Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition.

中文翻译：

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肝吸虫颗粒蛋白促进细胞外囊泡介导的串扰和有益于胆管癌的细胞微环境。

恶性细胞与邻近细胞之间的串扰有助于肿瘤生长。在东亚，肝吸虫感染是胆管癌（CCA）的主要危险因素。肝吸虫鬼臼单胞菌分泌一种称为肝吸虫颗粒蛋白的生长因子，这是人前颗粒蛋白的同系物，对胆道纤维化和发病率有显着贡献。在此，研究了暴露于肝吸虫颗粒蛋白后细胞外小泡（EV）介导的mRNA从人胆管细胞向未成熟受体细胞的转移。为了最大程度地减少内源性颗粒蛋白原的影响，使用基于CRISPR / Cas9的基因敲除将其同源基因失活。建立了几种耗尽谷粒蛋白的细胞系，称为ΔhuPGRN-H69。这些品系的特定转录本和颗粒蛋白原水平降低了> 80％，无论是在基因编辑的细胞中，还是在这些细胞释放的电动汽车中。来自ΔhuPGRN-H69的细胞外囊泡RNA（evRNA）具有CCA相关特征的特征表明，与细胞过程（包括致癌转化）相关的雌激素和Wnt信号通路，肽酶抑制剂和酪氨酸磷酸酶的转录物很少。通过暴露于肝吸虫颗粒蛋白可诱导包括MAPK / AKT途径成员在内的几种CCA特异性evRNA。相比之下，在暴露于肝吸虫颗粒蛋白的野生型H69细胞中，雌激素，Wnt / PI3K和TGF信号转导及其他CCA途径mRNA上调。其中，与CCA相关的evRNA修饰了与暴露于肝吸虫颗粒的ΔhuPGRN-H69细胞的EV共同培养的幼稚细胞中CCA的微环境，与对照受体细胞相比，原始受体细胞中MAPK磷酸化相关蛋白的表达和诱导翻译。响应于肝吸虫颗粒蛋白的外来体介导的串扰通过MAPK途径促进了CCA特异性程序，进而建立了CCA传导性。