Miyamoto Shingo, Nagamura Yuko, Nakabo Ayaka, Okabe Akira, Yanagihara Kazuyoshi, Fukami Kiyoko, Sakai Ryuichi, Yamaguchi Hideki
Department of Cancer Cell Research, Sasaki Institute, Sasaki Foundation, 2-2 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Department of Cancer Cell Research, Sasaki Institute, Sasaki Foundation, 2-2 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan; Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan.
Biochem Biophys Res Commun. 2018 Jan 8;495(2):1942-1947. doi: 10.1016/j.bbrc.2017.12.067. Epub 2017 Dec 13.
RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.
RhoA是Rho家族小GTP酶的成员，可调节多种细胞功能。最近的大规模测序研究已在弥漫型胃癌（DGC）中鉴定出RHOA的复发性体细胞突变，表明RHOA是DGC的驱动因子。在本研究中，我们调查了一组胃癌（GC）细胞系中RHOA可能存在的异常情况。下拉分析和免疫印迹分析表明，除了两个DGC细胞系HSC-59和GSU外，在所有测试的GC细胞系中均能检测到RhoA的活性和表达。RHOA编码区测序显示这些细胞系中发生了RHOA异常可变剪接。定量实时PCR分析表明，野生型RHOA的表达几乎无法检测到，而剪接变体几乎只在HSC-59和GSU细胞系中表达。然而，RHOA剪接变体的表达水平非常低，免疫印迹未检测到相应蛋白。此外，即使在细胞中异位表达，RhoA蛋白的剪接异构体也不能有效表达或激活。这些结果表明，RHOA的异常可变剪接导致其在DGC细胞中活性和表达丧失。
