Abstract:
Long non-coding RNAs (lncRNAs) are associated with the healing of burn wounds in the dermis. The present study aimed to probe the role and regulatory network of the lncRNA TPT1 antisense RNA 1 (TPT1-AS1) in human dermal fibroblasts (HDFs) following thermal injury. A model of thermally injured cells was constructed with HDFs. The levels of TPT1-AS1, microRNA (miR)-324-5p and cyclin-dependent kinase (CDK)16 were determined through reverse transcription-quantitative PCR. Cell viability, cell cycle distribution, cell apoptosis rate and extracellular matrix (ECM) synthesis were assessed with a series of in vitro gain-of-function experiments and MTT, flow cytometry and western blot analyses. The binding ability of miR-324-5p and TPT1-AS1 (or the 3\' untranslated region of CDK16) was identified via bioinformatics analysis and luciferase reporter assay. It was found that TPT1-AS1 and CDK16 were downregulated, but miR-324-5p was upregulated, in the HDFs after thermal injury. TPT1-AS1 elevation induced cell viability and ECM synthesis but attenuated cell cycle arrest at the G0/G1 stage and decreased the cell apoptosis rate of thermally injured HDFs. In addition, TPT1-AS1 sponged miR-324-5p to modulate CDK16 expression. Moreover, silencing CDK16 weakened the impacts of TPT1-AS1 upregulation on cell function and ECM synthesis in heat-treated HDFs. In summary, TPT1-AS1 relieved cell injury and induced ECM synthesis by sponging miR-324-5p and targeting CDK16 in the HDFs after thermal injury, implying a protective role for TPT1-AS1 in the burn wound healing process.
摘要:
长链非编码RNA(lncRNA)与真皮烧伤伤口的愈合有关。本研究旨在探讨lncRNATPT1反义RNA1(TPT1-AS1)在热损伤后人真皮成纤维细胞(HDF)中的作用和调节网络。用HDFs构建热损伤细胞模型。通过逆转录-定量PCR测定TPT1-AS1、微小RNA(miR)-324-5p和细胞周期蛋白依赖性激酶(CDK)16的水平。细胞活力,细胞周期分布,细胞凋亡率和细胞外基质(ECM)合成通过一系列体外功能获得实验和MTT,流式细胞术和蛋白质印迹分析。miR-324-5p和TPT1-AS1(或CDK16的3'非翻译区)的结合能力通过生物信息学分析和荧光素酶报告基因测定来鉴定。发现TPT1-AS1和CDK16下调,但miR-324-5p上调,在热损伤后的HDF中。TPT1-AS1升高可诱导细胞活力和ECM合成,但在G0/G1期减弱细胞周期停滞,并降低热损伤HDF的细胞凋亡率。此外,TPT1-AS1海绵化miR-324-5p以调节CDK16表达。此外,沉默CDK16减弱了TPT1-AS1上调对热处理HDFs细胞功能和ECM合成的影响。总之,TPT1-AS1通过在热损伤后的HDFs中形成miR-324-5p和靶向CDK16减轻细胞损伤并诱导ECM合成,暗示TPT1-AS1在烧伤伤口愈合过程中具有保护作用。