Genetic Heterogeneity of Single Circulating Tumour Cells in Colorectal Carcinoma
Abstract
:1. Introduction
2. Results
2.1. Validation of the Real-Time qPCR Experiment at the Single-Cell Level
2.2. Multi-marker mRNA Profiling of the Single CTCs, Matched Tumours and Colon Cancer Cell Lines
2.3. Genetic Heterogeneities of the Single CTCs
2.4. Correlation of the Gene Expression of Single CTCs and the Tumour Stages
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Patient Enrolment and Sample Processing
4.3. CTC Enrichment and Identification
4.4. Single CTC Isolation, RNA Extraction and Complementary DNA (cDNA) Conversion
4.5. RNA Extraction from Frozen Tumour Tissues and Reverse Transcription
4.6. Quantitative Real-Time Polymerase Chain Reaction (qRT PCR)
4.7. Statistical Analyses
Author Contributions
Funding
Conflicts of Interest
Abbreviations
CTCs | Circulating tumour cells |
CRC | Colorectal carcinoma |
qPCR | Quantitative polymerase chain reaction |
EMT | Epithelium–mesenchymal transition |
GAPDH | Glyceraldehyde 3-phosphate dehydrogenase |
mRNA | Messenger RNA |
PCA | Principal component analysis |
gDNA | Genomic DNA |
WNT | Wingless-related integration site |
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Genes | Accession no. | Forward (5ʹ-3ʹ) | Reverse (5ʹ-3ʹ) | Amplicon Size (*bp) | |
---|---|---|---|---|---|
Epithelial | EPCAM | NM_002354.3 | CGCAGCTCAGGAAGAATGTG | TGAAGTACACTGGCATTGACG | 88 |
CK20 | NM_019010.3 | CAGTCCCATCTCAGCATGAAAG | CCTCCAGAGAGCTCAACAGC | 109 | |
Oncogenes | KRAS | NM_033360.3 | AGGCCTGCTGAAAATGACTGAATAT | GCTGTATCGTCAAGGCACTCTT | 80 |
NRAS | NM_002524.4 | TTGAGGTTCTTGCTGGTGTG | TTAGCTGGATTGTCAGTGCG | 92 | |
HRAS | NM_005343.4 | TACCGGAAGCAGGTGGTCAT | GATGGCAAACACACACAGGA | 135 | |
Proliferation | BRAF | NM_004333.6 | TCTTCATGAAGACCTCACAGT | CCAGACAACTGTTCAAACTGA | 96 |
Cell survival | AKT1 | NM_005163.2 | AAGTACTCTTTCCAGACCC | TTCTCCAGCTTGAGGTC | 197 |
Tumour suppressor | TP53 | NM_000546.5 | ACCTATGGAAACTACTTCCTG | ACCATTGTTCAATATCGTCC | 99 |
FOXO3 | NM_001455.4 | GAATGTTGTTGGTTTGAACG | ATTTGGCAAAGGGTTTTCTC | 156 | |
*EMT | SNAI1 | NM_005985.4 | AACAATGTCTGAAAAGGGAC | ATAGTTCTGGGAGACACATC | 95 |
SLUG | NM_003068.5 | CATGCCTGTCATACCACAAC | GGTGTCAGATGGAGGAGGG | 169 | |
SOX2 | NM_003106.4 | GATCCTGGACTTCTTTTTGG | TCTATACAAGGTCCATTCCC | 87 | |
TWIST1 | NM_057179.3 | ATCATTTGTAACAACCCAGG | CAAATGATAGAGTCAGCACC | 160 | |
NANOG | NM_024865.4 | CTATCCATCCTTGCAAATGTC | GTTCTGGTCTTCTGTTTCTTG | 198 | |
Stemness | CD133 | NM_006017.3 | CACTACCAAGGACAAGGCGTTC | CAACGCCTCTTTGGTCTCCTTG | 151 |
CD44 | NM_000610.4 | CCAGAAGGAACAGTGGTTTGGC | ACTGTCCTCTGGGCTTGGTGTT | 151 | |
*ECM-degrading | MMP9 | NM_004994.3 | AAGGATGGGAAGTACTGG | GCCCAGAGAAGAAGAAAAG | 151 |
Cell cycle regulator | CDKN1A | NM_000389.5 | CAGCATGACAGATTTCTACC | CAGGGTATGTACATGAGGAG | 200 |
*WNT pathway regulator | APC | NM_001127511.3 | AGAGGTCATCTCAGAACAAG | CATGTTGATTTCTCCCACTC | 86 |
Housekeeping | GAPDH | NM_002046.7 | TGCACCACCAACTGCTTAGC | GGCATGGACTGTGGTCATGAG | 87 |
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Hamid, F.B.; Gopalan, V.; Matos, M.; Lu, C.-T.; Lam, A.K.-y. Genetic Heterogeneity of Single Circulating Tumour Cells in Colorectal Carcinoma. Int. J. Mol. Sci. 2020, 21, 7766. https://doi.org/10.3390/ijms21207766
Hamid FB, Gopalan V, Matos M, Lu C-T, Lam AK-y. Genetic Heterogeneity of Single Circulating Tumour Cells in Colorectal Carcinoma. International Journal of Molecular Sciences. 2020; 21(20):7766. https://doi.org/10.3390/ijms21207766
Chicago/Turabian StyleHamid, Faysal Bin, Vinod Gopalan, Marco Matos, Cu-Tai Lu, and Alfred King-yin Lam. 2020. "Genetic Heterogeneity of Single Circulating Tumour Cells in Colorectal Carcinoma" International Journal of Molecular Sciences 21, no. 20: 7766. https://doi.org/10.3390/ijms21207766