Association of Mutation and Methylation in the Promoter Region of TIF1γ 
with Non-small Cell Lung Cancer

Abstract

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. Conclusion 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.

Keywords

• Lung neoplasms
• TIF1γ
• Methylation
• Bisulfite-sequencing PCR (BSP)