Background: The EML4-ALK fusion gene is a newly discovered driver gene of non-small cell lung cancer and exhibits special clinical and pathological features. The JAK-STAT signaling pathway, an important downstream signaling pathway of EML4-ALK, is aberrantly sustained and activated in EML4-ALK-positive lung cancer cells fusion gene, but the underlying reason remains unknown. The suppressor of cytokine signaling (SOCS) is a negative regulatory factor that mainly inhibits the proliferation, differentiation, and induction of apoptotic cells by inhibiting the JAK-STAT signaling pathway. The aberrant methylation of the SOCS gene leads to inactivation of tumors and abnormal activation of the JAK2-STAT signaling pathway. The aim of this study is to investigate the methylation status of the SOCS3 promoter in EML4-ALK-positive H2228 cells and lung cancer tissues.
Methods: The methylation status of the SOCS3 promoter in EML4-ALK-positive H2228 lung cancer cells and lung cancer tissues was detected by methylation-specific PCR (MSP) analysis and verified by DNA sequencing. The expression levels of SOCS3 in H2228 cells were detected by Western blot and Real-time PCR analyses after treatment with the DNA methyltransferase inhibitor 5'-Aza-dC.
Results: MSP and DNA sequencing assay results indicated the presence of SOCS3 promoter methylation in H2228 cells as well as in three cases of seven EML4-ALK-positive lung cancer tissues. The expression level of SOCS3 significantly increased in H2228 cells after 5'-Aza-dC treatment.
Conclusions: The aerrant methylation of the SOCS3 promoter region in EML4-ALK (+) H2228 cells and lung cancer tissues may be significantly involved in the pathogenesis of EML4-ALK-positive lung cancer.
背景与目的 人类棘皮动物微管相关蛋白样4（echinoderm microtubule-associated-protein-like 4, EML4）和人类间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）融合基因EML4-ALK是新发现的非小细胞肺癌的驱动基因，患者具有独特的临床病理生理特征，ALK基因下游信号通路的异常持续激活是其重要的下游信号通路，而导致其下游基因持续激活的原因不明。细胞因子信号转导抑制因子（suppressor of cytokine signaling, SOCS）是一类在细胞信号转导过程中起重要作用的负调控因子，主要通过抑制JAK蛋白酪氨酸激酶（janus protein tyrosine kinase, JAK）信号传导和转录激活因子（signal transducer and activator of transcription, STAT）即JAK-STAT等信号通路来调控细胞的增殖、分化和凋亡。肿瘤中常存在SOCS基因的甲基化异常导致的失活，从而导致JAK2-STAT等信号通路持续异常活化。本研究的目的在于探讨EML4-ALK阳性H2228细胞和肺癌组织中SOCS3启动子区甲基化状态。方法 甲基化特异性PCR检测EML4-ALK阳性H2228肺癌细胞及肺癌组织中SOCS3启动子区的甲基化状态，并通过测序验证。DNA甲基转移酶抑制剂5’-Aza-dC处理H2228细胞，并通过Real-time PCR和Western blot检测SOCS3的表达水平变化。结果 MSP及测序分析发现EML4-ALK阳性细胞株H2228中存在SOCS3启动子区甲基化；7例EML4-ALK阳性肺癌组织中的3例存在SOCS3启动子区甲基化，H2228细胞经过5’-Aza-dC去甲基化处理后SOCS3的表达明显增加。结论 EML4-ALK（+）的H2228肺癌细胞及部分肺癌组织中存在SOCS3启动子区的异常甲基化，可能是EML4-ALK阳性肺癌的重要发病机制。.