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Homo sapiens

ABSTRACT: Quantitative Analysis of PPARD Transcriptomes in Colon Cancer Cells by Next Generation Sequencing (NGS)

PROVIDER: PRJNA353014 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5009350_1.fastq.gz	Fastqsanger.gz
	SRR5009350_2.fastq.gz	Fastqsanger.gz
	SRR5009351_1.fastq.gz	Fastqsanger.gz
	SRR5009351_2.fastq.gz	Fastqsanger.gz
	SRR5009352_1.fastq.gz	Fastqsanger.gz

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Project description:Purpose: NGS has revolutionized systems-based analysis of cell signaling pathways. The goal of this study is to determine the effects of PPARD in colon cancer cell transcriptomes in relation to the metastatic potential. Methods: NGS-derived colon cancer cell mRNA transcriptome profiles of HCT116 WT (HCT116) and HCT116 with genetic PPARD-knockout (KO1) cells were generated by deep sequencing, in quadruplicate, using Illumina HiSeq2000 .The transcriptomes of HCT116 and KO1 cells will be compared to determine the differentially expressed genes between HCT116 and KO1 cells. Differentially expressed genes will be examined in relation to the metastatic potential and validated by qRT-PCR. Results: Using an optimized data analysis workflow Tophat2, we mapped about 25 million sequence reads per sample to the human genome. Out of 22229 genes, we identified 12118 transcripts with >50 reads in at least one sample of HCT116 and KO1 cells with edgeR package and identified 6668 differentailly expressed genes with FDR 0.001 and P value cutoff 0.0022 using GLM tests fitted with BUM model. We further fltered the genes with both p-value and fold change and identified 416 genes with FDR 0.001 and fold change larger than 2. Among the differentially expressed genes, 311 were downregulated and 105 were upregulated in the KO1 cells compared with the WT cells. Twenty-three of the differentially expressed genes had significant association (i.e., a tendency towards co-occurrence) with PPARD expression (P < 0.05; log odds ratio > 1.5) in the TCGA colorectal adenocarcinoma database. Of these 23 genes, 7 were linked to metastasis by PubMed literature searches: GJA1, VIM, SPARC, NRG1, CXCL8 (IL-8), STC1, and SNCG, which were validated by q-RT-PCR. Conclusions: Our study represents the detailed analysis of PPARD transcriptomes in colon cancer cells, generated by mRNA-seq technology. Our results show that NGS offers a comprehensive and accurate quantitative and qualitative evaluations of mRNA contents in cells. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

Project description:Purpose: NGS has revolutionized systems-based analysis of cell signaling pathways. The goal of this study is to determine the effects of PPARD in gastric corpus epithenial cell transcriptomes in relation to gastric progenitor cell transfromation and gastric tumorigenesis. Methods: NGS-derived mRNA transcriptome profiles of gastric corpus epithelial cells from villin-PPARD and their age and sex matched WT mice at 10, 25 and 55 weeks were generated by deep sequencing (4-5 mice per group) using Illumina HiSeq4000 .The transcriptomes of villin-PPARD and WT mice were compared to determine the differentially expressed genes. Differentially expressed genes in the top canonical pathways will be examined and validated by qRT-PCR. Results: The raw data was aligned to the MM10 genome with Tophat2/2.0.14, and the number of reads per gene was counted with HTSeq/0.6.1.The read counts were normalized with "DESeq2". We used cutoff: FDR< 0.01 and fold change larger than 2 to identify 407 differentially expressed genes (DEGs) for 10 weeks, 717 DEGs for 25 weeks and 2694 DEGs for 55 weeks. Among those DEGs, 255 are shared by the 3 ages, in which 219 were upregulated and 36 were downregulated in villin-PPARD mice compared with the WT mice. Ingenuity Pathway Analysis for the 255 DGEs among the 3 age groups showed that IFN-g signaling was the top canonical pathway that PPAR-d activated, with an overlap ratio of 36.1% of the known genes involved in this pathway. The next two top canonical pathways, with overlap ratios of 23.7% and 15.9%, were the antigen presentation pathway and the pathway for activation of IRF by cytosolic pattern recognition receptors, respectively, both of which are related to IFN-g signaling pathway activation and immune inflammation. Conclusions: Our study represents the detailed analysis of PPARD transcriptomes in gastric corpus epithelial cells regulated by villin-promoter, generated by mRNA-seq technology. Our results show that NGS offers a comprehensive and accurate quantitative and qualitative evaluations of mRNA contents in cells. 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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Homo sapiens

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