Functional Analysis of Tumor Suppressor ING Family in Oral Cancer

1. 口腔癌是通过多种抑癌基因失活和致癌基因激活而发生的。在这项研究中，我们检测了ING家族成员基因ING1b、ING1c和ING3的功能，这些基因是我们之前发现的。剪接变体ING1b和ING1c的mRNA表达分析显示，与匹配的正常样本相比，肿瘤样本中ING1b和ING1c的mRNA表达减少了30%和58%。另一方面，50%的癌症样本中ING3 mRNA表达下调。基于限制性内切酶的甲基化分析显示，ING1b变体有51%的甲基化，而在ING1c中未检测到甲基化。这些结果表明，不同的剪接变体和成员在口腔癌中具有不同的作用和机制。构建标记ING1b、ING1c和ING3的表达载体进行体外功能分析。分别将这些表达载体单独或与p53表达载体联合转染到HEK293、SCCKN、HSC3、HSC3细胞系中，进行细胞增殖和凋亡实验。这些实验表明，INGlc-p53联合使用具有更好的细胞抑制作用。各基因及与p53的联合也导致细胞凋亡的改变。为了检测ING家族及促凋亡和抗凋亡基因对细胞毒性药物的反应，我们用5-FU、顺铂和依托泊苷处理细胞，并通过定量RT-PCR检测ING1lb、ING1c、ING3、Bax和Bc1-2 mRNA的表达。这些实验表明，每种细胞系对不同药物的反应不同，表明每种变体和成员的信号传导具有不同的机制。我们还检测了70多个癌症样本中ING3 mRNA的表达，并与临床病理变量进行了比较。本研究证实ING3 mRNA值可能是预后指标。ING3 mRNA表达正常的患者存活率为60%，而ING3 mRNA表达下调的患者存活率为40%，预后较差。未来的研究将包括siRNA对每个ING家族成员的抑制工作和动物实验结果的检验。识别ING家族的所有网络将导致包括口腔癌在内的各种癌症类型的新分子治疗的发展。少

1. Oral cancer occurs through inactivation of multiple tumor suppressor genes and activation of oncogenes. In this research, we examined the functions of the ING family members genes ING1b, ING1c and ING3, which we previously identified. mRNA expression analysis of splicing variants ING1b and ING1c showed 30% and 58% decrease in tumor samples as compared with the matched normal counterparts. On the other hand, ING3 mRNA was down-regulated in 50% of cancer samples. Restriction endonuclease-based methylation analysis demonstrated 51% methylation of ING1b variant, while it was not detected in ING1c. These results suggested that different splicing variants and members have various role and mechanisms in oral cancer.2. Flag-tagged expression vectors of ING1b, ING1c and ING3 were constructed for in vitro functional analysis. Each of these vectors alone and combination with p53 expression vectors were transfected into the cell lines of HEK293, SCCKN, HSC3, HSC3 and cell proliferation and apop … More tosis assay were performed. These assays showed that INGlc-p53 combination gave a better cell inhibitory effect. Each gene and combination with p53 also resulted in apoptotic changes.3. To examine the response of the ING family and proapoptotic as well as antiapoptotic genes to the cytotoxic drugs, we treated the cells with 5-FU, Cisplatin and Etoposide and mRNA expressions of ING1lb, ING1c, ING3, Bax and Bc1-2 were checked by quantitative RT-PCR. These experiments showed different reactions with various drugs in each cell line, suggesting that signaling of each variant and member has different mechanism.4. We also examined ING3 mRNA expression in over 70 cancer samples and compared with clinicopathological variables. This study demonstrated ING3 mRNA value as a possible prognostic marker. The samples with ING3 down-regulation had bad prognosis with a 40% survival rate as compared to 60% survival in normal ING3 mRNA expression. Future studies will include suppression work of each ING family member by siRNA and examination of the results in animal experiments. Identification of all networks of ING family will lead to development of novel molecular therapies In various cancer types including oral cancer. Less