Abstract
BackgroundBiliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation.MethodsA three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection.ResultsMicroarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p < 0.01, fold change > 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq.ConclusionsThese findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer.Graphical
Highlights
Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma
Poorly defined in detail, the molecular mechanism of cholangiocarcinogenesis is proposed as a multistep process: chronic inflammation of the bile ducts, cholangiocyte damage associated with reactive cell proliferation, consequent increases in genetic mutations and epigenetic changes, and dysregulation of apoptosis and neoangiogenesis [1]
As well as many individual cells, gradually merged into large cell aggregates at the center of each microwell surface. This spontaneous self-aggregation of H69 cells promoted bigger and denser pre-matured spheroid formation and eventually became tightly packed cholangiocyte spheroids with an average diameter of ~ 330 μm by about day 5 (Fig. 1a). These mature spheroids were incubated with conditional culture medium for 24 h, replaced with fresh conditional medium supplemented with excretory-secretory products (ESPs) (4 μg/ml), and consecutively cultivated for 5 and 10 days
Summary
Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. The established risk factors for CCA include primary sclerosing cholangitis, hepatolithiasis, and choledochal cystic disease Another critical factor is infection with liver flukes (Opisthorchis viverrini or Clonorchis sinensis), which accounts for the majority of cases of CCA in Southeast Asian countries [2, 3]. These liver flukes have been classified as definite biological carcinogens by the International Agency for Research on Cancer [4]
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