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.
Hyperproliferation of fibroblasts is the main cause of keloid formation. However, the pathogenesis of keloids has yet to be fully elucidated. Tumor necrosis factor (TNF)-α may play an important role in the formation and proliferation of keloids, as it is implicated in the pathogenesis of various fibrous disorders. In the present study, the expression level of TNF-α and its receptors, soluble TNF receptor (sTNFR)1 and sTNFR2, in the peripheral blood and skin tissues was detected by ELISA, reverse transcription-quantitative PCR or immunohistochemistry. There was no statistically significant difference in the expression of TNF-α and sTNFR2 in the peripheral blood and skin tissues between patients with keloids and healthy participants (P>0.05), while the sTNFR1 mRNA level in fibroblasts cultured
in vitro
and its protein level in keloid skin samples were significantly higher compared with those in normal skin (P<0.05). Subsequently, TNF-α recombinant protein was used to treat keloid-derived and normal skin fibroblasts, and it was observed that TNF-α promoted the proliferation of keloid fibroblasts (KFs), but had little effect on normal skin fibroblasts. Furthermore, it was observed that TNF-α stimulation led to the activation of the nuclear factor (NF)-κB, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways in KFs. In conclusion, KFs exhibited increased expression of sTNFR1, which may contribute to the increased sensitivity to TNF-α, resulting in low concentrations of TNF-α activating the NF-κB, JNK and p38 MAPK pathways, thereby promoting the sustained and excessive proliferation of KFs.
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