Prediction of in vitro response to interferon-alpha and acquisition of its susceptibility by gene and peptide transduction system

研究肾细胞癌（RCC）中干扰素-α (IFN-a)敏感性相关基因及其对IFN-α敏感性的调控具有重要意义。首先，我们在RCC细胞系中确定了与ifn -a反应相关的7个候选基因，然后利用ADFP、MITF、MTUS1和TNNT1 4个基因建立了预测公式。该预测模型在其他RCC细胞系中进行了验证，包括来自RCC患者的原代培养细胞。接下来，我们聚焦与IFN-α-敏感性相关的MITF基因，通过基因转染分析其表达与IFN-α-反应状态的关系。与野生型或模拟转染相比，MITF转染在IFN-α-抗性品系中表现出易感性。由于MITF是一个可以结合p16和HIF-1α的转录因子，提示MITF可能与RCC的发生和进展有关，加速细胞周期。因此，我们通过Wr-T转运体肽的肽转导系统来评估p16的表达和生物活性p16肽（p16- mis）的抗肿瘤作用。所有使用的RCC细胞系都缺乏p16蛋白，即使在IFN-α-抗性RCC mitf转染中也是如此。P16-MIS以浓度依赖的方式成功转移到RCC细胞的细胞质和细胞核中。有趣的是，在IFN-α耐药的RCC细胞系中，IFN-α-敏感性达到2.5 ~ 100倍。上述结果提示IFN-α-应答相关基因MITF及其相关细胞周期调节因子p16可能是RCC治疗策略的分子靶点。

It is important to investigate the interferon-alpha (IFN-a) sensitivity-related gene and regulation of susceptibility to IFN-α in renal cell carcinoma (RCC). First, we have identified seven candidate genes, which are associated with IFN-a-response and then established prediction formula using four genes, I.e. ADFP, MITF, MTUS1, and TNNT1, in RCC cell lines. Validation of this prediction model was performed in other RCC cell lines, including primary culture cells from patients with RCC. We next focused MITF gene, which individually correlated with IFN-α-sensitivity, to analyze relationship between its expression and IFN-α-response status by gene transfection. MITF transfectant demonstrated susceptibility in IFN-α-resistant line as compared with wild type or mock transfectant. Because MITF is one off transcription factor, which can bind to p16 and HIF-1α, it is suggested that MITF may be associated with development and progression of RCC acceleration of cell cycle. Thus, we evaluated expression of p16 and anti-tumor effect of biological active p16 peptide (p16-MIS) by peptide transduction system with Wr-T transporter peptide. All RCC cell lines used are absent for p16 protein, even in the IFN-α-resistant RCC MITF-transfectant. P16-MIS successfully transferred into cellular cytoplasm and nucleus of RCC cells by concentration dependent manner. Interestingly, IFN-α-susceptibility was acquired at 2.5 to 100 times intensity in IFN-α-resistant RCC cell line. These results suggested that IFN-α-response related gene, MITF and its related cell cycle regulator, p16 could be molecular targets in therapeutic strategy of RCC.