Hou Xiaobin, Li Tinghui, Ren Zhipeng, Liu Yang
Department of Thoracic Surgery, Chinese PLA General Hospital, Beijing, China.
Oncol Res. 2016;24(4):247-53. doi: 10.3727/096504016X14652175055765.
Mutation of breast cancer 2, early onset (BRCA2) has been identified as a vital risk factor for esophageal cancer (EC). To date, several proteins have been reported as BRCA2-interacting proteins and are associated with multiple biological processes. This study's aim was to identify a novel interactive protein of BRCA2 and to explore its functional roles in EC. A yeast two-hybrid screening was performed to identify a novel BRCA2-interacting protein. Glutathione-S-transferase (GST) pull-down analysis was performed to find out how the binding domain of BRCA2 interacts with LIM domains containing 1 (LIMD1). The interaction between LIMD1 and BRCA2 at the endogenous level was confirmed by using coimmunoprecipitation and immunobloting. Furthermore, two different sequences of short hairpin RNAs (shRNAs) against LIMD1 were transfected into the human EC cell line ECA109. Afterward, the effects of LIMD1 suppression on the centrosome localization of BRCA2 and cell division were analyzed using an immunofluorescence microscope. Results showed that LIMD1 was a novel BRCA2-interacting protein, and LIMD1 interacted with the conserved region of BRCA2 (amino acids 2,750-3,094) in vitro. Importantly, after interfering with the protein expression of LIMD1 in ECA109 cells, the centrosome localization of BRCA2 was significantly abolished and abnormal cell division was significantly increased. These results suggested that LIMD1 is a novel BRCA2-interacting protein and is involved in the centrosome localization of BRCA2 and suppression of LIMD1, causing abnormal cell division in EC cells.
乳腺癌2号基因（BRCA2）早期突变已被确定为食管癌（EC）的一个重要风险因素。迄今为止，已有几种蛋白质被报道为与BRCA2相互作用的蛋白质，并与多种生物学过程相关。本研究的目的是鉴定一种新的BRCA2相互作用蛋白，并探讨其在食管癌中的功能作用。通过酵母双杂交筛选来鉴定一种新的BRCA2相互作用蛋白。进行谷胱甘肽 - S - 转移酶（GST）下拉分析，以了解BRCA2的结合结构域如何与含1个LIM结构域的蛋白（LIMD1）相互作用。通过免疫共沉淀和免疫印迹在体内水平证实了LIMD1与BRCA2之间的相互作用。此外，将针对LIMD1的两种不同序列的短发夹RNA（shRNA）转染到人食管癌细胞系ECA109中。随后，使用免疫荧光显微镜分析LIMD1抑制对BRCA2中心体定位和细胞分裂的影响。结果表明，LIMD1是一种新的BRCA2相互作用蛋白，并且LIMD1在体外与BRCA2的保守区域（氨基酸2750 - 3094）相互作用。重要的是，干扰ECA109细胞中LIMD1的蛋白表达后，BRCA2的中心体定位明显消失，异常细胞分裂明显增加。这些结果表明，LIMD1是一种新的BRCA2相互作用蛋白，参与BRCA2的中心体定位，抑制LIMD1会导致食管癌细胞异常细胞分裂。
