Z-Y Chen1, X-F Yu, J-Q Huang, D-L Li. 1. Department of Burn Plastic Surgery, Fuling Central Hospital of Chongqing, Fuling, Chongqing, China. flzxmr@163.com.
Abstract
OBJECTIVE: To investigate the role of β-catenin siRNA on proliferation and apoptosis of keloid fibroblast cell. PATIENTS AND METHODS: Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to monitor the mRNA and protein expression levels of β-catenin in pathological scar tissue and adjacent normal tissue. Human keloid fibroblast cells (KFB) were isolated from the keloid's tissue by enzyme digestion assay and identified by immunocytochemistry assay. Keloid fibroblast cell lines in vitro were transfected with 3 pairs of specific β-catenin small interfering RNA (siRNA); RT-PCR and Western blot were performed to identify the best siRNA. The proliferation and apoptosis of KFB transfected with β-catenin were estimated by MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry (FCM). In addition, the expression levels of Bcl-2, p53, and active-caspase-3 were detected by Western blot. RESULTS: The RT-PCR and Western blot assay results showed that the expression levels of β-catenin mRNA and protein in pathological scar tissue were significantly higher than those in adjacent normal tissue (p<0.05). KFB were successfully separated from human pathological scar tissue, and immunofluorescence staining results showed that cells were spindle and positively stained with vimentin. The β-catenin siRNA2 remarkably inhibited the expression of β-catenin at mRNA and proteins levels in the human keloid fibroblasts. Compared with the control group, cell proliferation was decreased, and apoptotic rate was increased in β-catenin siRNA2 group. CONCLUSIONS: Knockdown of β-catenin significantly decreased the proliferation and increased apoptosis of KFB, which could inhibit the formation of pathological scar.
OBJECTIVE: To investigate the role of β-catenin siRNA on proliferation and apoptosis of keloid fibroblast cell. PATIENTS AND METHODS: Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to monitor the mRNA and protein expression levels of β-catenin in pathological scar tissue and adjacent normal tissue. Human keloid fibroblast cells (KFB) were isolated from the keloid's tissue by enzyme digestion assay and identified by immunocytochemistry assay. Keloid fibroblast cell lines in vitro were transfected with 3 pairs of specific β-catenin small interfering RNA (siRNA); RT-PCR and Western blot were performed to identify the best siRNA. The proliferation and apoptosis of KFB transfected with β-catenin were estimated by MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry (FCM). In addition, the expression levels of Bcl-2, p53, and active-caspase-3 were detected by Western blot. RESULTS: The RT-PCR and Western blot assay results showed that the expression levels of β-catenin mRNA and protein in pathological scar tissue were significantly higher than those in adjacent normal tissue (p<0.05). KFB were successfully separated from human pathological scar tissue, and immunofluorescence staining results showed that cells were spindle and positively stained with vimentin. The β-catenin siRNA2 remarkably inhibited the expression of β-catenin at mRNA and proteins levels in the human keloid fibroblasts. Compared with the control group, cell proliferation was decreased, and apoptotic rate was increased in β-catenin siRNA2 group. CONCLUSIONS: Knockdown of β-catenin significantly decreased the proliferation and increased apoptosis of KFB, which could inhibit the formation of pathological scar.
Authors: Hui Ma; Xingwu Duan; Runtian Zhang; Hang Li; Yang Guo; Ye Tian; Min Huang; Guangshan Chen; Zi Wang; Lingling Li Journal: Evid Based Complement Alternat Med Date: 2022-07-15 Impact factor: 2.650