Department of Dermatology, People's Hospital of Henan Province, Zhengzhou, Henan, China.
J Cell Biochem. 2019 Aug;120(8):13893-13902. doi: 10.1002/jcb.28662. Epub 2019 Apr 16.
This study aims to investigate whether terminal differentiation-induced ncRNA (TINCR) has an effect on apoptosis and autophagy induced by ALA-PDT in cutaneous squamous cell carcinoma (CSCC). A431 cells were treated with 5-aminolevulinic acid (ALA) solution at different concentrations and for different duration time. A431 cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, relative TINCR messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). A431 cell apoptosis was examined by flow cytometry. Relative apoptosis/autophagy-related protein expression was analyzed by Western blot analysis. The effect of TINCR on cell autophagy was detected by RFP-LC3 immunofluorescence assay. Reactive oxygen species concentration was detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Relative expressions of ERK1/2 and specificity protein 3 (Sp3) in A43 cells were detected by Western blot analysis and qRT-PCR. Sp3 binding sites were analyzed by ChIP-qPCR. The relative transcription activity was measured with luciferase reporter assay. ALA-PDT treatment at 3.2 mmol/L for 120 minutes significantly promoted TINCR expression in CSCC A431 cells, and TINCR promoted ALA-PDT-induced apoptosis and cell autophagy. Furthermore, ALA-PDT promoted TINCR expression through ERK1/2-SP3 pathway. Sp3 promoted TINCR transcription by binding TINCR promoters. Our data indicated that TINCR involves in ALA-PDT-induced apoptosis and autophagy in CSCC.
本研究旨在探讨终端分化诱导的非编码 RNA（TINCR）是否对 5-氨基酮戊酸（ALA）-PDT 诱导的皮肤鳞状细胞癌（CSCC）中的细胞凋亡和自噬有影响。用不同浓度和不同时间的 5-氨基酮戊酸（ALA）溶液处理 A431 细胞。用细胞计数试剂盒-8（CCK-8）检测 A431 细胞活力，用定量逆转录聚合酶链反应（qRT-PCR）检测相对 TINCR 信使 RNA 表达。用流式细胞术检测 A431 细胞凋亡。用 Western blot 分析检测相对凋亡/自噬相关蛋白表达。用 RFP-LC3 免疫荧光检测 TINCR 对细胞自噬的影响。用 2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探针检测活性氧浓度。用 Western blot 和 qRT-PCR 分析 A43 细胞中 ERK1/2 和特异性蛋白 3（Sp3）的相对表达。用 ChIP-qPCR 分析 Sp3 结合位点。用荧光素酶报告基因检测相对转录活性。3.2 mmol/L ALA-PDT 处理 120 分钟可显著促进 CSCC A431 细胞中 TINCR 的表达，TINCR 促进了 ALA-PDT 诱导的细胞凋亡和自噬。此外，ALA-PDT 通过 ERK1/2-SP3 通路促进 TINCR 表达。Sp3 通过结合 TINCR 启动子促进 TINCR 转录。我们的数据表明，TINCR 参与了 CSCC 中 ALA-PDT 诱导的细胞凋亡和自噬。
