Circular RNA circ-CPA4/ let-7 miRNA/PD-L1 axis regulates cell growth, stemness, drug resistance and immune evasion in non-small cell lung cancer (NSCLC)

Weijun Hong,Xiwen Gao,Jun Jiang,Yajuan Zhang,Min Xue

doi:10.1186/s13046-020-01648-1

Abstract

BackgroundNon-small cell lung cancer (NSCLC) cells derived intracellular and extracellular programmed cell death ligand 1 (PD-L1) promoted cancer progression and drug resistance, and facilitated tumor immune evasion. However, the detailed molecular mechanisms are still largely unknown. In the present study, we aimed to explore the role of circular RNA circ-CPA4/let-7 miRNA/PD-L1 axis in the regulation of NSCLC progression, drug resistance and tumor immune microenvironment.MethodsReal-Time qPCR and Western Blot analysis were conducted to examine gene expressions at transcriptional and translated levels, respectively. The regulatory mechanisms of circ-CPA4, let-7 miRNA and PD-L1 were validated by dual-luciferase reporter gene system and RNA pull-down assay. Cell growth and apoptosis were determined by CCK-8 assay, colony formation assay and Annexin V-FITC/PI double staining assay. Cell mobility was evaluated by transwell assay.ResultsCirc-CPA4 and PD-L1 were high-expressed, while let-7 miRNA was low-expressed in NSCLC cells and cancer tissues compared to the human bronchial epithelial (HBE) cells and their paired clinical normal adjacent tissues, respectively. Besides, knock-down of circ-CPA4 inhibited cell growth, mobility and epithelial-mesenchymal transition (EMT), and promoted cell death in NSCLC cells by downregulating PD-L1 through serving as a RNA sponge for let-7 miRNA. In addition, the NSCLC cells derived PD-L1-containing exosomes promoted cell stemness and increased resistance of NSCLC cells to cisplatin. Notably, by co-culturing the NSCLC cells with CD8+ T cells isolated from human peripheral blood mononuclear cells (hPBMCs) in a transwell co-culturing system, we found that NSCLC cells inactivated CD8+ T cells in a secreted PD-L1-dependent manner. Further results suggested that circ-CPA4 also positively regulated exosomal PD-L1, and the NSCLC cells with circ-CPA4 ablation re-activated CD8+ T cells in the co-culturing system.ConclusionTaken together, circ-CPA4 regulated cell growth, mobility, stemness and drug resistance in NSCLC cells and inactivated CD8+ T cells in the tumor immune microenvironment through let-7 miRNA/PD-L1 axis.

Highlights

Non-small cell lung cancer (NSCLC) cells derived intracellular and extracellular programmed cell death ligand 1 (PD-L1) promoted cancer progression and drug resistance, and facilitated tumor immune evasion
The results indicated that circ-CPA4 and PD-L1 mRNA were high-expressed, while let-7 miRNA was lowexpressed in the NSCLC tissues compared to the their
The levels of let-7 miRNA were negatively correlated with circ-CPA4 (Fig. 1f) and PD-L1 mRNA (Fig. 1g), and the levels of circ-CPA4 were positively correlated with PD-L1 mRNA (Fig. 1h) in NSCLC tissues

Summary

Introduction

Non-small cell lung cancer (NSCLC) cells derived intracellular and extracellular programmed cell death ligand 1 (PD-L1) promoted cancer progression and drug resistance, and facilitated tumor immune evasion. Among those drugs and therapies, programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) blockade was proved to be novel cancer immunotherapies for cancers treatment, and the associated drugs were developed to treat NSCLC in clinic [6, 7] These therapeutic drugs included PD-1 inhibitors Nivolumab (Opdivo) [8, 9] as well as Pembrolizumab (Keytruda) [10, 11], and PD-L1 inhibitors Atezolizumab (Tecentriq) [12], Durvalumab (Imfinzi) [13] and Avelumab (Bavencio) [14]. Previous data indicated that upregulation of PD-L1 promoted cancer cell growth and motility [22], and sustained stemness of breast cancer cells to facilitate drug resistance [20, 21, 23]

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