Transcriptomic profiling of three-dimensional cholangiocyte spheroids long term exposed to repetitive Clonorchis sinensis excretory-secretory products

Abstract

Background: Biliary 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. Methods: A 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. Results: Microarray 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. Conclusions: These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer. Graphic abstract: [Figure not available: see fulltext.]