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Next Generation Sequencing For Quantitative Analysis Of Hepatic Transcriptomes In Response To Erythrodiol

ABSTRACT: Purpose: The goal of this study was to determine erythrodiol effect on hepatic transcriptome by comparing high-throughput data for tested group vs. control group. Method: Hepatic mRNA profile of two Apoe deficient male mice groups fed over 12 weeks with either western diet or same diet enriched with 10 mg/kg erythrodiol was analysed by Transcriptome sequencing using BGISEQ-500 platform. Sequence reads that passed quality filters were mapped onto reference genome, followed by novel gene prediction, SNP & INDEL calling and gene-splicing detection. Results: When using optimized Bioinformatics Workflow, mapping ratio for both tested groups was about 74%. There was no significant difference in SNPs between both groups. Splicing patterns _including Skipped Exon (SE), Alternative 5' Splicing Site (A5SS), Alternative 3' Splicing Site (A3SS), Mutually exclusive exons (MXE) and Retained Intron (RI) _allowed the production of a variety of DSGs with a total of 14,920 novel transcripts, 13,439 coding and 1,481 non coding transcripts. Coding genes showed 11,605 previously unknown splicing events for known genes and 1,834 coding transcripts previously unknown. DEGs were 554 in control group vs. 488 in erythrodiol group. Applying a 2-fold change and P value <0.001 led to 68 up regulated genes and 124 down-regulated genes. Gene Ontology Analysis of DEG With the GO annotation and functional enrichment classified DEGs to 3 main classification with liver-dependent properties. Altered expression of 21 genes was confirmed with RT-qPCR, demonstrating the high degree of sensitivity and accuracy of RNA-seq technique Conclusions: This study represents the first analysis of hepatic transcriptome in respond to erythrodiol. Result data confirm that RNA-Sequencing is a sensitive and accurate tool that provides a framework for comparative investigations of known and novel DEGs.

ORGANISM(S): Mus musculus

PROVIDER: GSE155163 | GEO | 2020/10/28

REPOSITORIES: GEO

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Project description:MSI (Microsatellite Instability) colorectal cancer (CRC) show improved survival, are less prone to metastasis and show poor response to chemotherapy (compared to MSS tumors). The underlying reasons for these characteristics are still not understood and no specific therapeutic approach for MSI colon tumours (15% of CRC overall) has yet been developed. The MSI process is oncogenic when it affects DNA repeat sequences that have a functional role, e.g. Small Coding Repeats (SCR). MSI also frequently affects Long Non-Coding Repeats (LNCR) in tumour DNA. In contrast to SCR, only a few LNCR are endowed with biological activity. Consequently, this area has received very little attention. Our group recently identified HSP110 mutant chaperone protein in MSI CRC that was generated by somatic deletion of a LNCR. Of interest, HSP110 mutant (due to exon skipping) have anti-oncogenic properties and the survival of MSI CRC patients receiving chemotherapy is positively associated with HSP110 mutations in tumour DNA. The aim of the current project is to identify additional clinically relevant MSI-associated splicing aberrations due to mutations in LNCR located in splice acceptor sites. The four main steps are as follows: 1. To identify exon/intron sites affected by aberrant splicing events due to MSI in CRC . All RNASeq data will be exploited to identify recurrent splicing aberrations (mostly exon skipping) that occur specifically in MSI colon tumours; 2. To investigate for possible functional links between MSI and any detected aberrant splicing events . All specific aberrant splicing events detected by RNAseq in MSI CRC samples will be first confirmed (quantitative RT-PCR) in order to eliminate false positive cases. For validated exon candidates, the allelic profiles of adjacent intronic LNCR will be analysed (PCR and fluorescence genotyping) in CRC cell lines and primary tumours (MSI and MSS), as well as in matching normal mucosa samples in order to assess their polymorphic status; 3. To identify splicing events and LNCR mutations with clinical relevance in MSI CRC patients . All LNCR with a confirmed role in gene splicing in MSI CRC will be analysed. The clinical relevance of candidate genes will be assessed using multivariate survival regression models for Relapse- Free Survival, with interaction terms (response to chemotherapy); 4. To initiate functional studies on a limited number of clinically relevant, cancer-related genes whose splicing is perturbed in MSI cancer cells, and to develop biological tools to simplify screening in future clinical assays Similar to HSP110, we will focus on 4 or 5 mutant proteins that are promising drug therapeutic targets. Functional assays will be developed to further elucidate their role in the pathophysiology of MSI tumours. We also aim to develop biological tools for these candidate genes, such as the detection of wild-type or mutant proteins by immunohistochemistry.

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Next Generation Sequencing For Quantitative Analysis Of Hepatic Transcriptomes In Response To Erythrodiol

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