Silencing of p53 reduces cell migration in human Tenon's fibroblasts induced by TGF-β
Silencing of p53 reduces cell migration in human Tenon's fibroblasts induced by TGF-β
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DOI：
10.1007/s10792-020-01320-9
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发表时间：
2020-03-02
影响因子：
1.6
通讯作者：
Zhang, Ran
中科院分区：
文献类型：
--
作者：
Li, Ning;Zhang, Ran
目的生长因子被认为是参与纤维化形成的关键分子。本研究旨在阐明P53对转化生长因子-β(TGF-β)和纤维化形成的调节作用，并探讨其相关机制。方法用Vimentin对人Tenon‘s成纤维细胞进行鉴定。合成靶向p53的小干扰RNA(SiRNA)，并将其导入人成纤维细胞。实时定量聚合酶链式反应检测p53和microRNA-29b(miR-29b)的表达。用免疫细胞化学方法观察转化生长因子-β的表达。通过创面愈合实验评价HTF的迁移情况。用双荧光素酶分析法检测p53和miR-29b在HTF中的相互作用。结果Vimentin在HTFS细胞中广泛分布。细胞密度为5×10~(4)个/ml、培养6d的HTF生长最好。转化生长因子-β治疗组的P53水平显著高于空白对照组(P<；0.01)。靶向p53的siRNA组的miR-29b水平显著高于空白组(p<；0.01)。靶向p53的siRNA可显著抑制HTF的迁移，与单独应用转化生长因子-β处理组相比(p<；0.01)。与空载体pcDNA3.0相比，高表达p53的HTF细胞的相对荧光素酶活性显著增强(p<；0.01)。结论p53可能通过降低miR-29b的表达并与miR29b基因相互作用，从而抑制HTF中转化生长因子-β的表达，抑制HTF的迁移和生长。
Purpose Growth factors are considered as key molecules that participating in fibrosis formation. This research aimed to clarify potential effects of p53 on regulation of transforming growth factor beta (TGF-beta) and fibrosis formation and investigate the associated mechanisms. Methods Vimentin was examined to identify human Tenon's fibroblasts (HTFs). p53-targeting small interfere RNA (siRNA) was synthesis and transfected into HTFs. Real-time PCR assay was utilized to evaluate p53 and microRNA-29b (miR-29b) expression. Immunocytochemical assay was used to observe TGF-beta expression. The wound healing assay was conducted to evaluate migration of HTFs. Dual-luciferase assay was employed to identify interaction between p53 and miR-29b in HTFs. Results Vimentin was extensively distributed in HTFs cells. HTFs at density of 5 x 10(4) cells/ml and 6 days exhibited the best growth. The p53 level in TGF-beta treatment group was significantly higher compared to that in blank group (p < 0.01). miR-29b level in siRNA targeting p53 group was significantly increased compared to that in blank group (p < 0.01). siRNA targeting p53 could significantly inhibit HTFs migration compared to that in single TGF-beta treating HTFs group (p < 0.01). Relative luciferase activity was significantly increased in p53 overexpressed HTFs compared to that in cells transfected with empty pcDNA3.0 plasmid (p < 0.01). Conclusions p53 inhibited expression of TGF-beta, suppressed HTFs migration and inhibited HTFs growth, by reducing miR-29b expression and interacting with miR29b gene in HTFs.