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HNF4alpha P1 and P2 isoforms regulate different gene networks in the colorectal cancer cell line Caco2/15.

ABSTRACT: The nuclear receptor HNF4alpha is a master regulator of gene transcription in intestinal epithelial cells where P1 and P2 isoforms are expressed. HNF4alpha has been associated with colorectal cancer where its role remains controverisal since both tumor suppressor and oncogene characteristics have been observed. Evidences gathered from mouse models suggested however that this controversy on HNF4alpha function in colorectal cancer could be explained by the different functions of its two isoforms classes. In order to clarify this hypothesis in a human context, we downregulated the expression of HNF4alpha P1 or P2 isoforms using isoforms specific shRNA in the colorectal cancer cell line Caco2/15. Transcriptomic data were obtained by RNA-seq from these shRNA cell populations and compared to a non-target shRNA control.

ORGANISM(S): Homo sapiens

PROVIDER: GSE106378 | GEO | 2018/06/26

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study is to compare the NGS-derived from ventricular trascriptome profiling (RNA-seq) of 1- and 2-day old mouse hearts to gain insight into the molecular bases responsible for the decline in the heart regeneration competence between P1 and P2 mice Methods: Ventricular cardiac trascriptome profiling were generated by total RNA was extracted from only the ventricles of P1 (n=3) or P2 (n=3) hearts using Quiazol lysis reagent (Quiagen) and RNAeasy extraction kit (Qiagen) according to manufacturer’s instructions. Polyadenylated RNA was isolated from 1-5 μg of total RNA using Dynabeads mRNA purification kit (Invitrogen). Polyadenylated RNA was then fragmented, retro transcribed and finally re-synthetized to double strand DNA molecules using DNA polymerase I (Invitrogen). End-pair, A-tailing, adapter ligation and size selection were then performed. RNA-seq analysis was performed using the 50 nt long paired-end reads obtained from sequencing 6 samples of mice heart tissue at two different time points and using three biological replicates for each time point. Firstly, the quality of the reads was verified using FastQC v0.11.21. Then, the reads were mapped to Mus musculus reference genome vGRCm38 2, using tophat v2.0.143. Following mapping, the transcript and normalization counts were performed using cuffdiff v2.2.1 4. The differential expression analysis of normalized counts was performed using cummeRbund v2.12 5, a widely used R v3.2 package which works together with the cuffdiff software; the applied threshold on the parameter alpha in the function getSig of the package was <= 0.05. Results: We found that most differentially-expressed transcripts encode extracellular matrix components and structural constituents of the cytoskeleton. Conclusion: These results suggest that the stiffness of the local microenvironment, rather than cardiac cell-autonomous mechanisms, crucially determines the ability or inability of the heart to regenerate.

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HNF4alpha P1 and P2 isoforms regulate different gene networks in the colorectal cancer cell line Caco2/15.

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