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Studies on Transcriptomic Perturbation in Cancer Cells by Genome Wide Profiling

中山, 裕介 筑波大学 DOI:10.15068/00160452

2020.07.22

概要

Comprehensive transcriptomic profiling has given us numerous new biological insights and become an essential approach in biological research. However, it is still challenging to find meaningful information from transcriptome data, because cells in response to a stimulation show extremely complex transcriptomic perturbation, especially in cancer cells. Therefore, various bioinformatics approaches are necessary in the research of transcriptomic perturbation. In this study, I present usefulness of multi-profiling approach to understand transcriptional perturbation and generate valuable biological hypothesis.

In the first study, I investigated quantitative perturbation of mRNA in cancer cells with centromere protein E (CENP-E) inhibition. CENP-E is one of the core molecules in chromosome alignment in mitosis and has high potential as therapeutic target for cancer with chromosomal instability (CIN). However, molecular mechanism of anti-proliferative effect by CENP-E inhibition is unclear. To determine which signaling pathways contribute to the postmitotic effect of CENP-E inhibition, I performed comprehensive gene expression analysis using microarray for cancer cells with siCENP-E in the presence of siBubR1. Quantitative analysis revealed that genes related to p53 signaling and DNA damage response (DDR) are upregulated by CENP-E inhibition. The study suggested that induction of apoptosis by CENP-E inhibition could be led by activation of p53 signal and DDR.

In the second study, I investigated structural perturbation of mRNA in cancer cells with alternative polyadenylation modulator. Alternative polyadenylation (APA) plays a critical role in regulating gene expression. However, the balance between genome-encoded APA processing and autoregulation by APA modulating RNA binding protein (RBP) factors is not well understood. I discovered two potent small-molecule modulators of APA (T4, T5) that promote distal to proximal (DtoP) APA usage in multiple transcripts. Monotonically responsive APA events, induced by short exposure to T4 or T5, were defined in the transcriptome, allowing clear isolation of the genomic sequence features and RBP motifs associated with DtoP regulation. I found that longer vulnerable introns, enriched with distinctive A-rich motifs, were preferentially affected by DtoP APA, thus defining a core set of genes with genomically encoded DtoP regulation. Through APA response pattern and compound-siRNA epistasis analysis of APA-associated RBP factors, I further demonstrated that DtoP APA usage is partly modulated by altered autoregulation of polyadenylate binding nuclear protein-1 signaling.

Through the two studies, I demonstrated an effectiveness of genome-wide profiling approaches for elucidation of molecular mechanism of biological response in cells by generating practicable hypotheses. These approaches I presented here could be applicable for various biological scenarios, especially in oncology, which show complex transcriptomic perturbation.

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