Chapter 4: Global gene expression analysis of Caco-2 cells cultured in an
4.2 hypothesis and aim
4.4.2 Biological functions associated with differentially expressed
A log fold change cut-off of 1.25 (fold change approximately 2.38) was applied to reduce the number of differentially expressed genes to approximately 1000 (691 genes had increased expression and 239 genes had decreased expression), and the data were analysed using IPA.
The IPA functional analysis identified the biological functions that were most significantly over-represented among the list of differentially expressed genes. Based on the expression direction of the differentially expressed genes, 31 biological functions (grouped into 20 IPA categories) were predicted to be induced (Table 4.4), while three biological functions (grouped into three IPA categories) were predicted to be suppressed (Table 4.5) in Caco-2 cells cultured in the apical anaerobic model compared to those cultured in standard conditions for 12 hours.
The most significantly modulated functions included ‘cell survival’, where 65 of the 99 genes involved had an expression direction consistent with cell survival. The functions ‘proliferation of cells’, ‘cell cycle progression’ and ‘mitosis’ were also predicted to be induced. The ‘G2 phase’ and ‘G2/M phase’ functions were predicted to be suppressed, which together with the induced ‘cell cycle progression’ function, was consistent with a shortening of the G2/M phase and thus faster cell renewal. Furthermore, the function ‘cell death of epithelial cells’ was not significantly modulated.
Gene expression was also consistent with induced ‘development of blood
vessels’, ‘accumulation of blood cells’ and ‘cell movement’ functions, which are
potential mechanisms for obtaining more oxygen. Functional analysis also indicated an increase in ‘accumulation of leukocytes’ functions in the apical anaerobic model compared to Caco-2 cells cultured in standard conditions, implicating immune cell trafficking and local inflammatory responses.
Hypergeometric testing using the GO stats package328 identified 808 GO
biological processes significantly over-represented among the list of differentially expressed genes, the top 50 of which are shown on Table 4.6. Many GO biological
Table 4.4 Key IPA biological functions predicted to be induced in Caco-2 cells in the apical anaerobic environment (compared to Caco-2 cells in the co-culture chamber in a 5% CO2 incubator).
The P-value was calculated using the right-tailed Fisher Exact Test by considering the number of differentially expressed genes that participate in the function, and the total number of genes that are known to be associated with that function in the Ingenuity Knowledge Base. Given the observed differential regulation of a gene (up or down), the Z- score was used to determine the activation state of the biological function by the direction of effect associated with the relationship from the gene to the function. An absolute Z- score of ≥ 2 was considered significant (function activated if Z-score ≥ 2; inhibited if Z- score ≤ -2). ‘Genes’ indicates the number of differentially expressed genes associated with the function. *denotes functions that appear in more than one category.
Category Function P-value Z-score Genes
Cell Death apoptosis of kidney cell lines 1.11E-04 2.284 21 cell survival 5.47E-11 2.606 99
Cancer cancer 5.48E-19 2.902 262
metastasis 2.66E-07 2.937 46 metastasis of tumour 2.47E-04 2.226 13 neoplasia 3.45E-18 2.97 264 non-Hodgkin's disease* 2.34E-07 2.364 33 Cellular Growth and
