title: Regulation of the ITGA2 gene by epigenetic mechanisms in prostate cancer.
abstract: BACKGROUND
  Integrin alpha2 beta1 (α2 β1 ) plays an integral role in tumour cell invasion, metastasis and angiogenesis, and altered expression of the receptor has been linked to tumour prognosis in several solid tumours. However, the relationship is complex, with both increased and decreased expression associated with different stages of tumour metastases in several tumour types. The ITGA2 gene, which codes for the α2 subunit, was examined to investigate whether a large CpG island associated with its promoter region is involved in the differential expression of ITGA2 observed in prostate cancer.
  
  
  METHODS
  Bisulphite sequencing of the ITGA2 promoter was used to assess methylation in formalin-fixed paraffin-embedded (FFPE) prostate tumour specimens and prostate cancer cell lines, PC3, 22Rv1 and LNCaP. Changes in ITGA2 mRNA expression were measured using quantitative PCR. ITGA2 functionality was interrogated using cell migration scratch assays and siRNA knockdown experiments.
  
  
  RESULTS
  Bisulphite sequencing revealed strikingly decreased methylation at key CpG sites within the promoter of tumour samples, when compared with normal prostate tissue. Altered methylation of this CpG island is also associated with differences in expression in the non-invasive LNCaP, and the highly metastatic PC3 and 22Rv1 prostate cancer cell lines. Further bisulphite sequencing confirmed that selected CpGs were highly methylated in LNCaP cells, whilst only low levels of methylation were observed in PC3 and 22Rv1 cells, correlating with ITGA2 transcript levels. Examination of the increased expression of ITGA2 was shown to influence migratory potential via scratch assay in PC3, 22Rv1 and LNCaP cells, and was confirmed by siRNA knockdown experiments.
  
  
  CONCLUSIONS
  Taken together, our data supports the assertion that epigenetic modification of the ITGA2 promoter is a mechanism by which ITGA2 expression is regulated.
authors: Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, 7000, Australia.
authors: Suyin Paulynn
authors: SP
authors: Chin
authors: James R
authors: JR
authors: Marthick
authors: Alison C
authors: AC
authors: West
authors: Annabel K
authors: AK
authors: Short
authors: Jyoti
authors: J
authors: Chuckowree
authors: Andrea M
authors: AM
authors: Polanowski
authors: Russell J
authors: RJ
authors: Thomson
authors: Adele F
authors: AF
authors: Holloway
authors: Joanne L
authors: JL
authors: Dickinson
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D039081:Integrin alpha5beta1 / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D039081:Integrin alpha5beta1; D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: DNA methylation; epigenetics; integrins; prostate