Coordinated effects of microRNA-494 induce G₂/M arrest in human cholangiocarcinoma.
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.
Division of Gastroenterology and Hepatology, Johns Hopkins Hospital, Baltimore, MD, USA.
Sumitaka
S
Yamanaka
Nathaniel R
NR
Campbell
Fangmei
F
An
Scot C
SC
Kuo
James J
JJ
Potter
Esteban
E
Mezey
Anirban
A
Maitra
Florin M
FM
Selaru
D000951:Antigens, Neoplasm / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000378:metabolism / Q000473:pathology; D056744:Cyclin B1 / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D005434:Flow Cytometry; D059565:G2 Phase Cell Cycle Checkpoints; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D035683:MicroRNAs / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D064249:Securin; D020506:Untranslated Regions; D000097869:Polo-Like Kinase 1
D000951:Antigens, Neoplasm; D018797:Cell Cycle Proteins; D056744:Cyclin B1; D004250:DNA Topoisomerases, Type II; D004268:DNA-Binding Proteins; D035683:MicroRNAs; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D064249:Securin; D020506:Untranslated Regions; D000097869:Polo-Like Kinase 1; C547168:MIRN494 microRNA, human; C578002:pituitary tumor-transforming protein 1, human; C000618811:TOP2A protein, human