Interferon alpha induced gene expression in SOCS1 and SOCS3 overexpressing melanoma and hepatoma cell lines

Interferon-alpha (pegylated interferon and ribavirin) is used as standard-of-care therapeutic for chronic hepatitis C virus infection. Besides good cure in some patients other patients do not benefit from the treatment dependent on the virus type and host factors. One class of putative effector proteins is the family of Suppressors of cytokine signalling (SOCS). They act in a classical negative feedback-loop against the action of interferons and many other cytokines. It has been proven that some of them, in particular SOCS1 and SOCS3, inhibit the expression of interferon induced antiviral proteins. Their mode of action depends on the signal they are interfering with. In relation to the interferon-gamma pathway, they are thought to act on the interferon-alpha receptors by masking its recognition site for the Janus kinases (JAK), by blocking the kinase activity of the JAKs and coincidentally hindering STAT molecules from binding to the kinases. They are also thought to ubiquitinate the JAKs resulting in their proteosomal degradation. The function of SOCS proteins in suppressing the interferon-alpha pathway has not yet been characterized exhaustively. This study should unveil links to understand the resistance in interferon-alpha therapy. As results we got almost complete silencing of JAK-STAT signaling in SOCS1 over-expressing cells and tissue-dependent partially suppressed gene induction in SOCS3 over-expressing cell lines. Two human cancer cell lines (ME-15, HuH-7) were stably transfected with pcDNA3.1-SOCS plasmids in presence of geneticin and daughter cell lines were generated after singularization of cells. Next, original cell lines as well as SOCS1 and SOCS3 over-expressing cell lines were treated with 1000 U/ml interferon-alpha for 4 or 24 hours or in normal culture medium. Cells lines obtained from SOCS4 plasmid transfections were screened as additional control. Gene expression levels of cell cultured in control (0 for 4 hours, 2 for 24 hours) or interferon-alpha supplemented medium for 4 hours (1) or 24 hours (4) were analyzed. mRNA abundance was measured in triplicates using 12x8-sample commercial Illumina microarrays (HumanRef 8, version 3) and scanner system (iScan) as well as reagents recommend by Illumina (Illumina® TotalPrep Kit).

干扰素-α（Interferon-alpha，聚乙二醇干扰素联合利巴韦林）是慢性丙型肝炎病毒感染的标准治疗方案。尽管部分患者可获得良好的治愈效果，但另有部分患者无法从该治疗中获益，这一情况取决于病毒型别与宿主因素。细胞因子信号抑制因子（Suppressor of Cytokine Signaling, SOCS）家族是一类潜在的效应蛋白家族，它们通过经典的负反馈环路拮抗干扰素及多种其他细胞因子的生物学作用。已有研究证实，其中部分成员（尤其是SOCS1与SOCS3）可抑制干扰素诱导的抗病毒蛋白的表达，其作用模式取决于其所干扰的信号通路。针对干扰素-γ通路，现有研究认为此类蛋白可作用于干扰素-α受体：一是遮蔽其与贾纳斯激酶（Janus Kinases, JAK）的识别位点，二是阻断JAK的激酶活性，同时妨碍信号转导与转录激活因子（STAT）分子与JAK的结合；此外，这类蛋白还可对JAK进行泛素化修饰，使其经蛋白酶体途径降解。目前，SOCS蛋白在拮抗干扰素-α通路中的具体功能尚未被全面阐明。本研究旨在揭示相关机制，以阐明干扰素-α治疗的耐药性产生原因。研究结果显示：在过表达SOCS1的细胞中，JAK-STAT信号通路几乎完全被沉默；而在过表达SOCS3的细胞系中，基因诱导效应则呈现组织依赖性的部分抑制。本研究将pcDNA3.1-SOCS质粒稳定转染至两种人源癌细胞系（ME-15、HuH-7）中，通过遗传霉素（Geneticin）筛选并经单细胞克隆分离后获得子代细胞系。随后，将原始细胞系以及过表达SOCS1、SOCS3的细胞系分别置于含1000 U/ml干扰素-α的培养基中培养4小时或24小时，同时设置正常培养基培养的对照组；通过转染SOCS4质粒获得的细胞系被用作额外对照。本研究对不同处理条件下的细胞基因表达水平进行了分析：对照组细胞分别在正常培养基中培养4小时（标记为0）、24小时（标记为2）；干扰素-α处理组细胞则分别在含干扰素-α的培养基中培养4小时（标记为1）或24小时（标记为4）。采用12×8样本规格的商用Illumina微阵列芯片（HumanRef 8, 第3版）、iScan扫描系统，以及Illumina推荐的Illumina® TotalPrep试剂盒，对三组重复的mRNA丰度进行检测。