Proliferation
proliferation of tumour cell lines
3.79E-07 2.124 70 proliferation of tumour cells* 7.36E-04 2.011 20 Cellular Development differentiation of osteoclasts* 2.70E-06 2.095 20 Haematological
System Development and Function
accumulation of blood cells* 1.10E-05 2.142 25 accumulation of leukocytes* 1.28E-04 2.391 22 accumulation of mononuclear
leukocytes* 7.34E-04 2.457 12 Cellular Movement cell movement 3.74E-10 2.02 137
cell movement of tumour cell
lines 7.82E-06 2.355 51
invasion of cells 1.31E-09 2.63 61 invasion of tumour cell lines 6.19E-07 2.355 42 migration of tumour cell lines 2.62E-05 2.35 40 Tissue Development accumulation of blood cells* 1.10E-05 2.142 25 accumulation of cells 7.35E-05 2.656 28 accumulation of leukocytes* 1.28E-04 2.391 22
Category Function P-value Z-score Genes accumulation of mononuclear
leukocytes* 7.34E-04 2.457 12 adhesion of tumour cell lines* 3.12E-04 2.135 22 differentiation of osteoclasts* 2.70E-06 2.095 20 Cell Cycle cell cycle progression 1.94E-09 2.587 88 mitogenesis 1.33E-04 2.545 18
mitosis 3.18E-04 2.325 35
Organismal
Development development of blood vessel* vasculogenesis* 1.05E-08 1.55E-07 2.452 2.635 64 55 Connective Tissue
Development and Function
differentiation of osteoclasts* 2.70E-06 2.095 20 Cardiovascular
System Development and Function
angiogenesis 2.47E-05 2.768 47 development of blood vessel* 1.05E-08 2.452 64 vasculogenesis* 1.55E-07 2.635 55 Skeletal and Muscular
System Development and Function
differentiation of osteoclasts* 2.70E-06 2.095 20 Cellular Function and
Maintenance
autophagy of cells* 1.12E-03 2.562 17 cellular homeostasis 2.14E-05 2.169 90 Inflammatory
Response accumulation of leukocytes* accumulation of mononuclear 1.28E-04 2.391 22 leukocytes* 7.34E-04 2.457 12 Lymphoid Tissue
Structure and Development
development of lymphatic
system component 1.39E-04 2.072 30 Cell Morphology autophagy of cells* 1.12E-03 2.562 17 Hematological
Disease non-Hodgkin's disease* 2.34E-07 2.364 33 Immune Cell
Trafficking accumulation of leukocytes* accumulation of mononuclear 1.28E-04 2.391 22 leukocytes*
7.34E-04 2.457 12 Cell-To-Cell Signaling
and Interaction adhesion of tumor cell lines* 3.12E-04 2.135 22 Tumor Morphology proliferation of tumor cells* 7.36E-04 2.011 20
Table 4.5 Key IPA biological functions predicted to be supressed in Caco-2 cells in the apical anaerobic environment (compared to Caco-2 cells in the co-culture chamber in a 5% CO2 incubator).
The P-value was calculated using the right-tailed Fisher Exact Test by considering the number of differentially expressed genes that participate in the function, and the total number of genes that are known to be associated with that function in the Ingenuity Knowledge Base. Given the observed differential regulation of a gene (up or down), the Z- score was used to determine the activation state of the biological function by the direction of effect associated with the relationship from the gene to the function. An absolute Z- score of ≥ 2 was considered significant (function activated if Z-score ≥ 2; inhibited if Z- score ≤ -2). ‘Genes’ indicated the number of differentially expressed genes associated with the function.
Category Function P-Value Z-score Genes
Cell Death cell death of skin 1.01E-03 -2.056 10
Cancer G2 phase 6.94E-05 -2.451 23
Table 4.6 Gene ontology biological processes over-represented in differentially expressed genes from Caco-2 cells cultured in an apical anaerobic environment for 12 hours (compared to Caco-2 cells in the co-culture chamber in a 5% CO2 incubator).
