Supplementary Material for: Circular RNA hsa_circ_0068252 functions in cisplatin resistance and immune response via miR-1304-5p/PD-L1 axis in non-small cell lung cancer

Background: Research suggests that circRNAs play important roles in non-small cell lung cancer (NSCLC). The function of hsa_circ_0068252 in NSCLC, especially in cisplatin (DDP) resistance and the mechanisms, was explored in this study. Methods: NSCLC patient samples and two NSCLC cell lines along with corresponding DDP-resistant cell lines were used. Expression levels of circ_0068252 was detected. SiRNA for circ_0068252 and inhibitor for miRNA were used in all functional analysis. A co-culture system of NSCLC cells with CD8+ T cells was used. The cellular location of circ_0068252 was detected and its target miRNA was predicted and verified. Finally, the mechanism responsible for circ_0068252 function on PD-L1 was analyzed using luciferase reporter assay in the two DDP-resistant cell lines, as well as in the co-culture system. The cytotoxicity of T cells was detected by lactate dehydrogenase (LDH) assay. Results: Our findings revealed that high level of circ_0068252 was correlated with poor prognosis of NSCLC and DDP resistance. Knockdown of circ_0068252 could promote the sensitivity of DDP-resistant NSCLC cells to DDP. Moreover, knockdown of circ_0068252 could regulate the immune micro-environment which was mediated via CD8+ T cells. Finally, circ_0068252 could up-regulate PD-L1 expression by adsorbing miR-1304-5p. Conclusion: The circ_0068252/miR-1304-5p/PD-L1 signal axis participates in the regulation of DDP resistance and immune escape of NSCLC cells. Our results suggest that circ_0068252 may be a potential diagnostic marker and therapeutic target for DDP-resistant NSCLC.

背景：已有研究表明，环状RNA（circRNAs）在非小细胞肺癌（non-small cell lung cancer, NSCLC）中发挥重要调控作用。本研究探讨了hsa_circ_0068252在非小细胞肺癌中的功能，尤其是其在顺铂（cisplatin, DDP）耐药中的作用及潜在分子机制。方法：本研究采用非小细胞肺癌患者组织样本、两株非小细胞肺癌细胞系及其对应的顺铂耐药细胞系开展实验。检测了circ_0068252的表达水平。在所有功能验证实验中，均使用了针对circ_0068252的小干扰RNA（small interfering RNA, siRNA）以及microRNA（miRNA）抑制剂。构建了非小细胞肺癌细胞与CD8+ T细胞的共培养体系。检测了circ_0068252的细胞亚定位，并对其靶向miRNA进行预测与实验验证。最后，在两株顺铂耐药细胞系及上述共培养体系中，通过荧光素酶报告基因实验分析了circ_0068252调控程序性死亡配体1（PD-L1）功能的分子机制。采用乳酸脱氢酶（lactate dehydrogenase, LDH）实验检测CD8+ T细胞的细胞毒性。结果：本研究结果显示，高表达circ_0068252与非小细胞肺癌患者不良预后及顺铂耐药性显著相关。敲低circ_0068252可增强顺铂耐药非小细胞肺癌细胞对顺铂的化疗敏感性。此外，敲低circ_0068252可调控CD8+ T细胞介导的肿瘤免疫微环境。最终发现，circ_0068252可通过吸附miR-1304-5p上调PD-L1的表达水平。结论：circ_0068252/miR-1304-5p/PD-L1信号轴参与调控非小细胞肺癌的顺铂耐药与肿瘤免疫逃逸。本研究结果提示，circ_0068252有望成为顺铂耐药非小细胞肺癌的潜在诊断标志物及治疗靶点。