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Next Generation Sequencing Facilitates Quantitative Analysis of Control siRNA, RAB11A siRNA1 and RAB11A siRNA2 suppressed EBC-1 cell's Transcriptomes

ABSTRACT: This study aims to find cellular pathway changes in RAB11A suppressed in lung squamous cell carcinoma cell line EBC-1 compared to control and to evaluate downregulating genes by RAB11A suppression by siRNA. Total RNA was extracted from EBC-1 transfected with control siRNA and RAB11A specific siRNAs using the RNeasy mini kit (Qiagen, Hilden, Germany). CAGE library preparation, sequencing, mapping, and gene expression and motif discovery analysis were performed by DNAFORM (Yokohama, Japan). In brief, RNA quality was assessed by Bioanalyzer (Agilent) to ensure that RIN (RNA integrity number) is over 7.0 and A260/280 and 260/230 ratios are over 1.7. First-strand cDNAs were transcribed to the 5’end of capped RNAs, attached to CAGE “bar code” tags, and the sequenced CAGE tags were mapped to human hg19 genomes using BWA software (v0.5.9) after discarding ribosomal or non-A/C/G/T base-containing RNAs. For tag clustering, CAGE-tag 5’ coordinates were input for CAGEr clustering using Paraclu algorithm with default parameters. Using CAGE analysis, we could validate the downregulation of FGF/FGFR signal-related genes in RAB11A-suppressed EBC-1 cells compared to the control cells. Finally, this study showed that RAB11 was related to tumor aggressiveness and growth in vitro and in vivo via activation of FGFR signals.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178669 | GEO | 2021/06/23

REPOSITORIES: GEO

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Next Generation Sequencing Facilitates Quantitative Analysis of Control siRNA, RAB11A siRNA1 and RAB11A siRNA2 suppressed EBC-1 cell's Transcriptomes

Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Control siRNA, RAB11A siRNA1 and RAB11A siRNA2 suppressed EBC-1 cell's Transcriptomes

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Loss of Rab11 synergizes with oncogenic Ras to alter growth and metabolism in intestinal cancer models

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Genome-wide Analysis of Core Promoter Structures in Schizosaccharomyces pombe with DeepCAGE

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Cap analysis of gene expression (CAGE) of polysome-associated RNA against total RNA from human HEK293 cell line

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Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

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The pattern of gene expression in human CD34(+) stem/progenitor cells

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Project description:Purpose: MET is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). To understand the mechanisms of resistance to MET-TKIs and establish therapeutic strategies, we developed an in vitro model using capmatinib-resistant cell lines (EBC-CR1, CR2, and CR3) derived from the MET-amplified NSCLC cell line EBC-1. Methods: We established capmatinib-resistant NSCLC cell lines from the MET-amplified NSCLC cell line EBC-1 and identified alternative signaling pathways using 3’mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative PCR and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the EBC-CR1, -CR2, and -CR3 resistant cell lines. Results: We found that epidermal growth factor (EGFR) mRNA expression and protein activation were increased in EBC-CR1–3 cells compared to EBC-1 cells. EBC-CR1 cells showed EGFR-dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells, which overexpressed the EGFR-MET heterodimer, responded dramatically to the combination of capmatinib and the phosphoinositide-3 kinase catalytic subunit α (PIK3CA) inhibitor afatinib. In addition, EBC-CR3 cells, which had activated EGFR along with amplified PIK3CA, were sensitive to the combination of afatinib and the PI3Kα inhibitor. Conclusions: Our in vitro studies suggested that activation of EGFR signaling and/or genetic alteration of downstream effectors like PIK3CA were alternative resistance mechanisms used by capmatinib-resistant NSCLC cell lines. In addition, combined treatments with MET, EGFR, and PI3Kα inhibitors may be an effective therapeutic strategy in MET-TKI-resistant NSCLC patients.

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