Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/78127
Title: Investigate non-invasive early-diagnostic and prognostic biomarkers of colorectal cancer using targeted RNA sequencing
Authors: Xue, Weiwen
Advisors: Wong, Cesar (HTI)
Keywords: Colon (Anatomy) -- Cancer -- Diagnosis
Rectum -- Cancer -- Diagnosis
Issue Date: 2018
Publisher: The Hong Kong Polytechnic University
Abstract: This study aims to find circulating messenger RNA (mRNA) markers for early diagnoses of colorectal adenoma and diagnoses and potential prognoses of colorectal cancer (CRC) based on next-generation sequencing (NGS). In this study, the expression of a panel of 108 CRC-related genes was measured using targeted mRNA sequencing in plasma and tissue samples from normal, colorectal adenoma and CRC patients. Firstly, I studied how different centrifugal forces affect CRC-related mRNA in plasma samples. Targeted mRNA sequencing was used to profile gene expression in 18 healthy donors' plasma samples prepared by 1) 3,500g for 10 minutes at 4°C; and 2) 1,600g for 10 minutes at 4°C followed by 16,000g for 10 minutes at 4°C. Results showed that 75/108 (69.4%) and 86/108 (79.6%) CRC-related genes were detectable in 3,500g and 1,600g followed by 16,000g plasma samples, respectively. Besides, MYC and HIF1A as differential expressed genes (DEGs) were identified by a cut-off as fold change > 4 and adjusted P value < 0.05 based on sequencing data. Secondly, I studied non-invasive early-diagnostic markers for colorectal adenoma. Plasma samples from 39 normal and 40 colorectal adenoma patients were collected. Results showed that GSK3A as the only DEG was identified by a cut-off as fold change > 4 and adjusted P value < 0.05 based on sequencing data, and its expression in colorectal adenoma was significantly lower than those in normal plasma samples (0.01-fold with adjusted P < 0.05). Thirdly, I studied non-invasive diagnostic and potential prognostic markers for CRC. Plasma samples from 65 CRC patients were collected in multiple time points for targeted mRNA sequencing to find candidate genes as non-invasive diagnostic or potential prognostic markers. Results showed that in the comparison between before surgery CRC and normal plasma samples, 22 DEGs were identified by a cut-off as fold change > 4 and adjusted P value < 0.05 based on sequencing data. Among them, TWIST1 and DVL1 showed the most significant up- (41.150-fold change with adjusted P < 1×10⁻¹³) and down-regulation (0.004-fold change with adjusted P < 1×10⁻¹²) in before surgery CRC samples compared with normal samples, respectively. In the comparison between after surgery and before surgery CRC plasma samples, 4 DEGs were identified. All DEGs showed up-regulation in after surgery samples compared with before surgery samples, and FZD4 showed the most significant up-regulation (28.955-fold change with adjusted P < 1×10⁻⁶). In the comparison between before chemotherapy and after surgery CRC plasma samples, 3 DEGs were identified. All DEGs showed down-regulation in before chemotherapy samples compared with after surgery samples. Finally, I validated gene expression in 68 pairs of tumor and adjacent normal tissues to study whether differential expression of candidate markers was tumor-derived. Results showed that 23 DEGs were identified by a cut-off as fold change > 4 and adjusted P value < 1 × 10⁻¹⁰, and the differential expression of MYC and SOX9 in tumor and adjacent normal tissues was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Besides, the differential expression of WISP1 was confirmed by RT-qPCR in formalin-fixed, paraffin-embedded (FFPE) tissue samples from normal, colorectal adenoma with different grade of dysplasia and CRC. Information obtained in this study provides candidate non-invasive early-diagnostic markers for colorectal adenoma and diagnostic and potential prognostic markers for CRC, and it may promote the translation of targeted sequencing data of plasma samples into clinical practice.
Description: xx, 191 pages : color illustrations
PolyU Library Call No.: [THS] LG51 .H577P HTI 2018 Xue
URI: http://hdl.handle.net/10397/78127
Rights: All rights reserved.
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