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Gene expression profiles of NR1D1 knockout MDA-MB-231 cells treated with H2O2

ABSTRACT: To understand the role of NR1D1 in the progression of breast cancer, NR1D1 knockout (KO) MDA-MB-231 cells, established using the CRISPR/Cas9 system, were treated with 200 μM H2O2 for 72 h. Gene expression patterns were analyzed by microarray.

ORGANISM(S): Homo sapiens

PROVIDER: GSE231652 | GEO | 2023/06/27

REPOSITORIES: GEO

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Project description:Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidence for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L-serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L-serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L-serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L-serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions. Parental MDA-MB-231 cells and MDA-MB-231(SA) cells were cultured in cell culture flasks. RNA was isolated in order to compare the gene expression profiles of these cell variants. Total of two samples. No replicates.

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 CTU-treated MDA-MB-231 cell's 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: Purified RNA of CTU-treated MDA-MB-231 cells (10 μM, 6 h) were sent to the Australian Genome Research Facility for RNA-Seq analysis. Differential expression analysis was performed by edgeR and pathway enrichment was performed using the Reactome database as a reference.qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: RNA-Seq profiling identified a total of 14,951 genes in MDA-MB-231 cells, and detected 382 differentially expressed genes greater than or equal to (≥) 2-fold after 10 μM CTU treatment at 6 h. Genes whose expression was changed significantly were identified using the false discovery rate (FDR), an adjusted P-value for multiple testing. The expression of 250 and 132 genes was significantly up-regulated and down-regulated, respectively, to ≥2-fold of control in CTU-treated cells (FDR<0.05). Secondary pathway analysis was performed using the Reactome database to identify the principal biological pathways that were altered in CTU-treated MDA-MB-231 cells. This analysis implicated several mechanisms, including disruption of the tumour microenvironment, activation of cellular stress signalling and activation of cell cycle arrest. ER stress via the UPR survival mechanism was a major dysregulated pathway in MDA-MB-231 cells that had been treated with CTU (10 μM, 6 h) compared with control (FDR= 0.0001). Conclusions: Our study represents the first detailed analysis of CTU-treated MDA-MB-231 cells (10 μM, 6 h) l 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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Gene expression profiles of NR1D1 knockout MDA-MB-231 cells treated with H2O2

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