Background and objective: TIF1γ (transcription intermediary factor 1 gamma), which belongs to transcription intermediary factor 1 family, is an inhibitor of the TGF-β/Smad signaling pathway and could inhibit the signal transduction mediated by TGF-β. The deficiency of TIF1γ expression in a variety of tumor cells suggests that TIF1γ may play as a tumor suppressor gene in cancer development. The aim of this study is to confirm the relationship between TIF1γ and non-small cell lung cancer (NSCLC) through exploring the expression of TIF1γ in NSCLC cells and tissues, and investigate the regulation mechanism of TIF1γ expression in NSCLC cells.
Methods: Thirteen NSCLC and the paired corresponding para-cancerous lung tissue samples and three cell lines (a normal bronchial epithelial cell line HBE and two NSCLC cell lines A549 and 95C) were selected. The quantitative real-time PCR and Western blotting were used to determine the expression of TIF1γ, and ImageJ was used to evaluate the relative expression of TIF1γ. Direct sequencing was performed to detect mutations within the promoter region of the TIF1γ gene and then Bisulfite-sequencing PCR (BSP) and cloning sequencing were carried out to test the methylation status of the promoter region of TIF1γ gene in the selected cell lines.
Results: Both mRNA and protein expression of TIF1γ were found significantly decreased in A549 and 95C compared with those in HBE (P<0.05). And in 9 pairs (69.2%) of tissues among the 13 pairs, the mRNA expression of TIF1γ gene was lower in the cancer tissues than that in the paired paracancerous lung tissues (P<0.05). No abnormal mutation was found in the -287 to -5 region of the promoter of TIF1γ gene in the three cell lines. Moreover, five CpG sites (including -214 bp,-128 bp,-124 bp, -65 bp and -55 bp) were detected in the promoter of TIF1γ gene by using BSP, and the methylation profiles in these CpG sites showed similar pattern between NSCLC cells and HBE cells.
Conclusions: TIF1γ may play a tumor suppressor role in the progression of NSCLC. No mutation was found in the -287- -5 region of the promoter of TIF1γ gene in a normal bronchial epithelial cell line and two NSCLC cell lines. But there are five CpG sites which can be methylated located in this region.
背景与目的 TIF1γ（transcription intermediary factor 1 gamma）属于转录中介因子1家族的成员，可干扰TGF-β/Smad信号通路，抑制TGF-β介导的信号传导，且TIF1γ在多种肿瘤细胞的表达减弱或缺失表明TIF1γ在癌症的发生发展过程中起到抑癌的作用。本研究旨在探索TIF1γ在非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞与组织中的表达差异以确定TIF1γ与肺癌发生关系以及肺癌细胞中TIF1γ表达调控的潜在机制。方法 选取13例NSCLC患者癌组织以及对应的癌旁组织样本，培养正常支气管上皮细胞HBE和NSCLC细胞株：A549和95C。运用Real-time PCR和Western blot检测细胞与组织中TIF1γ基因的表达，并用ImageJ灰度扫描软件计算TIF1γ的相对表达量。通过DNA测序的方法检测TIF1γ基因启动子区域突变情况，并采用Bisulfite-sequencing PCR（BSP）克隆测序方法检测TIF1γ基因启动子区域甲基化情况。结果 相对于HBE，TIF1γ mRNA和蛋白在A549和95C中明显下调（P<0.05），13对组织样本中，9个（69.2%）癌旁组织样本里TIF1γ mRNA的表达量高于癌组织样本（P<0.05）。突变检测表明TIF1γ基因启动子区-287﹣-5在细胞株中没有突变发生。经BSP克隆测序方法分析发现在TIF1γ基因启动子-287﹣-5区域里存在5个可被甲基化的CpG位点（-214、-128、-124、-65和-55）。但这些CpG位点的甲基化频率在NSCLC细胞株中相比HBE没有明显差异。结论 TIF1γ在NSCLC的发生中可能起抑癌的作用，TIF1γ基因启动子区域-287﹣-5在正常细胞和NSCLC细胞中都没有发生突变，但在-287﹣-5区域中存在5个可被甲基化的CpG位点。