| Background:Keloid scar is a refractory disease commonly observed in dermatology and plastic surgical practice,which is a unique benign fibroproliferative tumor of the human skin.Currently,there still lacks of radical and effective treatment in the clinic,and the etiology and pathogenesis of keloid scar remain to be elucidated.Early growth response1（EGR1）,which encodes a zinc finger transcription factor,is regarded as a fibrotic factor and also expected to become a target for the prevention of keloid.EGR1 is expressed in many cell types and regulates many cell functions such as cell proliferation,apoptosis,migration,invasion.Micro RNAs（miRNAs）are noncoding RNAs with a length of 18-22 nucleotides that negatively regulate targeted gene expression by inhibiting translation levels,which plays a significant role in keloid pathogenesis.Combining the results of the miRNA microarray,miR-183-5p,predicted as a EGR1-targeted miRNA,was screened out from multiple miRNA databases.But no functional study of miR-183-5p/ EGR1 in keloid has been reported previously.Objective:We aimed to examine the effects of miR-183-5p and EGR1 on the phenotypes of KF,including migration,invasion,proliferation,apoptosis,and fibrosis in vitro and verify the targeted relationship between miR-183-5p and EGR1,so as to provide a theoretical basis for further research of precision medicine in keloid.Experimental method:In this study,human keloid fibroblasts are used as the research object by culturing primary cells from the tissue blocks of keloid samples and normal skin samples.miR-183-5p expression differences between KF and NF were determined using the quantitative polymerase chain reaction（RT-PCR）.The targeted relationship between miR-183-5p and EGR1 was verified by dual-luciferase reporter gene assay.Cells proliferation was examined by CCK-8 assay.Cellular migration and invasion were tested by wound healing and transwell assay.Cell apoptosis was assessed by flow cytometry assay.Western Blot and RT-PCR assay were used to detect m RNA and protein expression changes of EGR1 and other genes in keloids.Results:1.miR-183-5p was downregulated in keloid fibroblasts:Compared with normal skin tissues,a total of 247 known differentially expressed miRNAs（134 were up-regulated and 113 were down-regulated）were identified in keloid tissues（p＜0.05,fold change＜0.5）.Among these,miR-183-5p was downregulated.Then,total RNA of the KF and NF was extracted to evaluate the miR-183-5p expression by RT-PCR test.miR-183-5p was markedly downregulated in KF compared with that in NF（p＜0.001）.2.Effects of miR-183-5p on the phenotype of keloid fibroblasts:To assess the functional significance of miR-183-5p in keloid,we overexpressed miR-183-5p in KF by transient transfection of miRNA mimics and the transfection efficiency was verified by RT-PCR.The results revealed that miR-183-5p expression was upregulated following transfection with miR-183-5p mimics（p＜0.05）.CCK8 assay revealed that miR-183-5p overexpression was able to significantly reduce the viability of KF after 24,48,and 72 h post-transfection（p?＜?0.05）.Then,wound-healing assay showed that the wound-closure rate of KF was decreased by overexpression of miR-183-5p after 36 h post-transfection（p＜0.01）.The Transwell migration assay showed the results that the number of cells passed through transwell chamber was significantly decreased in miR-183-5p mimics group（p＜0.01）.Consistent results were observed in the transwell invasion assay（p＜0.001）.Conversely,the rate of apoptosis quantified by flow cytometry was increased in miR-183-5p mimics group（p＜0.05）.Western Blot results showed that the expression of cleaved-caspase3 was upregulated after transfection of miR-183-5p mimics（p＜0.05）.3.Effects of EGR1 silencing on the phenotype of keloid fibroblasts:To assess the functional significance of EGR1 in keloid,we silenced EGR1 expression in KF by transient transfection of small interfering RNA（si EGR1）and the transfection efficiency was verified by RT-PCR and Western Blot assay.The results revealed that EGR1 expression was downregulated following transfection with si EGR1（p＜0.001）.CCK8 assay revealed that EGR1 down-regulation was able to significantly reduce the viability of KF after 24,48,and 72 h post-transfection（p＜0.05）.Then,wound-healing assay showed that the wound-closure rate of KF was decreased by down-regulation of EGR1 after 36 h post-transfection（p＜0.05）.The transwell migration and invasion assay showed the results that the number of cells passed through transwell chamber was significantly decreased in si EGR1 group（p＜0.05）.In addition,the rate of apoptosis quantified by flow cytometry was decreased in si EGR1 group（p＜0.001）.Supportably,the protein expression levels of cleaved-caspase3 also decreased in si EGR1 group（p＜0.05）.RT-PCR and Western Blot results showed that the expression of fibrosis-related genes were downregulated after transfection of si EGR1.4.Targeting regulation relationship between miR-183-5p and EGR1:The binding sites of miR-183-5p in EGR1 m RNA were predicted using online bioinformatics databases.Then dual-Luciferase activity assay showed that miR-183-5p could bind with the 3′ UTR of EGR1 m RNA.Subsequently,RT-PCR and Western Blot results showed that the m RNA and protein expression of EGR1 was significantly reduced by miR-183-5p overexpression in KF.The above results confirmed that miR-183-5p targeted EGR1 and inhibited its expression.In order to further validate this conclusion,we carried out rescue experiment.We treated keloid fibroblasts with both miR-183-5p mimics and pc DNA3.1-EGR1.ECM production expression levels were quantified using RT-PCR and Western Blot analysis.Then we observed that the increased expression of ECM biomarkers were decreased after treatment with pc DNA3.1-EGR1 and miR-183-5p mimics co-transfection compared to pc DNA3.1-EGR1-only transfected cells.Subsequently,we treated keloid fibroblasts with both miR-183-5p inhibitors and si EGR1.The transwell assay showed that the inhibitory effects of si EGR1 on the KFs migratory ability was partially reversed by miR-183-5p inhibitors.Conclusion:1.miR-183-5p was downregulated in keloid fibroblasts and could inhibit proliferation,migration,and invasion of keloid fibroblasts,and promote the degree of apoptosis in vitro.2.EGR1 silencing inhibited the cell proliferation,migration invasion,fibrosis and apoptosis.3.miR-183-5p participated in keloid occurrence and development through downregulating EGR1 by directly targeting its 3′UTR. |
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