LncRNA NEAT1调节miR-128-3p/HNRNPL轴对非小细胞肺癌细胞增殖、凋亡及PD-1/PD-L1表达的影响

Effects of LncRNA NEAT1 on proliferation,apoptosis,and PD-1/PD-L1 expression in non-small cell lung cancer cells through miR-128-3p/HNRNPL axis

为探讨长链非编码RNA(long noncoding RNA,LncRNA)核富集转录本1(nuclear paraspeckle assembly transcript 1,NEAT1)在非小细胞肺癌(non-small cell lung cancer,NSCLC)免疫逃逸中的作用及机制,采集96例NSCLC患者的肿瘤及邻近正常组织,应用qRT-PCR检测NEAT1表达并分析其与NSCLC患者临床病理特征的相关性。以qRT-PCR检测人支气管上皮细胞16HBE和NSCLC细胞系中NEAT1、miR-128-3p、异质性胞核核糖核蛋白L(heterogeneous nuclear ribonucleoprotein L,HNRNPL)和PD-L1 mRNA表达。将NEAT1干扰质粒(si-NEAT1)、miR-128-3p inhibitor及其对照(si-NC、inhibitor-NC)转染A549细胞,分为正常对照组、si-NC组、si-NEAT1组、si-NEAT1+anti-NC组和si-NEAT1+anti-miR-128-3p组,随后检测HNRNPL、PD-L1 mRNA和蛋白表达,以RNA沉降等法验证NEAT1、miR-128-3p和HNRNPL的调控机制。A549与CD8^(+)T细胞共培养,验证敲低NEAT1在NSCLC免疫逃逸中的作用,并以ELISA检测PD-L1和IFN-γ水平。结果显示,NEAT1在NSCLC组织中高表达,且与NSCLC肿瘤大小、TNM分期和淋巴结转移显著相关(P<0.05)。NSCLC细胞中,NEAT1、HNRNPL和PD-L1显著过表达,而miR-128-3p显著低表达(均P<0.05)。敲低NEAT1可上调miR-128-3p,抑制HNRNPL和PD-L1表达,降低NSCLC细胞活力并诱导其凋亡(均P<0.05),而NEAT1可靶向结合NSCLC细胞中的miR-128-3p。共培养发现敲低NEAT1的A549细胞可激活CD8^(+)T细胞,抑制其凋亡并降低PD-L1和IFN-γ水平(均P<0.05),而下调miR-128-3p可增加HNRNPL和PD-L1表达,减弱NEAT1敲低对NSCLC细胞增殖、凋亡及CD8^(+)T细胞活化的影响(均P<0.05)。由此,NEAT1可能通过miR-128-3p/HNRNPL轴促进PD-L1表达,从而导致NSCLC的免疫逃逸。

To investigate the role and mechanism of long non-coding RNA(LncRNA)nuclear paraspeckle assembly transcript 1(NEAT1)in immune escape of non-small cell lung cancer(NSCLC),the tumor tissues and adjacent normal tissues of 96 NSCLC patients were collected in this study.qRT-PCR was used to detect the expression of NEAT1 in NSCLC tumors and normal tissues,and its correlation with the clinicopathologic feature of NSCLC patients was analyzed.In addition,qRT-PCR was used to detect the expression of NEAT1,miR-128-3p,heterogeneous nuclear ribonucleoprotein L(HNRNPL),and PD-L1 mRNAs in bronchial epithelial cells 16HBE and NSCLC cell lines.NEAT1 interference plasmid(si-NEAT1),miR-128-3p inhibitor,and controls(si-NC and inhibitor-NC)were transfected into A549 cells and divided into control group,si-NC group,si-NEAT1 group,si-NEAT1+anti-NC group,and si-NEAT1+anti-miR-128-3p group.The mRNA and protein expressions of HNRNPL and PD-L1 were measured and the regulatory mechanisms of NEAT1,miR-128-3p,and HNRNPL were verified by RNA pull-down experiment.A549 cells were co-cultured with CD8^(+)T cells to verify the role of NEAT1 knockdown in NSCLC immune escape.The levels of PD-L1 and IFN-γwere detected by ELISA.The results showed that NEAT1 was highly expressed in NSCLC tumor tissues,and it significantly correlated with NSCLC tumor size,TNM stage,and lymph node metastasis(P<0.05).In NSCLC tumor cells,NEAT1,HNRNPL,and PD-L1 were significantly overexpressed,while miR-128-3p was significantly underexpressed(all P<0.05).Knockdown of NEAT1 up-regulated miR-128-3p while inhibiting HNRNPL and PD-L1 expressions,which in turn significantly reduced NSCLC cell viability and induced NSCLC cell apoptosis(all P<0.05).NEAT1 targeted and bound miR-128-3p in NSCLC cells.In the co-culture system,knocking down NEAT1 in A549 cells could activate CD8^(+)T cells,inhibit their apoptosis and reduce the levels of PD-L1 and IFN-γ(all P<0.05).Down-regulation of miR-128-3p increased the expressions of HNRNPL and PD-L1,and attenuated the impact of NEAT1 knockdown on NSCLC cell proliferation,apoptosis,and CD8^(+)T cell activation(all P<0.05).Therefore,NEAT1 may promote PD-L1 expression through the miR-128-3p/HNRNPL axis,thereby leading to immune escape in NSCLC.