miR-454-3p通过外泌体途径介导T790M突变NSCLC传递EGFR-TKIs耐药性的作用机制研究

The response rate of the third generation of EGFR-TKIs in non-small cell lung cancer(NSCLC) patients with EGFR-T790M mutation is above 60%, while the mutation abundance of T790M is lower than 20%. Which indicates special mechanism exists in T790M induced EGFR-TKIs resistance. Research shows exosomes could transmit drug resistance between cells. Our preliminary explorations found that the exosomes from EGFR-T790M mutated cell line could reduce the sensitivity to EGFR-TKIs in EGFR sensitive mutated cell line, and that miR-454-3p was highly expressed in the exosomes of H1975 by sequencing analysis. Bioinformatics database indicated that BTG1 was one of the potential targets of miR-454-3p. Depending on above results, we assumed that miR-454-3p from exosomes could induce EGFR-TKIs resistance by targeting BTG1 and activating PI3K/AKT、MEK/ERK signal pathways and inhibiting apoptosis in recipient EGFR-TKIs sensitive cells. Based on previous research, this program plans to investigate the mechanism of miR-454-3p from EGFR-T790M mutant NSCLC through exosomes in transmitting resistance of EGFR-TKIs on molecular, cellular and animal levels. The results from above studies will be further verified in clinical specimens. This research might provide new strategy and potential target to overcome acquired resistance of EGFR-TKIs.

EGFR-T790M突变的NSCLC患者接受第三代靶向治疗有效率超过60%,而其突变丰度多低于20%,提示T790M突变介导EGFR-TKIs耐药机制独特。研究示外泌体可在细胞间传递耐药信息。前期研究发现EGFR-T790M突变细胞的外泌体可使敏感突变细胞对EGFR-TKIs敏感性下降,测序分析示H1975外泌体中miR-454-3p高表达,生信分析提示BTG1是miR-454-3p的潜在靶点,结合既往研究提出假说:外泌体中的miR-454-3p通过作用于靶点BTG1,进而激活PI3K/AKT、MEK/ERK信号通路及抑制细胞凋亡向敏感细胞传递EGFR-TKIs耐药。在前期研究基础上,本课题拟从分子、细胞、动物水平探讨T790M突变NSCLC中的miR-454-3p通过外泌体传递EGFR-TKIs耐药性的分子机制,并通过临床标本验证,为克服EGFR-TKIs耐提供新的思路和候选靶点。

第1代EGFR-TKIs治疗后出现EGFR T790M获得性耐药的NSCLC患者，可选择第3代EGFR-TKIs奥希替尼治疗。获得性T790M突变的肿瘤中仅1-20%的肿瘤细胞存在该突变，而T790M突变NSCLC患者接受奥希替尼治疗的有效率达61%-71%。提示T790M突变细胞可向非T790M突变细胞传递耐药性。.本研究旨在明确EGFR-T790M突变NSCLC外泌体在传递EGFR-TKIs耐药中的作用，探索miR-454-3p通过外泌体传递EGFR-TKIs耐药的机制，证实抑制miR-454-3p的表达是否可以阻断获得性耐药的传递，明确血清外泌体miR-454-3p表达是否可以作为生物标记物预测TKIs疗效。.本研究通过分子生物学、细胞学及动物实验证实PC9可以通过摄取H1975的外泌体获得EGFR-TKIs耐药性，而外泌体miR-454-3p是传递EGFR-TKIs耐药性的关键miRNA之一。生物信息学提示BTG1是miR-454-3p的靶点，本研究从分子、细胞及动物水平证实外泌体miR-454-3p通过作用于靶点BTG1,并激活下游的PI3K/AKT、MEK/ERK信号通路向敏感细胞传递TKIs耐药。细胞及动物实验证实Bcl2介导的抗凋亡途径在外泌体miR-454-3p传递EGFR-TKIs耐药中的作用较弱。动物实验发现抑制H1975细胞中miR-454-3p的表达，可减少H1975的外泌体向PC9传递EGFR-TKIs耐药性，提示抑制miR-454-3p的表达可以作为延缓EGFR-TKIs耐药的方法。在临床数据分析方面，本研究通过收集接受EGFR-TKIs治疗的EGFR敏感突变患者治疗前、部分缓解、进展时的血清标本，用real-time PCR法比较血清外泌体中miR-454-3p的含量，结果提示血清外泌体miR-454-3p表达量及动态变化均不能预测EGFR-TKIs耐药，外泌体miR-454-3p不是一个理想的预测EGFR-TKIs耐药的生物学标记物。.本课题首次研究外泌体miR-454-3p在传递EGFR-TKIs耐药性中的作用及机制，揭示晚期NSCLC中EGFR T790M突变的肿瘤细胞丰度低、使用奥希替尼有效率高的原因，证实了抑制外泌体miR-454-3p的表达可以减少耐药信息的传递，为逆转EGFR-TKIs耐药提供了潜在靶点。