Authors: Sumitaka Yamanaka, Nathaniel R. Campbell, Fangmei An, Scot C. Kuo, James J. Potter, Esteban Mezey, Anirban Maitra and Florin M. Selaru

Sumitaka Yamanaka

Division of Gastroenterology and Hepatology; Johns Hopkins Hospital; Baltimore, MD USA

Nathaniel R. Campbell

Division of Pathology; Johns Hopkins Hospital; Baltimore, MD USA

Fangmei An

Division of Gastroenterology and Hepatology; Johns Hopkins Hospital; Baltimore, MD USA; Department of Infectious Diseases; Ruijin Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, China

Scot C. Kuo

Biomedical Engineering and Cell Biology; Johns Hopkins University School of Medicine; Baltimore, MD USA

James J. Potter

Division of Gastroenterology and Hepatology; Johns Hopkins Hospital; Baltimore, MD USA

Esteban Mezey

Division of Gastroenterology and Hepatology; Johns Hopkins Hospital; Baltimore, MD USA

Anirban Maitra

Division of Pathology; Johns Hopkins Hospital; Baltimore, MD USA

Florin M. Selaru

Corresponding author: fselaru1@jhmi.edu
Division of Gastroenterology and Hepatology; Johns Hopkins Hospital; Baltimore, MD USA

Abstract:
MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G₂) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G₂/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G₂/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G₁) to synthesis (S), as described previously, as well as G₂ to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

Received: April 12, 2012; Accepted: June 12, 2012

Preview: