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
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-γ were 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-y(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.
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