MALAT1通过下调miR-125参与NSCLC EGFR-TKIs耐药的机制研究

Resistance against epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs) for NSCLCs with activating EGFR mutations becomes one of the biggest problems in clinical practice. Activation of PI3K/Akt downstream signal cascades is an important mechanism of acquired resistance to EGFR-TKIs, epithelial to mesenchymal transition (EMT) is another mechanism of acquired resistance as well. Recent evidence supports the role of microRNA-125 in modulating chemotherapeutic drug sensitivity, but its role in the resistance to EGFR-TKIs in NSCLC remains unclear. Many researchs shown that lncRNA could as a competing endogenous RNA (ceRNA) to regulate the biological activity of miRNAs. Our previous works found that miR-125 down-expression in EGFR-TKIs resistant NSCLC PC9/ER cell , and the expression of MALAT1 is significantly higher than PC9 cell. Therefore, we hypothesize that MALAT1 participate in resistance to EGFR-TKIs by modulating miR-125. In this study, we apply the lentiviral system to research the role of MALAT1 in resistance of EGFR-TKIs, and use the dual luciferase gene reporter to find the relationship between MALAT1 and miR-125. Interference MALAT1 to verify the impact on miR-125, PI3K/Akt pathway and EMT in vitro and vivo. Finally, we collect serum samples of patients who received EGFR-TKIs before and after resisitance to detect expresstion of MALAT1 and miR-125. This study would be helpful to elucidate the mechanism of MATAL1 participate NSCLC EGFR-TKIs resistance by down-regulating miR-125, and assessed whether MALAT1 as an effectiveness biomarker to beneficiaries of EGFR-TKIs treatments, also find its new intervention targets.

EGFR-TKIs获得性耐药是影响NSCLC分子靶向治疗临床疗效的重要原因，PI3K/Akt通路旁路激活及EMT的发生是其耐药的重要机制。研究发现miR-125通过调控PI3K通路参与该耐药，另研究发现MALAT1可调控miR-125。本课题组前期发现MALAT1在EGFR-TKIs耐药细胞中高表达而miR-125低表达。故推测MALAT1通过调控miR-125参与EGFR-TKIs耐药。本课题拟构建慢病毒干扰载体，采用免疫荧光、WB及qRT-PCR 等技术在细胞中探究MALAT1是否通过调控miR-125激活PI3K/Akt信号通路及EMT发生，导致EGFR-TKIs耐药。并用移植瘤确证MALAT1通过下调miR-125 参与NSCLC EGFR-TKI耐药；临床标本验证MALAT1和miR-125与EGFR-TKIs疗效的关系。以期为逆转NSCLC分子靶向药物耐药带来新的契机。

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）是EGFR突变晚期非小细胞肺癌的主要治疗手段，但获得性耐药是影响其临床疗效的重要原因，PI3K/Akt通路旁路激活及EMT的发生是其耐药的重要机制。课题组前期发现MALAT1在EGFR-TKIs耐药细胞中高表达而miR-125低表达,且与PI3K/AKT信号通路相关，故推测MALAT1通过调控miR-125参与EGFR-TKIs的耐药。课题组通过Crispr/Cas9技术构建慢病毒过表达及干扰载体，采用CCK8实验、流式细胞术、免疫荧光、WB及qRT-PCR 等技术发现：1.MALAT-1 在 TKI 耐药细胞株中呈高表达、miR-125a-5p呈低表达，且两者均与 TKI 耐药显著相关。2.miR-125a-5p是MALAT-1 作用的靶点，且 MALAT-1在转录后水平对 miR-125a-5p有抑制作用。3. 敲除MALAT-1 能逆转 TKI 的获得性耐药，其机制可能与抑制 miR-125a-5p/Rab25 及其下游PI3K/AKT 信号通路活化、抑制凋亡相关信号通路激活及抑制EMT的转化有关。动物实验发现MALAT-1过表达可以促进PC9细胞裸鼠移植瘤的生长，临床标本发现血清中MALAT-1的高表达与NSCLC的EGFR-TKIs耐药相关，提示其可作为EGFR-TKIs耐药的潜在标志物，还可作为EGFR-TKIs疗效预测的有效指标。