miR-20a/miR-106b-STAT3轴通过调控肺癌细胞自噬介导克唑替尼耐药的作用及其机制研究

Crizotinib has greatly improved the survival of non-small cell lung cancer patients harboring EML4-ALK fusion protein or MET amplifications. However, acquired resisitance of the initial therapy limits its application and the survival benefit of these patients. Our previous study showed that Crizotinib treatment mediated the downregulation of cytoplasmic total STAT3 expression, which in turn activated the pro-autophagic EIF2A pathway, and lead to drug-resistance. Employing microRNA microarrays, we discovered miR-20a and miR-106b was upregulated after Crizotinib treatment. Bioinformatic analysis revealed that STAT3 indeed was the target of miR-20a and miR-106b. Further dual-luciferase reporter assay system confirmed the binding of miR-20a and miR-106b to the 3' UTR of STAT3. Meanwhile miR-20a and miR-106b could induce autophagy on their own account. In light of the previous findings, we proposed the upregulation of miR-20a and miR-106b promoted autophagy by inhibiting cytoplasmic STAT3, ultimately lead to Crizotinib drug-resistance. Thus we plan to use luciferase reporter gene plasmids containing the promoter regions of miR-20a and miR-106b, combining with bioinformatic analysis, to determine the upstream regulators of miR-20a and miR-106b, and confirm the interaction using siRNA interference and ChIP analysis. miRNA antagomirs will be used to prove the downregulation of miR-20a and miR-106b could re-sensitize lung cancer cells to crizotinib treatment in vitro and in vivo. Meanwhile, we will collect clinical samples before Crizotinib treatment and after its resistance, to confirm the clinical significance between miR-20a/miR-106b-STAT3-autophagy axis and Crizotinib resistance. Our study might provide new targets to improve the clinical efficacy of Crizotinib therapy.

克唑替尼显著改善了目标肺癌患者的生存，但耐药问题限制了其疗效。我们的前期研究发现克唑替尼通过下调胞质中总STAT3表达继而上调EIF2A诱导自噬产生耐药。miRNA芯片分析显示克唑替尼诱导了miR-20a和miR-106b的显著上调，我们筛选并用双荧光素酶报告基因系统验证了miR-20a及miR-106b可以靶向STAT3，同时也可以诱导自噬发生。提示miR-20a/miR-106b-STAT3-自噬轴参与了克唑替尼的耐药。本研究拟在此基础上寻找并验证克唑替尼导致miR-20a及miR-106b升高的上游因子，在体内外实验中利用miR-20a及miR-106b的抑制剂证明下调其表达能够抑制STAT3介导的自噬，从而逆转肺癌细胞对克唑替尼的耐药。并收集临床样本，验证miR-20a/miR-106b-STAT3-自噬轴在克唑替尼耐药中的临床意义。本研究将为改善克唑替尼的临床有效性提供新的靶点。

克唑替尼的耐药问题限制了其疗效及患者的预后，我们发现克唑替尼通过在靶基因肺癌细胞中下调总STAT3并激活EIF2A通路诱导肺癌细胞自噬导致耐药，我们采用microRNA微阵列芯片检测克唑替尼处理EML4-ALK阳性和MET扩增的肺癌细胞前后的差异性miRNA表达，筛选发现miR-20和miR-106显著升高，并通过Dual-luciferase reporter assay发现，miR-20a和miR-106b能够靶向STAT3的3’UTR。用miR-20a mimic和miR-106b mimic转染SPC-A1、H2228、HCC827肺癌细胞，qPCR结果显示转染后STAT3水平显著下降，Western blot结果同样显示总STAT3蛋白水平明显下降。GFP-LC3质粒转染肺癌细胞，Confocal 观察miR-20a/106b mimic处理后LC3表达明显升高。使用miR-20a/106b mimic处理SPC-A1 , H2228等肺癌细胞后，电镜下可见自噬体形成，以miR-20a inhibitor和miR-106b inhibitor转染SPC-A1，H2228肺癌细胞后能够明显增敏克唑替尼，抑制肺癌细胞的增殖并增加凋亡。将SPC-A1细胞接种于免疫缺陷小鼠腋下后分别以对照组、Crizotinib组、miR-20 inhibitor组、miR-106 inhibitor组、miR-20 inhibitor联合Crizotinib组及miR-106 inhibitor联合Crizotinib组处理小鼠，IHC方法检测总STAT3表达和LC3表达，发现相对于Crizotinib处理组，联合应用miR-106 inhibitor和miR-20 inhibitor后总STAT3表达及LC3表达降低，自噬水平下降，相对于Crizotinib处理组，联用miR-106 inhibitor后可见肿瘤明显缩小。体现了miR-106 inhibitor在体内对克唑替尼的增敏作用。