Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human cancer cell lines HCT116, T47D and A549 in 3D collagen I microenvironments compared to 2D cell culture

ABSTRACT: The behavior of cancer cells strongly relies on the properties of the extracellular microenvironment. The aim was to compare the whole transcriptome of three non-invasive cancer cell lines, HCT-116, A549 and T-47D, embedded in collagen I matrices in a time-dependent manner to standard two-dimensional cell culture. For this purpose, human cancer cell lines (HCT-116, A549 and T-47D) were cultured in their recommended media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C, 5% CO₂ and 95% humidity. Cells were regularly tested negative for mycoplasma and authenticated by STR analysis. For 2D culture, cells were grown to 70–90% confluency and RNA was isolated. For 3D culture, single cells were embedded in 2.5 mg/mL collagen I matrices, which was polymerized at 37 °C, and cultured in medium. After 3, 5, 7, and 10 days, cells were released by collagenase IV digestion for RNA isolation.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: RGCC Central Europe GmbH

PROVIDER: E-MTAB-16838 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived HCT-116 cell transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods: HCT-116 cell mRNA profiles of HCT-116-GOLPH3-Vector and HCT-116-GOLPH3-Overepression were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: RNA sequencing (RNA-seq) was used to investigate gene expression in HCT-116-PMSCV-Vector and HCT-116-PMSCV GOLPH3 cells, while Gene Ontology (GO) was used to annotate the various functional genes. By comparing the differentially expressed genes, we noticed that GOLPH3 was associated with EMT. Gene set enrichment analysis (GSEA) analysis of the RNA-seq results based on the GSE77953 dataset was performed to investigate the biological functions of HCT-116-PMSCV-Vector and HCT-116-PMSCV-GOLPH3 cells. The findings suggested that GOLPH3 expression was positively associated with colon cancer cell autophagy. Signal pathway enrichment was next analyzed based on the differential expression of genes between HCT-116-PMSCV-Vector and HCT-116-PMSCV-GOLPH3 cells, as examined by RNA-seq. Notably, GOLPH3 has correlated with the PI3K/Akt signaling pathway. Conclusions: Our study represents the first detailed analysis of HCT-116 cell transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

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RNA-seq of human cancer cell lines HCT116, T47D and A549 in 3D collagen I microenvironments compared to 2D cell culture

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