EGR1基因敲除Hela细胞

The stress-activated protein kinase (SAPK) family consists of three c-Jun N-terminal kinase (JNK) and four p38 members. To explore the isotype-specific or overlapping roles of SAPK members, HeLa-derived multiplex SAPK-KO cells, such as JNK1/2/3-triple KO, p38α/β/γ/δ-quadruple KO, and JNK1/2/3/p38α/β/γ/δ-septuple KO cells, were generated using the CRISPR-Cas9 method. Also, "sole survivor" (ss)-hextuple KO cells, in which only one of seven SAPK genes remains intact, were generated. Western blot analyses using phospho-specific antibodies for SAPK substrates showed that serum- or anisomycin-induced phosphorylation of MAPKAPK2, MSK1, Mnk1, and CREB (cyclic AMP response element-binding protein)/ATF-1 largely depended on p38, whereas anisomycin-induced phosphorylation of c-Jun/JunD depended on JNK. Similar analyses using the ss-hextuple KO cell lines revealed that JNK1 rather than JNK2 contributed to the c-Jun/JunD phosphorylation, whereas p38α was the primary species phosphorylating the examined p38 substrates. Expression analyses of three typical immediate-early genes, c-Jun, EGR1, and c-Fos, demonstrated that JNK1 and JNK2 are responsible for c-Jun expression induced by interleukin-1β, tumor necrosis factor-α, UV-C, and heat shock (HS), whereas p38 is predominant in EGR1 expression induced by UV and HS and in c-Fos expression induced by the cytokines, UV, and HS. On the other hand, neither JNK nor p38 contributed significantly to the cytokine-induced EGR1 expression, suggesting complicated SAPK-signaling mechanisms that regulate immediate-early gene expression. Together, these results demonstrate the utility of the comprehensive multigene KO and ss-KO strategy in dissecting intracellular signaling pathways consisting of multiple family members.

Ganglioside GM2 plays a critical role in cancer cell migration and invasion, although the intricate molecular mechanism remains elusive. This study provides a novel insight on the underlying signaling pathways and their cross talk involved in GM2-mediated tumorigenesis. Transcriptome sequencing displayed differential expression of ERK (extracellular signal-regulated kinase) target genes in GM2-treated HeLa cells. Results further showed significant upregulation of the ERK target gene expression in HeLa, MCF7 and SK-RC-45 cells in the presence of exogenous GM2. Inhibition of the MAPKK (mitogen-activated protein kinase kinase), MEK pathway with small molecule inhibitor U0126 abrogated target gene expression through the reduction in the phosphorylation level of ERK1/2 and caused functional reduction of GM2-induced migration and invasion of HeLa cells. CRISPR-Cas9-mediated knockout of ERK1 and ERK2 in HeLa cells rendered the downregulation of ERK-target gene expression in response to exogenous GM2, confirming the involvement of MEK/ERK pathway in the regulation of GM2-mediated oncogenesis. Moreover, functional knockout of Egr1 (early growth response protein 1, an ERK-target) caused significant reduction in the GM2-mediated migration/invasion of HeLa cells and induction in expression of its targets, Tgfβ1 (transforming growth factor beta 1) and Pai-1 (plasminogen activator 1). Finally, Egr1 KO in HeLa cells further reduced the induction of mesenchymal marker expression in the presence of GM2, thereby confirming the role of Egr1 in GM2-induced epithelial-mesenchymal transition (EMT) process. Taken together, this study identified MEK-ERK-Egr1 axis as an important regulatory signaling in GM2-mediated EMT and pro-tumorigenic functions.