Background and objective: It has been reported that defective expression of TGFBR3 was found in non-small cell lung cancer (NSCLC). However, its molecular mechanisms remain unclear. The aim of this study is to investigate expression of TGFBR3 in NSCLC cell lines and normal human bronchial epithelial cell (HBEpiC), and to explore potential molecular mechanisms underlying inactivation of TGFBR3 gene.
Methods: Western blot was performed to determine the expression of TGFBR3 in HBEpiC and NSCLC cell lines. Automatic image analysis was carried out to estimate relative expression of TGFBR3 protein. We screened for mutation of the promoter region of TGFBR3 gene using DNA direct sequencing. Bisulfite-sodium modification sequencing was used to detect the methylation status of TGFBR3 promoter.
Results: TGFBR3 protein level was abnormally reduced in NSCLC cell lines as compared with HBEpiC. There was significant difference in TGFBR3 expression between the highly metastatic cell line 95D and non-metastatic cell lines, including LTEP-alpha-2, A549 and NCI-H460. No mutation and methylation was found in upstream sites -165 to -75 of the proximal promoter of TGFBR3 in HBEpiC and NSCLC cell lines. Hypermethylation was shown in upstream sites -314 to -199 of the distal promoter of TGFBR3 in HBEpiC and NSCLC cell lines.
Conclusion: Reduced expression of TGFBR3 was observed in NSCLC cell lines, especially in 95D, suggesting that TGFBR3 might play an important role in development and progression of NSCLC and correlate with NSCLC invasion and migration. The methylation event occurring at TGFBR3 promoter is not a major cause for reduction of TGFBR3 expression.
背景与目的: 国外有研究表明，非小细胞肺癌（non-small cell lung cancer, NSCLC）中转化生长因子受体Ⅲ（TGFBR3）存在缺陷表达，但是分子机制尚未明确。本研究以正常支气管上皮细胞（human bronchial epithelial cell, HBEpiC）为对照，分析NSCLC细胞株中TGFBR3基因的表达情况，并探讨TGFBR3基因表达失活的分子机制。
方法: 采用Western blot检测HBEpiC和NSCLC细胞株中TGFBR3的表达情况并做相对定量分析；采用DNA直接测序检测TGFBR3基因启动子基本转录元件区的突变情况；应用亚硫酸氢钠处理后测序法（bisulfite sequence-PCR, BSP）检测TGFBR3基因启动子区甲基化状态。
结果: NSCLC细胞株中TGFBR3表达水平明显低于HBEpiC；高转移细胞株95D明显低于非转移细胞株LTEP-α-2、A549、NCI-H460；HBEpiC与NSCLC细胞株中TGFBR3基因近端启动子区-165到-75区域无遗传突变，且未见甲基化，远端启动子区-314到-199区域均为高甲基化。
结论: TGFBR3基因在NSCLC细胞株中表达下调，在高转移细胞株95D中尤其明显，提示该基因的表达缺陷对NSCLC发生发展起重要作用，可能与NSCLC的侵袭和转移相关；然而，TGFBR3基因启动子区重要转录元件区域的甲基化状态并不是导致TGFBR3基因表达下调的主要原因。