Pang Kun, Dong Yang, Hao Lin, Shi Zhen-Duo, Zhang Zhi-Guo, Chen Bo, Feng Harry, Ma Yu-Yang, Xu Hao, Pan Deng, Chen Zhe-Sheng, Han Cong-Hui
Department of Urology, Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical College, Jiangsu, China.
STEM Academic Department, Wyoming Seminary, Kinston, PA, United States.
Front Oncol. 2022 Jun 14;12:871687. doi: 10.3389/fonc.2022.871687. eCollection 2022.
There is a lack of research on the molecular interaction of the enhancers of rudimentary homolog (ERH) in bladder cancer (BC) cells. This study aimed to determine the interacting proteins of ERH in human T24 cells.
First, the gene was overexpressed in human T24 cells. Coimmunoprecipitation (co-IP) and shotgun mass spectrometry (MS) analyses were performed to obtain a list of proteins that interact with ERH. Subsequently, bioinformatic analyses with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) studies were performed to analyze the ERH-interactive protein list (ERH-IPL). Then, we selected one of the interacting proteins, EIF2α for verification. An immunofluorescence colocalization assay was performed to validate the co-expression of the selected protein, and the binding sites of the two proteins were predicted by ZDOCK technology. Finally, PCR analysis on the downstream molecules of the interacting protein was performed for verification.
ERH protein was successfully overexpressed in human T24 cells. We obtained a list of 205 proteins that might directly or indirectly interact with the ERH protein by mass spectrometric analysis. The bioinformatic analysis showed that ERH-interacting proteins were related to "ribonucleoprotein complex", "ATPase activity", "nuclear speck", and "translation factor activity, RNA binding". We further identified one of the key genes, EIF2S1, and confirmed that the corresponding protein EIF2α is co-expressed and may bind with ERH in human T24 cells. The mRNA levels of molecules ATF4 and CHOP were found to be upregulated by ERH.
ERH protein affects "ribonucleoprotein complex", "ATPase activity", "nuclear speck", and "translation factor activity, RNA binding". The ERH protein can interact with EIF2α and regulate the EIF2α-ATF4/CHOP signaling pathway in human T24 cells.
关于膀胱癌（BC）细胞中原始同源物增强子（ERH）的分子相互作用研究较少。本研究旨在确定人T24细胞中ERH的相互作用蛋白。
首先，在人T24细胞中过表达该基因。进行免疫共沉淀（co-IP）和鸟枪法质谱（MS）分析以获得与ERH相互作用的蛋白质列表。随后，进行基因本体论（GO）、京都基因与基因组百科全书（KEGG）和蛋白质-蛋白质相互作用（PPI）研究的生物信息学分析，以分析ERH相互作用蛋白列表（ERH-IPL）。然后，我们选择其中一种相互作用蛋白EIF2α进行验证。进行免疫荧光共定位分析以验证所选蛋白的共表达，并通过ZDOCK技术预测两种蛋白的结合位点。最后，对相互作用蛋白的下游分子进行PCR分析以进行验证。
ERH蛋白在人T24细胞中成功过表达。通过质谱分析，我们获得了一份可能直接或间接与ERH蛋白相互作用的205种蛋白质的列表。生物信息学分析表明，ERH相互作用蛋白与“核糖核蛋白复合物”、“ATP酶活性”、“核斑点”和“翻译因子活性，RNA结合”有关。我们进一步鉴定了关键基因之一EIF2S1，并证实相应蛋白EIF2α在人T24细胞中共表达且可能与ERH结合。发现分子ATF4和CHOP的mRNA水平受ERH上调。
ERH蛋白影响“核糖核蛋白复合物”、“ATP酶活性”、“核斑点”和“翻译因子活性，RNA结合”。ERH蛋白可与人T24细胞中的EIF2α相互作用并调节EIF2α-ATF4/CHOP信号通路。
