Screening of STAT3⁃binding elements within human PD⁃L1 promoter
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摘要:
目的：检查信号转导与转录激活因子3（signal transducer and activator of transcription 3，STAT3）对人胶质瘤细胞程序性死亡配体1（programmed death-ligand 1，PD-L1）基因转录和表达的影响，同时筛选PD-L1基因启动区可能的STAT3结合元件。方法：培养U251细胞，给予STAT3抑制剂（BP-1-102）或DMSO处理，9 h后用流式细胞术检查细胞表面PD-L1蛋白的表达情况。将PD-L1基因启动子全长荧光素酶报告质粒（pGL3-PD-L1-FL）转染U251细胞，再给予BP-1-102或DMSO处理，9 h后检查细胞内荧光素酶活性。将PD-L1基因启动子全长和各截短（pGL3-PD-L1-1~4）荧光素酶报告质粒和STAT3过表达质粒（pCMV-STAT3）共转染HEK293细胞，再测定荧光素酶活性，初步确定STAT3的结合区域。应用生物信息学软件预测PD-L1基因启动子上STAT3的结合元件，并开展结合元件突变实验确定有效性。结果：BP-1-102可明显下调U251细胞中PD-L1的基因转录和蛋白表达。此外，将pGL3-PD-L1-FL、pGL3-PD-L1-1~4和pCMV-STAT3共转染HEK293细胞后，pGL3-PD-L1-4的启动活性显著低于其他质粒，提示STAT3可能结合于PD-L1基因启动子-200~0 nt区域。生物信息学软件预测发现，此区域含有2个STAT3的结合元件，分别位于-194~-184 nt和-135~-125 nt部位。进一步研究揭示突变-194~-184 nt和-135~-125 nt元件均可显著下调pGL3-PD-L1-FL的荧光素酶活性，联合突变更为明显。结论：本研究成功筛查出STAT3在人PD-L1基因启动子上的可能结合区域，为后续研究胶质瘤细胞中STAT3相关的PD-L1基因转录调控奠定了基础。
Abstract:
Objective：This study aims to examine the effects of signal transducer and activator of transcription 3（STAT3） on the transcription and expression of programmed death ligand 1（PD-L1）gene in human glioma cells and screen the possible STAT3-binding elements within PD-L1 gene promotor. Methods：U251 cells were cultured and treated with STAT3 inhibitor（BP-1-102） or DMSO. The expression of PD-L1 protein on the surface of U251 cells was examined by flow cytometry after 9 h. The luciferase reporter plasmid of full-length PD-L1 gene promotor（pGL3-PD-L1-FL）was transfected into U251 cells，and then cells were treated with BP-1-102 or DMSO. The luciferase activity in U251 cells was examined after 9 h. The luciferase reporter plasmids of full-length or truncated PD-L1 promoter（pGL3-PD-L1-FL，pGL3-PD-L1-1，pGL3-PD-L1-2，pGL3-PD-L1-3，pGL3-PD-L1-4）and the plasmid of pCMV-STAT3 were co-transfected into HEK293 cells. Then，the luciferase activity was detected to screen the STAT3-binding elements. Furthermore，bioinformatics software was used to predict the STAT3-binding elements in the promoter of PD-L1 gene，and mutation experiments were carried out to determine the validity of these binding elements. Results：BP-1-102 could significantly down-regulate the gene transcription and protein expression of PD-L1 in U251 cells. In addition，the plasmids of pGL3-PD-L1-FL or pGL3-PD-L1-1~4 and pCMV-STAT3 were co-transfected into HEK293 cells，and then the luciferase activity in different groups was determined. The result displayed that the activity of pGL3-PD-L1-4 was much lower than that in others，indicating that the region of PD-L1 promoter（-200~0 nt）might contain STAT3-binding elements. Bioinformatics software predicted that the region might contain two STAT3-binding elements，that are located at -194~-184 nt and -135~-125 nt. Further studies revealed that mutation of -194 ~-184 nt and -135~-125 nt elements especially the combined mutation could significantly down-regulate the luciferase activity of pGL3-PD-L1-FL. Conclusion：STAT3-binding elements within PD-L1 gene promotor were successfully screened out，which could be beneficial to further studies about the STAT3-related transcription regulation of PD-L1 gene in glioma cells.
