lncRNA HOTAIR介导的Wnt/β-catenin与EGFR信号通路对话调控肺腺癌侵袭的机制研究

Secondary resistance of EGFR tyrosine kinase inhibitors (EGFR-TKIs) is a bottleneck in EGFR-TKIs targeted therapy of lung cancer. ADAM17 plays an important role in shedding of extracellular domain of EGFR ligand, but its role in targeted therapy of lung cancer is still needed to be studied. Intersection between Wnt signal and EGFR signaling pathway participates in the regulation of tumor invasion. HOTAIR is a long non-coding RNA (lncRNA), first to be discovered to have function of trans-transcriptional regulation, which may regulate development of esophageal cancer through Wnt/β-Catenin signal pathway. However, its involvement in regulation of lung adenocarcinoma invasion remains to be studied. We have found that EGFR phosphorylation was increased in A549 cells, lentivirus mediated ADAM17 RNAi could significantly inhibit the expression. Thus, in the present study, we will explore the regulatory mechanism of lncRNA HOTAIR mediated Wnt/β-catenin and EGFR signal pathway in lung adenocarcinoma invasivsion in vitro and in vivo and whether lentivirus mediated ADAM17 RNAi can reverse the secondary resistance of lung adenocarcinoma to EGFR-TKI, which could provide a new strategy to solve the secondary drug resistance of lung adenocarcinoma to EGFR-TKI.

EGFR酪氨酸激酶抑制剂（EGFR-TKIs）继发性耐药是制约肺腺癌EGFR-TKIs靶向治疗的瓶颈。ADAM17在EGFR配体胞外域脱落中起着重要作用，但目前仍缺乏ADAM17在肺腺癌靶向治疗中的作用研究。Wnt信号与EGFR信号通路存在交汇对话,共同参与调控肿瘤侵袭过程。HOTAIR是第一个被发现具有反式转录调控作用的长非编码RNA（lncRNA),可通过Wnt/β-Catenin信号通路调控食管癌进展。但其参与调控肺腺癌侵袭的作用和机制仍有待于研究。我们已发现A549细胞中EGFR磷酸化水平升高，慢病毒介导的ADAM17 RNAi可抑制其表达。为此，本项目将研究lncRNA HOTAIR介导的Wnt/β-catenin与EGFR信号通路对话调控肺腺癌侵袭过程的作用机制及慢病毒介导的ADAM17 RNAi能否逆转肺腺癌EGFR-TKIs耐药，为治疗肺腺癌EGFR-TKI耐药提供新靶点。

肺癌是全球发病率及病死率最高的恶性肿瘤。EGFR酪氨酸激酶抑制剂（EGFR-TKIs）在肺腺癌EGFR 19外显子缺失或21外显子L858R 突变患者取得显著疗效，但是随后对EGFR-TKI继发性耐药不可避免。EGFR-TKIs继发性耐药制约肺癌靶向治疗的瓶颈。因此，寻找能够逆转肺腺癌对EGFR-TKIs继发耐药的新方法至关重要。本项目首先利用吉非替尼浓度递增法诱导肺癌PC-9细胞，通过体外细胞实验筛选吉非替尼继发性耐药的肺腺癌细胞RPC-9。并构建长非编码RNA HOTAIR 和ADAM17的慢病毒干扰载体、包装重组慢病毒，通过下调RPC-9细胞中的HOTAIR 和ADAM17，体内外实验分析对吉非替尼耐药逆转的影响及其相应的分子机制。体内外实验研究表明：慢病毒介导肺腺癌RPC-9的HOTAIR RNAi可下调HOTAIR表达，逆转RPC-9对吉非替尼的继发性耐药，并能够促进吉非替尼对其杀伤作用。其主要表现在：G1期细胞阻滞、细胞凋亡和抑制皮下成瘤能力。进一步机制研究表明，可能与其能够抑制RPC-9细胞中EGFR通路重新活化有关，而对WNT通路未见相关性。同样慢病毒介导肺腺癌RPC-9的ADAM17 RNAi可下调ADAM17的表达,也能够显著抑制RPC-9细胞的增殖及实验裸鼠皮下移植的成瘤能力。通过本项目研究，在体外成功诱导筛选了一株耐吉非替尼的肺癌RPC-9细胞株，并在此基础上，并发现慢病毒介导的HOTAIR和ADAM17 RNA干扰能逆转RPC-9的耐药，促进吉非替尼的抗肿瘤作用。本研究结果为肺腺癌EGFR-TKIs 的继发性耐药提供潜在的治疗靶点。