Decreased DEPTOR expression is involved in enhanced cell motility and invasion capability of ASS1 (Argininosuccinate synthetase 1) -KO cells. Homo sapiens

To elucidate the mechanisms underlying the enhanced sensitivity to arginine in the absence of ASS1 expression in endometrial cancer cells, we performed DNA microarray-based gene expression profiling using HEC1B harboring the empty vector (EV) and ASS1-knockout (KO) HEC1B cells. Since accumulating evidence has shown that arginine regulates mTORC1 activity, we focused on the expression patterns of genes related to the mTOR complex. ASS1-KO HEC1B cells had lower DEPTOR transcript abundance than EV cells, while no significant difference was observed in the expression levels of genes coding for other mTOR complex components such as mTOR, RAPTOR, or RICTOR. Indeed, immunoblot analysis confirmed that the expression level of DEPTOR protein was significantly decreased in ASS1-KO HEC1B cells, which were cultured in arginine-replete conditions. Overall design: The labeled cRNAs were hybridized on a Whole Human Genome Oligo Microarray ver. 2 (G4845A) in two dye swap experiments, resulting in four individual microarrays.

为阐明子宫内膜癌细胞缺失ASS1表达时对精氨酸敏感性增强的潜在机制，我们采用携带空载体（empty vector, EV）的HEC1B细胞与ASS1敲除（ASS1-knockout, KO）的HEC1B细胞，开展了基于DNA微阵列的基因表达谱分析。鉴于越来越多研究证据表明精氨酸可调控mTORC1的活性，我们重点关注了与mTOR复合物相关的基因表达模式。与EV组细胞相比，ASS1敲除的HEC1B细胞中DEPTOR的转录本丰度更低，但编码mTOR、RAPTOR及RICTOR等其他mTOR复合物组分的基因的表达水平无显著差异。免疫印迹分析进一步证实，在精氨酸充足的培养条件下，ASS1敲除的HEC1B细胞中DEPTOR蛋白的表达水平显著降低。整体实验设计：将标记好的互补RNA（complementary RNA, cRNA）与全人类基因组寡核苷酸微阵列版本2（Whole Human Genome Oligo Microarray ver. 2, G4845A）进行杂交，共开展2次染料互换实验，最终得到4张独立的微阵列芯片。