title: miR-34a-dependent overexpression of Per1 decreases cholangiocarcinoma growth.
abstract: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small non-coding RNAs that trigger mRNA translation inhibition. We aimed to evaluate the role of Per1 and related miRNAs in cholangiocarcinoma growth.
  
  
  
  The expression of clock genes was evaluated in human cholangiocarcinoma tissue arrays and cholangiocarcinoma lines. The rhythmic expression of clock genes was evaluated in cholangiocarcinoma cells and H69 (non-malignant cholangiocytes) by qPCR. We measured cell proliferation, cell cycle and apoptosis in Mz-ChA-1 cells after Per1 overexpression. We examined tumor growth in vivo after injection of Per1 overexpressing cells. We verified miRNAs that targets Per1. The circadian rhythm of miR-34a was evaluated in cholangiocarcinoma and H69 cells. We evaluated cell proliferation, apoptosis and invasion after inhibition of miR-34a in vitro, and the potential molecular mechanisms by mRNA profiling after overexpression of Per1.
  
  
  
  Expression of Per1 was decreased in cholangiocarcinoma. The circadian rhythm of Per1 expression was lost in cholangiocarcinoma cells. Decreased cell proliferation, lower G2/M arrest, and enhanced apoptosis were shown in Per1 overexpressing cells. An in vivo study revealed decreased tumor growth, decreased proliferation, angiogenesis and metastasis after overexpressing Per1. Per1 was verified as a target of miR-34a. miR-34a was rhythmically expressed in cholangiocarcinoma cells and H69. The inhibition of miR-34a decreased proliferation, migration and invasion in cholangiocarcinoma cells. mRNA profiling has shown that overexpression of Per1 inhibits cell growth through regulation of multiple cancer-related pathways, such as cell cycle, cell growth and apoptosis pathways.
  
  
  
  Disruption of circadian rhythms of clock genes contribute to the malignant phenotypes of human cholangiocarcinoma.
  
  
  
  The current study is about how biological clock and its regulators affect the bile duct tumor growth. The disruption of biological clock has a negative impact in different cancers. Per1 is a gene that is involved in maintaining the biological clock and show 24h oscillation. Reduced levels of Per1 and disruption of 24h circadian rhythm was found in bile duct cancer cells. Therefore, a genetic modified bile duct cancer cells was created. It has a higher level of Per1 expression and partially recovered circadian rhythm. Those genetic modified cells also displayed slower cell growth or higher rate of cell death. We also used mice model that lack of immune system to show that our genetic modified bile duct cells form smaller tumor. In addition, we tried to see how Per1 is communicating with other genes in regarding of controlling the tumor growth. We found Per1 is regulated by microRNA-34a, a small non-coding RNA that directly binds to genes and inhibit gene expression. Decreased level of miR-34a has also significantly reduced tumor growth through controlling the cell growth and cell death balance. Therefore bile duct cancer patients may be treated with miR-34a inhibitor or Per1 stimulator in the future.
authors.affiliation: Research, Central Texas Veterans Health Care System, United States; Department of Medicine, Texas A&M Health Science Center, United States.
authors.forename: Yuyan
authors.initials: Y
authors.lastname: Han
authors.affiliation: Research, Central Texas Veterans Health Care System, United States; Baylor Scott & White Digestive Disease Research Center, United States; Baylor Scott & White Academic Operations, Baylor Scott & White Healthcare, United States.
authors.forename: Fanyin
authors.initials: F
authors.lastname: Meng
authors.affiliation: Department of Medicine, Texas A&M Health Science Center, United States.
authors.forename: Julie
authors.initials: J
authors.lastname: Venter
authors.affiliation: Department of Medicine, Texas A&M Health Science Center, United States.
authors.forename: Nan
authors.initials: N
authors.lastname: Wu
authors.affiliation: Baylor Scott & White Digestive Disease Research Center, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wan
authors.affiliation: Research, Central Texas Veterans Health Care System, United States.
authors.forename: Holly
authors.initials: H
authors.lastname: Standeford
authors.affiliation: Research, Central Texas Veterans Health Care System, United States; Baylor Scott & White Digestive Disease Research Center, United States; Baylor Scott & White Academic Operations, Baylor Scott & White Healthcare, United States.
authors.forename: Heather
authors.initials: H
authors.lastname: Francis
authors.affiliation: Department of Medical Physiology, Texas A&M Health Science Center, United States.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Meininger
authors.affiliation: Pathology, Baylor Scott & White, Temple, TX 76504, United States.
authors.forename: John
authors.initials: J
authors.lastname: Greene
authors.affiliation: Department of Medical Physiology, Texas A&M Health Science Center, United States.
authors.forename: Jerome P
authors.initials: JP
authors.lastname: Trzeciakowski
authors.affiliation: Department of Medicine, Texas A&M Health Science Center, United States.
authors.forename: Laurent
authors.initials: L
authors.lastname: Ehrlich
authors.affiliation: Research, Central Texas Veterans Health Care System, United States; Baylor Scott & White Digestive Disease Research Center, United States; Department of Medicine, Texas A&M Health Science Center, United States.
authors.forename: Shannon
authors.initials: S
authors.lastname: Glaser
authors.affiliation: Research, Central Texas Veterans Health Care System, United States; Baylor Scott & White Digestive Disease Research Center, United States; Department of Medicine, Texas A&M Health Science Center, United States; Department of Medical Physiology, Texas A&M Health Science Center, United States. Electronic address: galpini@medicine.tamhsc.edu.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Alpini
mesh_terms: D000818:Animals; D001650:Bile Duct Neoplasms / Q000473:pathology*; D056926:CLOCK Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018281:Cholangiocarcinoma / Q000473:pathology*; D002940:Circadian Rhythm; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000502:physiology*; D009361:Neoplasm Invasiveness; D056950:Period Circadian Proteins / Q000502:physiology*
chemical_list: C522425:MIRN34 microRNA, human; D035683:MicroRNAs; C539022:PER1 protein, human; D056950:Period Circadian Proteins; D056926:CLOCK Proteins; C538943:CLOCK protein, human
keywords: Biliary epithelium; Cholangiocarcinoma; Circadian rhythm; Clock genes; microRNAs