title: Up-regulation of CRKL by microRNA-335 methylation is associated with poor prognosis in gastric cancer.
abstract: BACKGROUND
  MicroRNAs have been suggested to play a vital role in regulating carcinogenesis, tumor progression and invasion. MiR-335 is involved in suppressing metastasis and invasion in various human cancers. However, the mechanisms responsible for the aberrant expression of miR-335 in gastric cancer (GC) remain unknown.
  
  
  METHODS
  Expression of miR-335 in four GC cell lines and 231 GC tissues was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). DNA methylation status in the CpG islands upstream of miR-335 in GC cell lines and tissues was determined by methylation-specific PCR and bisulfite sequence-PCR. The effects of the demethylating agent 5-aza-2'-deoxycytidine on cell proliferation, apoptosis, cell cycle, migration, and invasion were investigated in GC cell lines.
  
  
  RESULTS
  Cancer-specific methylation was detected in the upstream CpG-rich regions of miR-335, which dramatically silenced its transcriptional activity in GC cell lines and tissues. Low levels of miR-335 expression and high levels of miR-335 methylation in GC tissues were associated with poor clinical features and prognosis. Restoration of miR-335 expression in GC cells promoted cell apoptosis, inhibited tumor cell migration, invasion, and proliferation, and arrested the cell cycle at G0/G1 phase. Overexpression of miR-335 significantly reduced the activity of a luciferase reporter containing the 3' untranslated region of V-crk avian sarcoma virus CT10 oncogene homolog-like (CRKL).
  
  
  CONCLUSIONS
  MiR-335 functions as a tumor suppressor and may be silenced by promoter hypermethylation. It plays a role in inhibiting tumor cell migration, invasion, and proliferation, arresting the cell cycle at G0/G1 phase, and promoting apoptosis in GC cells through targeting CRKL.
authors.affiliation: Department of Gastroenterological Surgery, The Fourth Affiliated Hospital of China Medical University, 4 Chongshan Road, Shenyang, 110032 People's Republic of China.
authors.forename: Jia-Kui
authors.initials: JK
authors.lastname: Zhang
authors.affiliation: Department of Gastroenterological Surgery, The Fourth Affiliated Hospital of China Medical University, 4 Chongshan Road, Shenyang, 110032 People's Republic of China.
authors.forename: Yong-Shuang
authors.initials: YS
authors.lastname: Li
authors.affiliation: Department of Gastroenterological Surgery, The Fourth Affiliated Hospital of China Medical University, 4 Chongshan Road, Shenyang, 110032 People's Republic of China.
authors.forename: Chun-Dong
authors.initials: CD
authors.lastname: Zhang
authors.affiliation: Department of Gastroenterological Surgery, The Fourth Affiliated Hospital of China Medical University, 4 Chongshan Road, Shenyang, 110032 People's Republic of China.
authors.forename: Dong-Qiu
authors.initials: DQ
authors.lastname: Dai
keywords: Gastric cancer; Methylation; MiR-335; MicroRNA; Prognosis