Mechanism Study of LncRNA LINC01137 Promoting the Progression of Non-small Cell Lung Cancer by Inducing CD8+T Cell Exhaustion
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目的 研究长链非编码RNA(long non-coding RNA,LncRNA)LINC01137 在非小细胞肺癌(non-small cell lung cancer,NSCLC)免疫逃逸中的生物学功能及其潜在的调节机制.方法 采集 24 例健康志愿者和24 例NSCLC患者血液样本,并收集NSCLC肿瘤组织和癌旁组织检测LINC01137 水平.利用Starbase数据库预测LINC01137 与miR-22-3p的结合位点,荧光素酶报告基因分析进行验证.采用A549 细胞来源的外泌体和/或sh-LINC01137 干扰序列转染A549 细胞,检测细胞增殖和侵袭能力;收集转染后的细胞上清液培养CD8+T细胞,检测CD8+T细胞耗竭标志物干扰素-γ(interfereron-γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、颗粒霉素B(granzyme B)和白细胞介素-2(interleukin-2,IL-2)水平,以及PD-1+Tim3+CD8+T细胞百分比.采用外泌体和/或miR-22-3p模拟物(miR-22-3p mimic)转染CD8+T细胞,检测PD-1 蛋白水平.结果 与癌旁组织相比,NSCLC肿瘤组织中LINC01137 表达(3.357±0.548 vs 1.011±0.371)明显升高;与健康志愿者相比,NSCLC患者外周血LINC01137 表达(3.216±0.342 vs 1.007±0.313)亦明显升高,差异具有统计学意义(t=-17.367,-17.147,均P<0.001).肿瘤组织LINC01137 表达与外周血中LINC01137 表达呈正相关(r=0.755,P<0.05).在A549 细胞来源的外泌体中LINC01137 显著富集.与Exo+sh-NC组相比,Exo+sh-LINC01137 组细胞活力(65.852%±4.715%vs 100.153%±11.934%)及细胞侵袭(21.464%±3.481%vs 43.126%±1.447%)能力显著降低,差异具有统计学意义(t=4.630,9.953,均P<0.01).NSCLC患者外周血中LINC01137 表达和CD8+T细胞百分比呈负相关(r=-0.520,P＜0.05).与Exo+sh-NC组相比,Exo+sh-LINC01137 组IFN-γ(3 865.314±543.852 pg/ml vs 1 786.971±105.982 pg/ml),TNF-α(4 631.930±510.715pg/ml vs 1 973.242±379.623pg/ml),Granzyme B(3 876.496±312.438pg/ml vs 1 879.439±287.584pg/ml)和IL-2 mRNA水平(3.286±0.437 vs 1.015±0.314)升高,PD-1+Tim3+CD8+T细胞百分比(7.680%±2.185%vs 18.952%±3.216%)降低,差异具有统计学意义(t=-6.497,-7.237,-8.146,-7.310,5.021,均P<0.01).miR-22-3p是LINC01137 的 靶 基 因.与Exo+NC mimic组 相 比,Exo+miR-22-3p组PD-1 蛋白水平(0.384±0.087 vs 1.003±0.147)显著降低,差异有统计学意义(t=6.277,P<0.01).结论 NSCLC患者肿瘤组织及外周血中LINC01137 表达显著上调;NSCLC细胞来源的外泌体中LINC01137 通过靶向CD8+T细胞中miR-22-3p并抑制其表达,诱导CD8+T细胞耗竭,促进NSCLC细胞免疫逃逸.
Objective To investigate the biological function of long non-coding RNA(LncRNA)LINC01137 in immune escape of non-small cell lung cancer(NSCLC)cells and its potential regulatory mechanisms.Methods The blood samples of 24 healthy volunteers and 24 NSCLC patients were collected.The tumor tissues and paracancerous tissues of 24 NSCLC patients were collected,and the levels of LINC01137 were detected.The binding sites of LINC01137 and miR-22-3p were predicted by Starbase database and verified by the luciferase reporter gene analysis.A549 cells were transfected with exosomes derived from A549 cells and/or sh-LINC01137 interference sequence to detect cell proliferation and invasion.The supernatant of A549 cells were collected to culture CD8+T cells,and the levels of CD8+T cell exhaustion markers,including interfereron-γ(IFN-γ),tumor necrosis factor-α(TNF-α),granzyme B and interleukin-2(IL-2),and the percentage of PD-1+Tim3+CD8+T cells were detected.CD8+T cells were transfected with exosomes and/or miR-22-3p mimics to detect the protein level of PD-1.Results The expression of LINC01137 in tumor tissues of patients with NSCLC was increased compared with paracancerous tissues(3.357±0.548 vs 1.011±0.371),while the expression of LINC01137 in peripheral blood of patients with NSCLC was increased compared with healthy volunteers(3.216±0.342 vs 1.007±0.313),with statistically significant differences(t=-17.367,-17.147,all P<0.001).There was a positive correlation between the expression of LINC01137 in tumor tissue and peripheral blood(r=0.755,P<0.05).LINC01137 was significantly enriched in exosomes derived from A549 cells.Compared with Exo+sh-NC group,the cell viability(65.85%±4.71%vs 100.15%±11.93%)and cell invasion(21.46%±3.48%vs 43.12%±1.44%)in Exo+sh-LINC01137 group were decreased,and the differences were statistically significant(t=4.630,9.953,all P<0.01).The expression of LINC01137 in peripheral blood of NSCLC patients was negatively correlated with the percentage of CD8+T cells(r=-0.520,P<0.05).Compared with Exo+sh-NC group,the IFN-γ(3 865.31±543.85 pg/ml vs 1 786±105.98 pg/ml),TNF-α(4 631.93±510.71 pg/ml vs 1 973.24±379.62 pg/ml),Granzyme B(3 876.49±312.43 pg/ml vs 1 879.43±287.58 pg/ml),and IL-2 mRNA levels(3.286±0.437 vs 1.015±0.314)were increased,and the percentage of PD-1+Tim3+CD8+T cells(7.68%±2.18%vs 18.95%±3.21%)was decreased in Exo+sh-LINC01137 group,with statistical significances(t=-6.497,-7.237,-8.146,-7.310,5.021,all P<0.01).Our results showed that miR-22-3p was the target gene of LINC01137.Compared with Exo+NC mimic group,the level of PD-1 protein in Exo+miR-22-3p group(0.384±0.087 vs 1.003±0.147)was significantly decreased,and the difference was statistically significant(t=6.277,P<0.01).Conclusion The expression of LINC01137 was significantly up-regulated in tumor tissues and plasma of NSCLC patients.Exosomes LINC01137 derived NSCLC cell induces CD8+T cell exhaustion by targeting miR-22-3p and inhibiting its expression,and thus promoting NSCLC cell immune escape.
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