LINC00346通过miR-148a-3p/PD-L1轴调控前列腺癌免疫逃逸  

LINC00346 regulates prostate cancer immune escape through miR-148a-3p/PD-L1 axis

作　　者：汤磊[1] 李志坤[1] 赵亚伟[1] 马柳疆 马旺[1] 王新会[1] 蒋凤君[1] 李前跃[1] Tang Lei;Li Zhikun;Zhao Yawei;Ma Liujiang;Ma Wang;Wang Xinhui;Jiang Fengjun;Li Qianyue(Department of Urology,Xinjiang Production and Construction Corps Hospital,Urumqi 830002,China)

机构地区：[1]新疆生产建设兵团医院泌尿外科,乌鲁木齐830002

出　　处：《国际泌尿系统杂志》2024年第6期961-967,共7页International Journal of Urology and Nephrology

基　　金：2021年度兵团财政科技计划项目(2021AB036);2019年度兵团财政科技计划项目(2019AB033)。

摘　　要：目的探讨LINC00346在前列腺癌细胞免疫逃逸中的作用及相关分子机制。方法体外培养前列腺癌细胞DU145、PC3和LNCaP,以人正常前列腺上皮细胞RWPE-1作为对照。将PC3细胞分为NC-siRNA组、LINC00346-siRNA组、NC inhibitor组、miR-148a-3p inhibitor组、NC mimic组、miR-148a-3p mimic组、LINC00346-siRNA+NC inhibitor组、LINC00346-siRNA+miR-148a-3p inhibitor组及LINC00346-siRNA+pcDNA-PD-L1组,采用Lipofectamine 2000试剂分别进行相应转染处理。采用实时荧光定量PCR实验(qRT-PCR)检测各组PC3细胞中LINC00346和miR-148a-3p的表达,CCK-8实验和流式细胞术分别检测各组PC3细胞的增殖和凋亡能力。采用生物信息学方法和双荧光素酶报告基因实验分别预测和验证LINC00346与miR-148a-3p以及miR-148a-3p与程序性死亡配体1(PD-L1)的靶向关系,采用qRT-PCR和Western blot分别检测PC3细胞中PD-L1 mRNA和蛋白的表达。采用CCK-8实验和流式细胞术分别检测CD8^(+)T细胞的增殖和凋亡能力;采用ELISA实验检测CD8^(+)T细胞中干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)水平。结果细胞功能实验结果表明,与NC-siRNA组比较,LINC00346-siRNA组PC3细胞中LINC00346表达降低,细胞增殖能力降低,细胞凋亡率升高(均P<0.05)。双荧光素酶报告基因实验结果表明,与NC mimic组相比,转染miR-148a-3p mimic组细胞中LINC00346-WT和PD-L1-WT荧光素酶活性明显降低(均P<0.05)。与NC-siRNA组比较,LINC00346-siRNA组细胞中miR-148a-3p表达显著升高(P<0.05)。与NC-siRNA组比较,LINC00346-siRNA组PC3细胞增殖能力降低,细胞凋亡率显著升高(均P<0.05);与LINC00346-siRNA+NC inhibitor组比较,LINC00346-siRNA+miR-148a-3p inhibitor组PC3细胞中miR-148a-3p水平和细胞凋亡率降低,细胞增殖能力升高(均P<0.05)。与NC inhibitor组比较,miR-148a-3p inhibitor组细胞中PD-L1 mRNA表达水平升高(P<0.05)。与NC-siRNA组比较,LINC00346-siRNA组PC3细胞中PDL1 mRNA和蛋白水平降低(均P<0.05);与LINC00346-siRNA+NC Objective To investigate the role of LINC00346 in the immune escape of prostate cancer cells and its related molecular mechanism.Methods Prostate cancer cells DU145,PC3 and LNCaP were cultured in vitro,and human normal prostate epithelial cell RWPE-1 was used as a control.PC3 cells were divided into NC-siRNA group,LINC00346-siRNA group,NC inhibitor group,miR-148a-3p inhibitor group,NC mimic group,miR-148a-3p mimic group,LINC00346-siRNA+NC inhibitor group,LINC00346-siRNA+miR-148a-3p inhibitor group and LINC00346-siRNA+pcDNA-PD-L1 group were transfected with Lipofectamine 2000 reagent,respectively.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression of LINC00346 and miR-148a-3p in PC3 cells.CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of PC3 cells in each group,respectively.Bioinformatics methods and dual luciferase reporter gene assay were used to predict and verify the targeting relationship between LINC00346 and miR-148a-3p and between miR-148a-3p and programmed death ligand 1(PD-L1),respectively.qRT-PCR and Western blot were used to detect the expression of PD-L1 mRNA and protein in PC3 cells,respectively.The transfected PC3 cells were co-cultured with CD8^(+)T cells,and the proliferation and apoptosis of CD8^(+)T cells were detected by CCK-8 assay and flow cytometry,respectively.The levels of IFN-γand TNF-αin CD8^(+)T cells were measured by ELISA.Results Compared with human normal prostate epithelial cell RWPE-1,LINC00346 expression was significantly increased in prostate cancer cells DU145,PC3 and LNCaP(all P<0.05),and the expression level of LINC00346 was the highest in PC3 cells.Compared with the NC-siRNA group,the LINC00346 level and proliferation ability of PC3 cells were significantly decreased,and the apoptosis rate was significantly increased in the Linc00346siRNA group(all P<0.05).The results of dual luciferase reporter gene assay showed that compared with the NC mimic group,the luciferase activities of LINC00346-WT and PD-L1-WT in th

关 键 词：前列腺肿瘤 LINC00346 miR-148a-3p 程序性细胞死亡配体1 肿瘤逃逸 

分 类 号：R737.25[医药卫生—肿瘤]