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Next Generation Sequencing (RNA-Sequencing) for the analysis of RUNX3 targets in H460, H460-ERT2-RUNX3 WT and H460-ERT2-RUNX3 MT(K94/171R mutation)

ABSTRACT: Purpose: The goal of this study is to compare the gene expression profiles of H460, H460-ERT2-RUNX3-WT and H460-ERT2-RUNX3-MT(K94/171R) cells. Methods: H460, H460-ERT2-RUNX3 WT, and H460-ERT2-RUNX3-MT(K94/171R) cells were serum-starved for 24 hr, and then stimulated with 10% serum or 10% serum + 1 uM 4-OHT for 0, 8, or 16 hr. RNA was extracted from the cells. Isolated total RNA was processed for preparation of an RNA-seq library using the Illumina TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA). Quality and size of libraries were assessed using the Agilent 2100 Bioanalyzer DNA kit (Agilent, Santa Clara, CA, USA). All libraries were quantified by qPCR using a CFX96 Real Time System (Bio-Rad, Hercules, CA, USA) and sequenced on NextSeq500 sequencers (Illumina). Sequencing adapters and low-quality bases in the raw reads were trimmed using the Cutadapt software. The cleaned high-quality reads were mapped to the human reference genome hg19 (https://genome.ucsc.edu) using STAR software. Genes differentially expressed between two selected biological conditions were identified by Cuffdiff in the Cufflinks package (http://cole-trapnell-lab.github.io/cufflinks/papers/). Results: RNA-Sequencing Data suggest that the R-point defends against oncogenic K-RAS–induced tumorigenesis not only by regulating intracellular programs (cell cycle, apoptosis, and metabolic pathways), but also by regulating extracellular programs (inflammatory response and immune response).

ORGANISM(S): Homo sapiens

PROVIDER: GSE128174 | GEO | 2019/03/13

REPOSITORIES: GEO

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Project description:BACKGROUND: Cancer stem-like cells were isolated from human H460 non-small cell lung cancer cells by limiting dilution assays and by their holoclone morphology, followed by assessment of their self-renewal capacity, tumor growth, vascularity, and tumor blood perfusion. H460 holoclones were used to implant H460 holoclone-derived tumors, which grew slower than parental H460 tumors, but displayed significant increases in microvessel density and tumor blood perfusion. Microarray analysis identified differentially regulated genes in the holoclone-derived tumors, of which many were associated with angiogenesis. The differentially regulated genes include several small leucine-rich proteoglycans that may modulate angiogenesis and serve as novel therapeutic targets for inhibiting cancer stem cell-driven angiogenesis.(Cancer Letters Vol 346, Issue 1, 28 April 2014, Pages 63M-bM-^@M-^S73; PMID: 24334139; PMCID: PMC3947657). Implanted tumors derived from H460 parental cells, or from each of four independent H460 holoclones, were implanted subcutaneously in scid immunodeficient mice. Tumors derived from the 4 holoclones (designated H460/2E1, H460/2H3, H460/3F1, and H460/2E7) were used for global transcriptome/microarray analysis in direct comparison to H460 tumors grown from parental H460 cells. For each H460 holoclone-derived tumor line or H460 parental cell-derived tumor, total RNA was prepared from n=7 to n=10 individual tumors and used to prepare two independent pools of tumor RNA (each pool comprised of RNA isolated n=3-5 tumors) for use in a two color microarray analysis. Tumor RNA pools were prepared by combining equal amounts of RNA from each individual tumor to minimize the impasct of inter-tumoral variation on gene expression profiles. All RNAs had an RNA integrity number >8.0, determined using an Agilent Bioanalyzer 2100 instrument. cDNAs transcribed from pools of RNA for each holoclone-derived tumor line and for each parental H460 cell-derived tumor pool, were labeled with Alexa 647 or Alexa 555 dyes in a fluorescent reverse pair (dye swap) design for competitive hybridization to Agilent Whole Human Genome Microarrays.

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Next Generation Sequencing (RNA-Sequencing) for the analysis of RUNX3 targets in H460, H460-ERT2-RUNX3 WT and H460-ERT2-RUNX3 MT(K94/171R mutation)

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