miR-577靶向Ku80调控小细胞肺癌细胞中PD-L1表面表达的机制研究  

miR-577 regulates PD-L1 surface expression in small cell lung cancer cells by targeting Ku80

作　　者：何淼[1] 杨桄权 黄虹超 李超[1] 张友才 HE Miao;YANG Guangquan;HUANG Hongchao;LI Chao;ZHANG Youcai(Department of Oncology,People's Hospital of Deyang City,Deyang 618000,China)

机构地区：[1]德阳市人民医院肿瘤科,618000

出　　处：《免疫学杂志》2021年第12期1058-1064,共7页Immunological Journal

摘　　要：目的探讨DNA损伤引起小细胞肺癌细胞NCI-H1688中PD-L1表面表达的潜在分子机制。方法使用DNA损伤诱导剂ETP、CPT、APH处理小细胞肺癌细胞NCI-H1688后,检测细胞中PD-L1的蛋白水平和mRNA水平。通过高通量测序检测ETP、CPT、APH处理小细胞肺癌细胞NCI-H1688前后miRNA表达差异。过表达差异miRNA,检测细胞中PD-L1的mRNA水平。通过miRDB在线分析和荧光素酶报告实验寻找miRNA的靶标。结果ETP、CPT、APH诱导小细胞肺癌细胞NCI-H1688DNA损伤后,PD-L1的表达在mRNA水平和蛋白水平上均上升(P<0.05)。将ETP、CPT、APH处理小细胞肺癌细胞NCI-H1688后的高通量测序结果取交集后,共有11种基因的表达受到调控。敲低上述基因后发现,敲低Ku80时,PD-L1的表达上升(P<0.05)。过表达Ku80后,PD-L1的表达下降(P<0.05)。ETP、CPT、APH处理小细胞肺癌细胞NCI-H1688后,Ku80的mRNA水平下降(P<0.05),但是Ku80的启动子活性没有显著变化(P<0.05)。miRNA mimics过表达miRDB在线分析Ku80的潜在miRNA后,发现10种miRNA能够显著降低Ku80的表达(P<0.05)。ETP、CPT、APH处理小细胞肺癌细胞NCI-H1688后,miR-577的表达水平显著上升(P<0.05)。过表达miR-577后,Ku80的m RNA和蛋白水平均上升(P<0.05);敲低miR-577后,Ku80的mRNA和蛋白水平均下降(P<0.05)。过表达miR-577后,PD-L1的mRNA和蛋白水平均下降(P<0.05);敲低miR-577后,PD-L1的mRNA和蛋白水平均上升(P<0.05)。结论miR-577通过靶向DNA损伤应答蛋白Ku80 mRNA的3端非编码区以影响其翻译水平,最终提高了PD-L1在小细胞肺癌细胞NCI-H1688中的表达水平。This study aims to explore the potential molecular mechanism of DNA damage-caused PD-L1 surface expression o in small cell lung cancer cells(NCI-H1688).NCI-H1688 was treated with DNA damage inducers ETP,CPT and APH,then the protein and mRNA levels of PD-L1 in the cells were detected.Highthroughput sequencing was used to detect the miRNA expression difference of NCI-H1688 in small-cell lung cancer cells before and after ETP,CPT and APH treatment.Then the differential miRNA was over expressed and the mRNA level of PD-L1 in cells was observed.Online miRDB analysis and luciferase reporting assay were used to search for miRNA targets.Data showed that after DNA damage of NCI-H1688 cells induced with ETP,CPT and APH,the expression of PD-L1 was increased at both mRNA and protein levels(P<0.05).The high-throughput sequencing results showed the after DNA damage,there are 11 genes were regulated,among which gene KU80 was negatively related with the expression of PD-L1(P<0.05).After ETP,CPT and APH treatment,the mRNA level of KU80 in small-cell lung cancer cells NCI-H1688 decreased(P<0.05),but the promoter activity of KU80 showed no significant change(P<0.05).Online miRDB analysis showed that there are 10 miRNAs could significantly reduce the expression of KU80(P<0.05),among which,miR-77 was positively related with KU80 expression(P<0.05).Therefore,miR-577 overexpression could down-regulate the mRNA and protein levels of PD-L1(P<0.05),while miR-577 knockdown could up-regulate the mRNA and protein levels of PD-L1(P<0.05).Taken together,miR-577 affects the translation level of the DNA response protein KU80 mRNA by targeting the 3-terminal non-coding region,and ultimately increases the expression level of PD-L1 in small-cell lung cancer cells NCI-H1688.

关 键 词：miR-577 KU80 小细胞肺癌 NCI-H1688 PD-L1 

分 类 号：R734.2[医药卫生—肿瘤]