result of excessive hyperplasia of fibroblasts and collagen caused by the continuous hyperactivity of collagen anabolism during wound healing [1]. MiRNAs that are involved in keloid formation and growth have been identified, and have differential expression in keloid tissues and keloidderived fibroblasts [7]. MiRNAs have been characterized as novel regulators of cellular processes, including fibroblast proliferation and extracellular matrix (ECM) deposition, which are related to wound healing [8]. Yao et al determined that overexpression of miR-1224-5p led to the inhibition of proliferation, migration, invasion and promotion of apoptosis in keloid fibroblasts (KFs) [9]. Overexpression of miR-181a was shown to enhance keloid fibroblast DNA synthesis, proliferation and inhibit apoptosis, inducing the progression of keloids [11]. MicroRNA-4328 (miR4328), a member of the miRNA family, was first determined to cause aberrant expression in keloids [12]; the
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