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Published on: December 9, 2016
Application of RNA-sequencing to identify transcriptome modification by DCLK1 in colorectal cancer cells
Lianna Li1, Hao Mei2, Abednego Nii Adom Commey3
1Biology Department, Tougaloo College, 500 West County Line Road, Tougaloo, MS, 39174, USA. lli@tougaloo.edu.
Abstract:
Doublecortin-like kinase 1 (DCLK1) is a cancer stem cell marker for the colorectal cancer (CRC). It plays critical roles in the oncogenesis, progression, and metastasis of CRC. DCLK1 can be an intriguing therapeutic target for CRC treatment. However, the molecular mechanism of how DCLK1 functions is unclear currently. In our research, we aim to apply RNA-Sequencing (RNA-Seq) technology, a high throughput massively Next-generation sequencing approach, to monitor transcriptome changes due to DCLK1 overexpression in the CRC cells. In order to achieve our goal, RNA from quadruplicate samples from two clones of isogenic DCLK1 stable overexpression cells and the parental wild-type HCT116 cells was sent for RNA-Seq on the Illumina NextSeq500 platform. Differentially expressed (DE) genes were evaluated by t-test (P < 0.05 and fold-change ± 1.5 or greater) using two methods: (1) FWER; and (2) Benjamani and Hochberg FDR (false discovery rate) which corrects for multiple comparisons. Gene networks and functional analysis were evaluated using Ingenuity Pathways Analysis (IPA). We identified 1463 DE genes common for both DCLK1 overexpression clone A and clone B cells. IPA results indicated that 72 canonical pathways were significantly modified by DCLK1 overexpression (P < 0.05), among which nine out of the top ten pathways are involved in the cell cycle regulation, indicating that DCLK1 might play its tumorigenesis role via activation of pathways facilitating cell proliferation, repression of pathways inhibiting cells proliferation, and function against pathways facilitating cell apoptosis. Cell cycle analysis results confirmed the IPA findings, which demonstrated that DCLK1 overexpression cells had much less G0/G1 cells but much more S and G2/M cells (P < 0.05). In conclusion, DCLK1 overexpression significantly modified transcriptome profile of CRC cancer cells. Control of the cell cycle regulation might be one of the critical mechanism for DCLK1 function. Our findings provide more direct evidence for the development of DCLK1 as a therapeutic target for CRC treatment, and will be of great benefit for the discovery of novel therapeutic target within the DCLK1 molecular network for the treatment of colorectal cancer patients.
Insights
Doublecortin-like kinase 1 (DCLK1) overexpression in colorectal cancer cells alters gene expression, primarily impacting cell cycle regulation. This suggests DCLK1 drives tumor growth by promoting proliferation and inhibiting apoptosis, highlighting its therapeutic potential.
Area of Science:
- Molecular Biology
- Oncology
- Genomics
Background:
- Doublecortin-like kinase 1 (DCLK1) is a marker for colorectal cancer (CRC) stem cells, implicated in tumor growth, progression, and metastasis.
- The precise molecular mechanisms underlying DCLK1's function in CRC remain largely unelucidated, necessitating further investigation.
- DCLK1 presents a promising therapeutic target for colorectal cancer treatment.
Purpose of the Study:
- To investigate the global transcriptome changes induced by DCLK1 overexpression in colorectal cancer cells using RNA-Sequencing (RNA-Seq).
- To elucidate the molecular pathways and networks affected by DCLK1 overexpression.
- To provide evidence supporting DCLK1 as a therapeutic target for CRC.
Main Methods:
- RNA-Sequencing (RNA-Seq) was performed on isogenic DCLK1-overexpressing HCT116 colorectal cancer cells and parental wild-type cells.
- Differential gene expression analysis was conducted using t-test with adjustments for multiple comparisons (FWER and Benjamini-Hochberg FDR).
- Gene network and functional pathway analysis were performed using Ingenuity Pathway Analysis (IPA).
Main Results:
- A total of 1463 differentially expressed genes were identified common to both DCLK1 overexpression clones.
- IPA revealed significant modification of 72 canonical pathways, with nine of the top ten pathways related to cell cycle regulation.
- Cell cycle analysis confirmed that DCLK1 overexpression led to a decrease in G0/G1 phase cells and an increase in S and G2/M phase cells.
Conclusions:
- DCLK1 overexpression significantly alters the transcriptome profile of colorectal cancer cells.
- Regulation of the cell cycle appears to be a critical mechanism through which DCLK1 exerts its oncogenic functions.
- These findings strengthen the rationale for developing DCLK1 as a therapeutic target for colorectal cancer.
