LncRNA MALAT1通过下调p53促进非小细胞肺癌吉非替尼耐药的机制研究

The clinical effect of gefitinib on patients with EGFR mutational advanced non-small cell lung cancer (NSCLC) was remarkable, but was inevitably impaired by progressive drug resistance. Currently, the specific mechanism of gefitinib resistance in NSCLC patients remained unexplained. Prior studies uncovered the high expression state of MALAT1 in gefitinib resistant NSCLC cells， and such resistance tended to fade as expression of MALAT1 decreased. Furthermore, we found that tumor suppressor gene p53 may be a key target gene of MALAT1 through TCGA database and KEGG pathway enrichment analysis. It has been reported that the p53 pathway plays an important role in gefitinib resistance in NSCLC cells. The project sets out to verify the specific functional properties of MALAT1 in gefitinib resistance on levels of cellular, animal study and clinical samples, and further explore the possibility that MALAT1 contributes to gefitinib resistance depends on p53 through “rescue” experiments. Finally, the detailed mechanisms will be investigated through RNA pull down assay, protein mass spectrometry and RNA binding protein immunoprecipitation (RIP), promising a new theoretical basis for gefitinib resistant NSCLC patients’ treatment.

吉非替尼用于治疗EGFR基因突变的进展期非小细胞肺癌（NSCLC）取得了惊人的效果，但不可避免的耐药严重影响了其疗效。而吉非替尼耐药的机制尚不完全清楚。我们前期研究发现：在吉非替尼耐药的NSCLC细胞中，MALAT1显著高表达，低表达MALAT1能够恢复细胞对吉非替尼的敏感性；利用TCGA数据库资源，结合KEGG通路富集分析，我们发现了MALAT1下游的关键靶基因之一p53，而后者在NSCLC吉非替尼耐药中发挥着重要的作用。基于此，本项目拟从临床样本，结合细胞实验和动物实验三个层面验证MALAT1在NSCLC吉非替尼耐药中的作用；进一步，通过“抑制-恢复”实验证实MALAT1调控NSCLC吉非替尼耐药是否依赖于p53；最后，通过RNA pull down、蛋白质谱分析、RIP等实验技术探索MALAT1调控p53的具体机制，为NSCLC吉非替尼耐药患者的治疗提供新的理论依据。

吉非替尼用于治疗EGFR基因突变的进展期非小细胞肺癌（NSCLC）取得了惊人的效果，但不可避免的耐药严重影响了其疗效。通过本项目的实施，我们发现了调控NSCLC吉非替尼耐药的新机制。通过体内及体外实验，我们发现MALAT1在NSCLC吉非替尼耐药中发挥着重要的作用。过表达MALAT1能够诱导NSCLC对吉非替尼耐药，而低表达MALAT1能够恢复耐药细胞对吉非替尼敏感性。进一步，我们发现了MALAT1下游重要靶基因p53也参与了NSCLC吉非替尼耐药。低表达p53能够诱导NSCLC吉非替尼耐药，而过表达p53能够恢复耐药细胞对吉非替尼的敏感性。同时，我们证明了MALAT1调控p53的分子生物学机制。我们发现：过表达MALAT1能够明显抑制NSCLC中p53的表达，而低表达MALAT1能够促进p53的表达；进一步，我们发现MALAT1能够通过结合EZH2蛋白，p53启动子区调控H3K36的甲基化，抑制p53启动子活性，从而调控p53蛋白水平及mRNA水平的表达。最后，我们在体内及体外实验中证实了MALAT1调控NSCLC细胞吉非替尼耐药依赖于p53介导的信号通路。项目的实施，阐明了MALAT1调控NSCLC吉非替尼耐药的具体机制，可能为NSCLC吉非替尼耐药患者的治疗提供新的理论依据。