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. 2016 Sep 20;19(9):565-70.
doi: 10.3779/j.issn.1009-3419.2016.09.01.

[Methylation Status of the SOCS3 Gene Promoter in H2228 Cells and EML4-ALK-positive Lung Cancer Tissues]

[Article in Chinese]
Chunlai Liu  1 , Yongwen Li  2 , Yunlong Dong  1 , Hongbing Zhang  1 , Ying Li  2 , Hongyu Liu  2 , Jun Chen  3
Affiliations

[Methylation Status of the SOCS3 Gene Promoter in H2228 Cells and EML4-ALK-positive Lung Cancer Tissues]

[Article in Chinese]
Chunlai Liu et al. Zhongguo Fei Ai Za Zhi. .

Abstract
in English, Chinese

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。.

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Figures

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MSP方法检测肺癌细胞株及EML4-ALK阳性肺癌样品中SOCS3基因启动子甲基化状态。M：甲基化特异；U：非甲基化特异 MSP assay for SOCS3 promoter in different lung cancer cell lines and EML4-ALK (+) lung cancer tissues. M:methylated-specific; U: unmethylated-specific.
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H2228细胞SOCS3启动子MSP分析后测序验证 The MSP result of SOCS3 promotor were verified by DNA sequencing in H2228 cells
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Real-time PCR检测5'-Aza-dC处理不同肺癌细胞后，SOCS3表达水平的变化情况。*P < 0.05 The analysis of SOCS3 expression by Real-time PCR in human lung cancer cell lines after 5'-Aza-dC treatment. *P < 0.05
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Western blot检测肺癌细胞株经5'-Aza-dC处理后SOCS3表达水平的变化 The analysis of SOCS3 expression by Western blot in human lung cancer cell lines after 5'-Aza-dC treatment
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