The 50 most significantly over-represented biological processes are shown. ‘P-value’ indicates significance of over-representation. ‘Exp Count’ indicates the expected number of differentially expressed genes associated with the biological process through chance alone, while ‘Count’ indicates the actual number of expressed genes. ‘Size’ indicates the total number of genes in the genome associated with the biological process
GO ID Term P-value Exp
Count Count Size GO:0044237 cellular metabolic process <0.001 513 625 7431 GO:0008152 metabolic process <0.001 582 687 8439 GO:0009987 cellular process <0.001 802 875 11620 GO:0006915 apoptosis <0.001 78 136 1133 GO:0012501 programmed cell death <0.001 79 136 1141 GO:0044260 cellular macromolecule
metabolic process
<0.001 393 487 5699 GO:0008219 cell death <0.001 86 144 1250 GO:0016265 death <0.001 86 144 1253 GO:0044238 primary metabolic process <0.001 526 615 7629 GO:0051716 cellular response to stimulus <0.001 76 124 1102 GO:0042221 response to chemical stimulus <0.001 105 159 1516 GO:0042981 regulation of apoptosis <0.001 61 105 889 GO:0010941 regulation of cell death <0.001 62 106 904 GO:0043067 regulation of programmed cell
death
<0.001 62 105 897 GO:0043170 macromolecule metabolic
process <0.001 433 514 6274 GO:0010033 response to organic substance <0.001 62 105 903 GO:0008283 cell proliferation <0.001 81 128 1168 GO:0048523 negative regulation of cellular
process <0.001 131 188 1895 GO:0034641 cellular nitrogen compound
metabolic process
<0.001 316 391 4584 GO:0033554 cellular response to stress <0.001 48 85 698 GO:0006807 nitrogen compound metabolic
GO ID Term P-value Exp Count
Count Size GO:0050794 regulation of cellular process <0.001 430 506 6236 GO:0051098 regulation of binding <0.001 15 36 211 GO:0048522 positive regulation of cellular
process <0.001 141 196 2042 GO:0031325 positive regulation of cellular
metabolic process
<0.001 69 110 999 GO:0042127 regulation of cell proliferation <0.001 59 97 852 GO:0009893 positive regulation of
metabolic process <0.001 73 114 1052 GO:0043687 post-translational protein
modification <0.001 104 152 1509 GO:0048008 platelet-derived growth factor
receptor signaling pathway <0.001 2 10 22 GO:0006793 phosphorus metabolic process <0.001 90 135 1308 GO:0006796 phosphate metabolic process <0.001 90 135 1308 GO:0048519 negative regulation of
biological process <0.001 142 196 2064 GO:0043066 negative regulation of
apoptosis
<0.001 28 55 404 GO:0060548 negative regulation of cell
death <0.001 29 56 415
GO:0090304 nucleic acid metabolic process <0.001 257 322 3724 GO:0043069 negative regulation of
programmed cell death <0.001 28 55 409 GO:0042326 negative regulation of
phosphorylation <0.001 4 16 59 GO:0009891 positive regulation of
biosynthetic process <0.001 52 87 759 GO:0019222 regulation of metabolic
process <0.001 280 345 4060 GO:0048518 positive regulation of
biological process <0.001 155 208 2243 GO:0006916 anti-apoptosis <0.001 16 37 236 GO:0006950 response to stress <0.001 131 181 1905 GO:0050789 regulation of biological process <0.001 454 523 6579 GO:0051101 regulation of DNA binding <0.001 11 29 166 GO:0009892 negative regulation of
GO ID Term P-value Exp Count
Count Size GO:0010629 negative regulation of gene
expression
<0.001 40 69 573 GO:0006139 nucleobase, nucleoside,
nucleotide and nucleic acid metabolic process
<0.001 296 360 4294 GO:0010563 negative regulation of
phosphorus metabolic process <0.001 4 16 63 GO:0045936 negative regulation of
phosphate metabolic process <0.001 4 16 63 GO:0006464 protein modification process <0.001 122 169 1768
processes share similarities with the IPA biological functions, such as ‘cell death’ and
‘cell proliferation’, and ‘cellular processes’ (Table 4.6), which include ‘cell adhesion’,
‘cell communication’, ‘cell cycle’, and ‘cell junction organisation’. GO processes shared with IPA biological functions (not shown in Table 4.6) include ‘blood vessel
development’ and ‘immune response-’ related functions. Other GO processes included
‘response to stress’, and ‘metabolic process’, which include‘cellular metabolic process’
and ‘nitrogen compound metabolic process’.