Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.
OBJECTIVE
To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product.


METHODS
NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR.


RESULTS
Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05).


CONCLUSIONS
O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.
Thuwajit
Chanitra
C
Department of Biochemistry, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. tchani@kku.ac.th
Thuwajit
Peti
P
Uchida
Kazuhiko
K
Daorueang
Daoyot
D
Kaewkes
Sasithorn
S
Wongkham
Sopit
S
Miwa
Masanao
M
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology; D000818:Animals; D000947:Antigens, Helminth / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D018920:Coculture Techniques; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000502:physiology; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004815:Epidermal Growth Factor / Q000235:genetics / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000469:parasitology / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D015801:Helminth Proteins / Q000032:analysis / Q000494:pharmacology*; D008103:Liver Cirrhosis; D051379:Mice; D041681:NIH 3T3 Cells; D020411:Oligonucleotide Array Sequence Analysis; D009889:Opisthorchiasis / Q000378:metabolism; D009891:Opisthorchis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000947:Antigens, Helminth; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; C083430:Eps8 protein, mouse; D015801:Helminth Proteins; D016212:Transforming Growth Factor beta; C097418:excretory-secretory protein, Ostertagia; D004815:Epidermal Growth Factor