Knockdown of EIF3G inhibits the intracellular protein translation of H1299 cells. Knockdown of EIF3G inhibits the intracellular protein translation of H1299 cells

eIF3 is the largest translation initiation factor in mammalian cells, consisting of 13 subunits. This translation initiation factor is involved in multiple processes of protein translation in cells, including translation initiation, termination, and ribosome recycling. Several studies have reported that multiple subunits of eIF3 exhibit abnormal expression in tumor cells and play an important role in the occurrence and development of tumors. In this study, it was found that changes in the expression level of EIF3G significantly affected the growth of non-small cell lung cancer. Knockdown of EIF3G inhibited the intracellular protein translation process of non-small cell lung cancer cells H1299. Through the study of translatome, it was found that knockdown of EIF3G significantly affected the cell cycle processes in H1299 cells. In vitro cell experiments also showed that changes in the expression level of EIF3G influences the cell cycle distribution of H1299 cells. This study is the first to explore the impact of knockdown of EIF3G on the translatome of H1299 cells. Overall design: The H1299 cells treated with siEIF3Gs or siCtrl were lysed, and the cell supernatants were collected after centrifugation. Ten percent of the total lysate was taken as input control. The cell lysate supernatants were then subjected to sucrose density gradient centrifugation, and the resulting products were separated using Gradient108 and Fractionator (BIOCOMP, Canada). The centrifuged products of the polysomal fraction were collected for RNA extraction. Subsequently, both the input control and the mRNA bound to the polysomes were extracted and sequenced.

真核翻译起始因子3（eIF3）是哺乳动物细胞中最大的翻译起始因子，由13个亚基组成。该翻译起始因子参与细胞内蛋白质翻译的多个核心过程，包括翻译起始、终止及核糖体循环回收。多项研究已证实，eIF3的多个亚基在肿瘤细胞中存在异常表达，并在肿瘤的发生与发展中发挥关键作用。本研究发现，EIF3G的表达水平变化可显著影响非小细胞肺癌的生长。敲低EIF3G可抑制非小细胞肺癌H1299细胞内的蛋白质翻译进程。通过翻译组学（translatome）分析，本研究发现敲低EIF3G可显著干扰H1299细胞的细胞周期进程。体外细胞实验亦证实，EIF3G的表达水平变化可影响H1299细胞的细胞周期分布。本研究首次探讨了敲低EIF3G对H1299细胞翻译组的影响。实验整体设计如下：将经siEIF3Gs或siCtrl处理的H1299细胞裂解，离心后收集细胞上清液；取总裂解液的10%作为输入对照。随后将细胞裂解液上清进行蔗糖密度梯度离心，采用Gradient108与Fractionator（加拿大BIOCOMP公司）分离所得产物。收集多糖体组分的离心产物用于RNA提取。随后分别对输入对照以及与多聚核糖体结合的mRNA进行提取并测序。