title: Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients.
abstract: BACKGROUND
  Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease.
  
  
  METHODS
  Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease.
  
  
  RESULTS
  U6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization.
  
  
  CONCLUSIONS
  Elevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose.
authors.affiliation: Department of Surgery, Indiana University School of Medicine, West Walnut Street, Indianapolis, IN 46202, USA. hnakshat@iupui.edu
authors.forename: Hitesh N
authors.initials: HN
authors.lastname: Appaiah
authors.forename: Chirayu P
authors.initials: CP
authors.lastname: Goswami
authors.forename: Lida A
authors.initials: LA
authors.lastname: Mina
authors.forename: Sunil
authors.initials: S
authors.lastname: Badve
authors.forename: George W
authors.initials: GW
authors.lastname: Sledge
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Liu
authors.forename: Harikrishna
authors.initials: H
authors.lastname: Nakshatri
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D001943:Breast Neoplasms / Q000097:blood* / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D018572:Disease-Free Survival; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D012342:RNA, Small Nuclear / Q000097:blood* / Q000235:genetics; D020537:RNA, Small Nucleolar / Q000097:blood* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012016:Reference Values
chemical_list: D014408:Biomarkers, Tumor; C524942:MIRN155 microRNA, human; C505362:MIRN16 microRNA, human; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; C405486:U6 small nuclear RNA