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Nascent RNA profiles of WT and MED23-KD 293T cells

ABSTRACT: To profile the nascent RNAs, we performed 4SU-seq. Briefly, WT or MED23-KD 293T cells were seeded at 50% confluence in a 10 cm dish and cultured overnight. The newly transcribed RNAs in living cells were then transiently labelled with 4SU at a final concentration of 1 mM for 15 minutes. The labelled cells were harvested immediately with TRIzol. Total RNA was extracted with phenol/chloroform/isoamyl alcohol. The 4SU-labelled RNAs were biotinylated and enriched using streptavidin beads. Finally, the enriched nascent RNAs were subjected to library construction using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina for sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE282498 | GEO | 2025/05/28

REPOSITORIES: GEO

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Project description:K-RAS activating mutations occur frequently in non-small cell lung cancer (NSCLC), leading to aberrant activation of Ras-MAPK signaling pathway that contributes to the malignant phenotype. However, the development of Ras-targeted therapeutics remains challenging. Here, we show that MED23, a component of the multisubunit Mediator complex that is known to integrate signaling and gene activities, is selectively important for Ras-active lung cancer. By screening a large panel of human lung cancer cell lines with or without a Ras mutation, we found that Med23 RNAi specifically inhibits the proliferation and tumorigenicity of lung cancer cells with hyperactive Ras activity. Med23-deficiency in fibroblasts selectively inhibited the oncogenic transformation induced by Ras but not by c-Myc. Transcription factor ELK1, which is phosphorylated by MAPK for relaying the Ras signaling to MED23, was also required for the Ras-driven oncogenesis. Transcriptiome analysis revealed that MED23 and ELK1 co-regulate a common set of target genes enriched in regulating cell cycle and proliferation to support the Ras-dependency. Furthermore, correlated with the strength of Ras signaling as indicated by the ELK1 phosphorylation level, MED23 was up-regulated by Ras-transformation, and was found to be overexpressed in both Ras-mutated lung cancer cell lines and primary tumor samples. Remarkably, lower Med23 expression predicts better survival in Ras-active lung cancer patients and xenograft mice. Collectively, our findings demonstrate a critical role for MED23 in enabling the 'Ras-addiction' of lung carcinogenesis, thus providing a vulnerable target for the treatment of Ras-active lung cancer. To gain a genome-wide understanding of how MED23 and ELK1 control gene expression in Ras-active lung cancer cells, we performed gene profiling experiments to analyze the transcriptomes from control, si-Med23, or si-Elk1 A549 cells. The si-Ctrl, si-Med23 and si-Elk1 A549 cells were cultured in the normal condition. Then the cells were harvested for RNA extraction and hybridization on Affymetrix microarrays. The analysis contain 9 samples. si-Ctrl cells have three replicates (si-Ctrl#1, si-Ctrl#2 and si-Ctrl#3), and the si-Med23 or si-Elk1 group contains three different cell lines that harbor three different RNAi oligos against Med23 or Elk1 (si-Med23A, B, C and si-Elk1A, B, C).

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Nascent RNA profiles of WT and MED23-KD 293T cells

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