646 Translational Control of the Circadian Rhythms by MicroRNA-34a in Human Cholangiocarcinoma

Abstract

Background & aims: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that trigger mRNA translation, repression or degradation. Core clock genes are the molecular basis of circadian rhythms, which include BMAL1, CLOCK, Per1/2/3 and Cry1/2. We have previously demonstrated the downregulation of Per1 expression in human cholangiocarcinoma (CCA) that was predicted as the direct target of miR-34a. Thus, we aimed to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Methods: Human intraand extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hr before stimulation with 50% serum for 2 hours. The 24-hour rhythmic expression of core clock genes, such as Per1,2/3, CLOCK, BMAL1 and Cry1/2 was evaluated in CCA and H69 cells by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting the Mz-ChA-1 cells with Per1 cDNA and empty vector. In parallel, we also silenced miR-34a expression with lenti-miR-34a ZIP with relative controls. A stably overexpressing Per1 cell line and silencing of miR-34a were established, respectively. Then, wemeasured: (i) cell proliferation byMTS assays and PCNA immunoblots; (ii) cell cycle by BD Cycle test Plus reagent kit; (iii) apoptosis by Annexin V-PE apoptosis detection kit; and (iv) cell migration and invasion by commercially available kits. We utilized luciferase assay to demonstrate that Per1 act as a target of miR-34a. Results: The 24-hour rhythmical expression of Per1 was abolished in all CCA cell lines but not in H69 cells. The rhythmic expression of BMAL1, CLOCK, Per2/3, Cry1/2 was also lost in some of the CCA cell lines tested. After overexpression of Per1, the extrahepatic CCA cell line, Mz-ChA-1, showed: (i) reduced cell proliferation (by MTS assays and PCNA immunoblots); (ii) higher G0/G1 arrest and lower G2/M arrest; (iii) enhanced apoptosis; and (iv) decreased cell invasion and migration compared to control cells. Interestingly, miR-34a was rhythmically expressed in CCA cell lines and H69. The expression level of miR-34a was higher in CCA cell lines compared to H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in CCA cell lines. Dual luciferase reporter assay demonstrated that miR-34a directly targets Per1. Summary and conclusions: Disruption of circadian rhythms, miR-34a and Per1 expression may contribute to the malignant phenotypes of human cholangiocarcinoma. Our findings suggest that modulation of miR-34a-dependent Per1 expression may represent a novel therapeutic approach for human CCA.