miR-125调控非小细胞肺癌细胞RAB-25-PI3K/AKT通路在NSCLC-TKIs耐药中的作用研究

Resistance against epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs) is one of the biggest obstacles in clinical practice.In previous studies have found that RAB25 which is closely related to tumor invasiveness and metastasis was markedly up-regulated in human lung cancer cells. 3' UTR sequence complementarity to the seed region of RAB25 was found in miR-125 molecule by bio-information analysis. And PI3K/AKT are the key downstream molecules of RAB25 signal pathway. Therefore we hypothesize that miR-125 was involved in the EGFR-TKIs resistance in lung cancer cells by regulating RAB25-PI3K/AKT pathway. In this study, on the early basis of induction and culture of erlotinib resistance lung cancer cells,we plan to use dual luciferase reporter assay system to verify whether miR-125 can regulate RAB25.The expression of miR-125 was up-regulated or down-regulated to testify the effection of miR-125 on expression of RAB25 and PI3K/AKT pathway.Further evidence that miR-125 regulates RAB25-PI3K/AKT pathway would get by respectively blockades of expression of RAB25 and PI3K/AKT in erlotinib resistance lung cancer cells in vitro and in vivo.Finally,LNA-ISH and IHC will be used to find the relationship between miR-125,RAB25 and clinical and pathological indicators of the lung cancer. This study would be helpful to elucidate the mechanism of lung cancer resistance against EGFR-TKIs, and assessed whether miR-125 is an effective biomarker to identify beneficiaries of EGFR TKIs treatment ,also find its new intervention targets.

耐药是影响表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）临床疗效的主要因素之一。研究发现RAB25表达增加与肿瘤的侵袭和转移密切相关，分子信息学提示miR-125有与RAB25结合的种子序列，PI3K/AKT是RAB25的关键下游信号通路，在TKIs耐药中起重要作用。我们因此推测miR-125可通过调控RAB25-PI3K/AKT通路导致肺癌EGFR-TKIs耐药。本课题拟在诱导厄洛替尼耐药细胞基础上验证miR-125调控RAB25；上、下调miR-125验证其对RAB25表达及PI3K/AKT通路的影响；分别阻断RAB25、PI3K/AKT反证miR-125对该通路的调控作用；移植瘤等体内外实验证实miR-125调控NSCLC RAB25-PI3K/AKT通路导致对EGFR-TKIs耐药，并通过临床标本加以验证。以评估miR-125能否作为判断EGFR-TKIs疗效的生物标记物

肺癌是最常见的恶性肿瘤之一，占癌症死因的第一位。近年来肺癌的分子靶向治疗使EGFR突变阳性患者的生存显著获益，但靶向药物最终会产生耐药而影响疗效，因而探讨其耐药机制成为研究的热点。研究显示PI3K/AKT通路异常激活是EGFR-TKIs获得性耐药的重要机制，前期研究证实在PC9/ER细胞中存在PI3K/AKT信号通路的激活；同时还发现在PC9/ER细胞中存在RAB25高表达，进一步通过生物信息学分析Rab25是miR-125a-5p的靶基因。因此，本课题从细胞水平探讨miR-125a-5p及Rab25在EGFR-TKIs耐药中的作用，并验证miR-125a-5p与Rab25的关系，进而探讨miR-125a-5p对PI3K/AKT信号通路及对PC9/ER细胞耐药性的影响；体内研究探讨miR-125a-5p在人肺腺癌细胞裸鼠移植瘤EGFR-TKIs治疗中的作用；临床标本验证miR-125a-5p在NSCLC患者EGFR-TKIs耐药中的作用。结果从细胞水平上证实了miR-125a-5p 在厄洛替尼耐药细胞中呈低表达、Rab25 在耐药细胞中呈高表达，且两者均与耐药显著相关；Rab25 基因是miR-125a-5p 直接作用的靶基因，且 miR-125a-5p在转录后水平对 Rab25 基因有直接抑制作用； miR-125a-5p 能逆转 TKI 获得性耐药，其机制可能与抑制 Rab25 及其下游PI3K/AKT 信号通路活化及凋亡相关信号通路激活有关。体内实验验证了过表达miR-125a-5p可以抑制PC9/ER细胞裸鼠移植瘤的生长，且用厄洛替尼治疗后裸鼠肿瘤体积缩小更显著；其机制可能是miR-125a-5p通过影响Rab25/PI3K/AKT信号通路抑制肿瘤的生长。临床血液标本检测发现miR-125a-5p的低表达可能与NSCLC的EGFR-TKIs耐药相关，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；EGFR-TKIs耐药患者血清中miR-125a-5p在T790M突变组和未突变组中表达无显著差异。通过本课题深入研究了miR-125a-5p在NSCLC TKIs耐药中的调控作用，它可能会成为预测肺癌患者EGFR-TKIs耐药及疗效的潜在标志物，并有望成为逆转肺腺癌分子靶向药物的靶点。