DAGL is the principal synthesizing enzyme of 2-AG and promotes aggressive phenotype of intrahepatic cholangiocarcinoma via AP-1/DAGL/miR4516 feedforward circuitry

The endocannabinoid system (ECS) is dysregulated in various liver diseases. Previously, we had shown that the major endocannabinoid 2-arachidonoyl glycerol (2-AG) promoted tumorigenesis of intrahepatic cholangiocarcinoma (ICC). However, biosynthesis regulation and clinical significance of 2-AG remain elusive. In the present study, we quantified 2-AG by gas chromatography/mass spectrometry (GC/MS) and showed that 2-AG was enriched in patients with ICC samples as well as in thioacetamide-induced orthotopic rat ICC model. Moreover, we found that diacylglycerol lipase beta (DAGL beta) was the principal synthesizing enzyme of 2-AG that significantly upregulated in ICC. DAGL beta promoted tumorigenesis and metastasis of ICC in vitro and in vivo and positively correlated with clinical stage and poor survival in patients with ICC. Functional studies showed that activator protein-1 (AP-1; heterodimers of c-Jun and FRA1) directly bound to the promoter and regulated transcription of DAGL beta, which can be enhanced by lipopolysaccharide (LPS). miR-4516 was identified as the tumor-suppressing miRNA of ICC that can be significantly suppressed by LPS, 2-AG, or ectopic DAGL beta overexpression. FRA1 and STAT3 were targets of miR-4516 and overexpression of miRNA-4516 significantly suppressed expression of FRA1, SATA3, and DAGL beta. Expression of miRNA-4516 was negatively correlated with FRA1, SATA3, and DAGL beta in patients with ICC samples. Our findings identify DAGL beta as the principal synthesizing enzyme of 2-AG in ICC. DAGL beta promotes oncogenesis and metastasis of ICC and is transcriptionally regulated by a novel AP-1/DAGL beta/miR4516 feedforward circuitry. NEW & NOTEWORTHY Dysregulated endocannabinoid system (ECS) had been confirmed in various liver diseases. However, regulation and function of 2-arachidonoyl glycerol (2-AG) and diacylglycerol lipase beta (DAGL beta) in intrahepatic cholangiocarcinoma (ICC) remain to be elucidated. Here, we demonstrated that 2-AG was enriched in ICC, and DAGL beta was the principal synthesizing enzyme of 2-AG in ICC. DAGL beta promotes tumorigenesis and metastasis in ICC via a novel activator protein-1 (AP-1)/DAGL beta/miR4516 feedforward circuitry.