Functional Role of microRNAs in Patient‐Derived Xenograft Models of Human Cholangiocarcinoma

Abstract

BACKGROUND & AIMSPatient‐derived xenograft (PDX) models are now being widely used in cancer research including liver and biliary carcinogenesis, and have the potential to feedback and improve the therapeutic effects in cancer patients. MicroRNAs (miRNAs) are highly conserved 19–25 nucleotide non‐coding small RNAs which are primarily involved in gene silencing. Our aim was to characterize the functional roles of microRNA regulated protein translocations in patient‐derived cholangiocarcinoma xenograft tumors.METHODSmiRNA expression in human cholangiocarcinoma cancer tissues, patient‐derived cholangiocarcinoma xenograft tissues, human extrahepatic cholangiocarcinoma cancer stem cells (GB‐CSCs)/Mz‐ChA‐1 extrahepatic cholangiocarcinoma cancer cells and their derived xenograft tumor tissues, as well as H69 non‐malignant cholangiocytes was assessed using a hybridization based microarray and was further evaluated by Taqman real‐time PCR analysis. Promoter methylation, matrix metalloproteinase (MMP) 1, 2, 3 and 9 mRNA expression was quantitated by real‐time PCR and western blot analysis.RESULTSWe identified miR‐200 family including miR‐200a, miR‐200b and miR‐200c that are differentially expressed in doxorubicin‐enriched cell fractions in a patient derived mouse xenograft tumor model of extrahepatic cholangiocarcinoma. Members of the miR‐200 family were notably more silenced in GB‐CSCs compared with their parental extrahepatic cholangiocarcinoma cancer cells (GBCs) and Mz‐ChA‐1 cells. Expression of miR‐200 family microRNAs and transmembrane protein MMP‐2 and MMP‐9 were substantially enhanced concomitant with GBCs differentiation and loss of self‐renewal, whereas forced expression of a polycistron construct encoding miR‐200 miRNAs significantly decreased chemoresistance to doxorubicin. Methylation‐specific PCR detected that miR‐200b and miR‐200c promoter was hypermethylated in GB‐CSCs, GBCs as well as Mz‐ChA‐1 cells. Cell migration and invasion were significantly decreased by miR‐200 precursor introduction in GB‐CSCs and Mz‐ChA‐1 cells. Bioinformatics and dual‐luciferase reporter assays identified ZEB1 and ZEB2 as the direct targets of miR‐200 family.CONCLUSIONOur results define a novel oncogenic regulatory mechanism of specific microRNA group in patient derived cholangiocarcinoma xenograft models and suggesting that miR‐200 cluster may be the molecular targets for eradication of human cholangiocarcinoma.Support or Funding InformationTGEN grant from Translational Genomics Research InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.