The Effect of Disrupted Circadian Rhythm and Associated microRNA on Biliary Injury and Malignant Transformation

Cholangiocarcinoma (CCA) is a devastating tumor characterized by late presentation of symptoms with limited treatment options. 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. The aim of the study was to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Human intra- and extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hours before stimulation with 50% serum for 2 hours. The 24-hours rhythmic expression of core clock genes, such as Per1/2/3, CLOCK, Bmal1, Cry1/2 and two clock-controlled genes (CCGs) WEE1 and DBP, was evaluated in the selected CCA cells and H69 controls by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting Mz-ChA-1 CCA cells with Per1 or empty vector. In parallel studies, we silenced miR-34a expression with anti-miR-34a inhibitor. Then, we measured: (i) cell proliferation by MTS assays and PCNA immunoblots; (ii) cell cycle; (iii) apoptosis; and (iv) cell migration and. We used luciferase assay to demonstrate whether Per1 acts as a direct target of miR-34a. Finally, we maintained CCA xenograft nude mice in complete dark or light/dark cycle for up to 40 days before evaluating tumor growth. We found the 24-hours rhythmical expression of Per1 was abolished in all CCA cell lines. The rhythmic expression of Bmal1, CLOCK, Per2/3, Cry1/2, WEE1 and DBP was also lost in some of the CCA cell lines tested. After overexpression of Per1, Mz-ChA-1 showed: (i) reduced cell proliferation; (ii) higher G0/G1 arrest and lower G2/M arrest and (iii) enhanced apoptosis. miR-34a was rhythmically expressed in CCA cell lines and H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in the selected CCA cell lines. Per1 was verified as a target of miR-34a. However, prolonged darkness therapy did not inhibit the CCA xenograft growth in vivo. Summary and conclusions: Disruption of circadian rhythms contributes to the malignant phenotypes of human CCA, and may serve as novel prognostic or therapeutic targets for CCA.