Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product

Table 1 List of signal transduction genes¹ validated the expression by real-time RT-PCR, their corresponding PCR primers², annealing temperature and product sizes

Group of stimulating Gene growth factor		Primer sequence	Annealing temperature (°C)/ product size (bp)
PDGF/EGF	abl 1	Forward: 5’-TGAAGTTGGTGGGCTGCA-3’	52/148
		Reverse: 5’-TTTTCACTGGGCCCGCA-3’
	pkC	Forward: 5’-GGCATGCCTTGTCCTGGAGA-3’	55/149
		Reverse: 5’-TGTAGCCCTGCCTCGAGAGA-3’
	jak 1	Forward: 5’-CAGCCTCGCTCGTCCTT-3’	58/147
		Reverse: 5’-CCAAGCCCTTCAGAGCT-3’
	raf 1	Forward: 5’-TTTCCCGGATGCCTGCTA-3’	62/147
		Reverse: 5’-TCCTGCTGTCCCATGCA-3’
	h ras	Forward: 5’-TGGTGGGCAACAAGTGTGA-3’	65/150
		Reverse: 5’-CCGCAATTTATGCTGCCGAA
PDGF	pdgfrα	Forward: 5’-TGCGGGGAAGGACTGGA-3’	58/122
		Reverse: 5’-GTGAGGAGACAGCTGAGGA-3’
EGF	eps 8	Forward: 5’-CCGCTCCGTGGGTATGGA-3’	54/146
		Reverse: 5’-ACGTCCGACACACTGCTGA-3’
TGF-β	tgfβ 1i4	Forward: 5’-GCAAGTGTGGTAGCTATCGA-3’	52/136
		Reverse: 5’-CAGATTGTTCTCCTGCTCC-3’
	strap	Forward: 5’-GCCGGGAGATACAGGAGA-3’	52/153
		Reverse: 5’-TGCTTATGAGCCGGGTCA-3’

¹They are classified based on their functions as the downstream signaling molecules of the specific growth factor. ²Primers for the internal control (β₂m) are: 5’-CATGGCTCGCTCGGTGA-3’ (forward) and 5’-AATGTGAGGCGGGTGGAA-3’ (reverse). The annealing temperature and the product size of RT-PCR for β₂m are 60°C and 148 bp, respectively. PDGF: platelet-derived growth factor; EGF: epidermal growth factor; TGF-β: transforming growth factor-β.

Table 2 Proliferation-related genes with 2-fold and more¹ up-regulation in fibroblast cell proliferation activated by O. viverrini ES product compared to the negative control (not exhaustive)

A Energy and metabolism	Glyceraldehyde-3-phosphate dehydrogenase (gapd), cyto-chrome C oxidase, alpha-enolase, ATP synthase beta sub-unit2, thioredoxin, aldehyde dehydrogenase
B Signal transduction	Transforming growth factor beta-1-induced transcript 4 (tgfβ 1i4), tyrosine kinase (abl 1), interleukin 1 receptor-associated kinase, MAP kinase phosphatase (mkp 6), colony stimulating factor 3 receptor, protein kinase C, Janus kinase 2 (Jak 2), casein kinase I alpha (csnk 1a1), c-myc
C Protein synthesis and translation	Polyubiquitin C (Ubc) gene, ribosomal protein S27a2, ribosomal protein L27, elongation factor Tu, RNA polymerase 1-2, translation initiation factor 4 gamma2
D Matrix and structural protein	Lysophospholipase 1 (lypla 1)
E Transcription control	Transformation/transcription domain-associated protein (trrap)2, telomeric repeat binding factor 1 (terf 1), transcription factor EB (tcfeb), DEAD-box RNA helicase (ddx 21)2
F Cell cycle	Kinesin family member 3a (kif 3a), mitotic kinesin-linked protein 12, cyclin-dependent kinase 4 (cdk 4), cdk 5, cyclin B1
G DNA replication	RPII215 polymerase II large sub-unit, topoisomerase II alpha, DNA primase p58 subunit (prim 2), ribonucleotide reductase

¹Sequence in each group is ordered from high to low expression increases;

²represents human gene.

Table 3 Up-regulation of signal transduction-related genes in NIH-3T3 treated with O. viverrini, categorized in groups according to the response to the specific growth factor

Related growth factor	Gene name/production	Folding
PDGF/EGF
AW544655	Tyrosine kinase (v abl)	7.12
C80388	Protein kinase C (pkC)	4.23
AU042564	Casein kinase I-alpha isoform (csnk1α1)1	3.81
C87299	Casein kinase I-epsilon (csnk1ε)	2.88
AU046274	Ras-GTPase activating protein SH3-domain binding protein (c3bp-pending)	2.73
C87788	JAK1 protein tyrosine kinase	2.56
AU017366	H ras	2.26
AW552623	N ras	2.01
PDGF
AW537708	PDGF receptor, alpha-polypeptide (pdgfrα)	2.64
EGF
C88280	Epidermal growth factor receptor pathway substrate (eps 8)	2.32
TGF- β
AW546174	Transforming growth factor beta-1-induced transcript 4 (tgf β 1i4)	8.24
AU016757	c myc	3.70
C79202	Serine/threonine kinase receptor-associated protein (strap)	3.40

¹Represents human gene.

Table 4 cDNA array analysis showing up-regulation of genes encoded for the kinase receptors

Receptor kinases	Gene names (accession number)	Folding
Serine-threonine kinase	Serine/threonine kinase 11 (C85710)	4.42
	Serine/threonine kinase 19 (AW557191)	3.28
	Serine/threonine kinase 4 (AU020804)	2.88
Tyrosine kinase	TYRO3 protein tyrosine kinase 3 (AW556118)	2.60
	JAK1 protein tyrosine kinase (C87788)	2.56
	Protein tyrosine kinase 9 (AW544421)	1.83
	Downstream of tyrosine kinase 1 (AW557123)	1.59