UCA1/microRNAs轴向调控PD-L1表达在NSCLC EGFR-TKIs耐药中的分子机制

The mechanisms of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) resistance in non-small cell lung cancer (NSCLC) have not been yet fully elaborated. Our research group in previous studies found that long non-coding RNA (lncRNA) UCA1 over-expression mediated EGFR-TKIs non-T790M acquired resistance in EGFR mutations via regulating epithelial-mesenchymal transition (EMT). Moreover, we found that lncRNA UCA1 could target the 3’-untranslated regions (3’-UTR) of programmed cell death ligand 1 (PD-L1) through microRNAs by bioinformatic analysis. The activation of PD-L1 could help NSCLC cells evade immune system destruction. In our pre-experiment, we analyzed the expression of PD-L1 in both EGFR-TKIs sensitive and resistant cell lines, and found resistant cell lines had higher level of PD-L1 expression than sensitive cell lines. Mesenchymal NSCLC cell lines had higher level of PD-L1 expression than epithelial NSCLC cell lines. After gefitinib treatment, the level of PD-L1 expression decreased in sensitive cell lines. However, the mechanism remains undetermined. In the current project, we will systematically investigate the role of lncRNA UCA1/microRNAs in the regulation of PD-L1 expression by using the transgenic mouse model of primary lung cancer, gene transfection, etc., and deeply elaborate the mechanisms of EGFR-TKIs resistance from immune escape.

EGFR-TKIs 耐药机制未完全阐明。我们前期研究证实UCA1通过调控EMT介导EGFR突变型NSCLC 的获得性耐药。进一步采用生物信息学分析发现UCA1可通过microRNAs间接结合PD-L1的3'-UTR调控其表达。活化的PD-L1通路可引起免疫逃逸而参与靶向耐药。我们预实验结果显示：在EGFR-TKIs获得性耐药细胞株中PD-L1的表达显著高于敏感株；间质表型细胞株PD-L1的表达显著高于上皮型细胞株。吉非替尼处理后，敏感株PD-L1表达下降。因此，我们推测UCA1/microRNAs可能通过轴向调控PD-L1表达而介导NSCLC的EGFR-TKIs耐药，但具体机制不明。本课题将承前启后，采用转基因小鼠原发肺癌模型、基因转染等技术深入探讨UCA1/microRNAs调控PD-L1表达介导NSCLC EGFR-TKIs耐药的分子机制，从免疫逃逸角度深度解析EGFR-TKIs耐药。

本项目探讨了UCA1通过调控PD-L1从而在EGFR-TKI获得性耐药的作用机制的假说，通过实验发现UCA1在PD-L1表达调控过程中的作用有限，但是UCA1能够促进细肿瘤细胞的侵袭和转移；采用体外实验和构建动物模型，课题组发现外泌体可作为传递耐药性的介质，EGFR-TKIs耐药细胞H1975来源的外泌体具有传递耐药性至EGFR-TKIs敏感细胞株的作用，通过miRNA测序和后续实验明确了H1975细胞株分泌的外泌体内的miR-522-3p是介导和传递TKI耐药的关键miRNA，可能通过激活PI3K/AKT信号而发挥作用。另外，课题组在体外诱导建立了三代EGFR-TKI获得性耐药肺腺癌细胞株PC9R/OR，并成功筛选出与三代TKI耐药相关的miRNA，再进一步研究目标miRNA是否通过影响肿瘤微环境而介导三代EGFR-TKI耐药。