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Next Generation Sequencing Facilitates Quantitative Analysis of MED23-knockdown and Control HUVEC Cells Transcriptomes

ABSTRACT: Purpose: RNA-seq analysis to evaluate the impact of MED23 knockdown on the transcriptome of HUVEC cells.Methods: Total RNA was isolated with TRIzol from Med23 knockdown HUVECs (n = 3) or control cells (n = 3). cDNA sequencing libraries were prepared with an NEBNext® Ultra™ RNA Library Prep Kit for Illumina. The RNA-Seq FASTQ raw data were trimmed to remove low-quality reads and adapters using Trimmomatic. The trimmed reads were aligned to the mouse reference genome UCSC GRCh37/hg19 with HISAT2. Gene and transcript quantification was performed using Cutdiff. The results of the mapping of the RNA-seq reads, transcript assembly and abundance estimation were reported as fragments per kilobase of exon per million fragments mapped (FPKM).

ORGANISM(S): Homo sapiens

PROVIDER: GSE186112 | GEO | 2021/10/21

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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Next Generation Sequencing Facilitates Quantitative Analysis of MED23-knockdown and Control HUVEC Cells Transcriptomes

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