title: Epigenetic profiling of gallbladder cancer and gall stone diseases: Evaluation of role of tumour associated genes.
abstract: BACKGROUND
  As on today, the global mortality rate of gallbladder cancer is still very high. Both genetic and epigenetic alterations play pivotal roles in the development of cancer. We selected seven tumour associated genes, implicated in other cancers, to assess their methylation status in gallbladder cancer and gallstone diseases.
  
  
  OBJECTIVE
  To study the promoter methylation of certain tumour associated genes in the molecular pathogenesis of gallbladder cancer and gall stone diseases.
  
  
  METHODS
  Methylation specific PCR for seven tumour associated genes, viz., MASPIN, 14-3-3 sigma gene, THBS1, FLNC, HLTF, COX-2 and SOCS1, was performed in 50 gallbladder cancer (GBC), 30 gall stone diseases (GSD) and their respective adjacent control tissues. Semi-quantitative PCR and immunohistochemistry was carried out to check the expression level. Student's t-test was carried out to compare the differences in the methylation and expression patterns between cases and control tissues.
  
  
  RESULTS
  We observed methylation of CpG islands in seven of the studied markers, but, the frequency of methylation was found varying among different samples. Of them, 14-33 sigma showed methylation in 45 GBC (90%; p=0.0001) and 25 GSD (86.66%; p=0.001), MASPIN in 35 GBC (70%; p=0.0008) and 18 GSD (51.43%; p=0.040), FLNC in 16 GBC (32%; p=0.0044) and 9 GSD (25.71%; p=ns), THBS1 in 26 GBC (52%; p=0.0009) and 10 GSD (28.57%; p=0.0505), HLTF in 8 GBC (16%; p=ns) and 2 GSD (5.71%; p=ns), COX2 in 10 GBC (20%; p=ns) and 6 GSD (17.14%; p=ns) and SOCS-1 in 3 GBC samples only (6%; p=ns), but not in GSD. Semi-quantitative PCR revealed down regulation in MASPIN, 14-3-3 sigma, THBS1, HLTF, COX2 and SOCS1 in advanced gallbladder cases. Immunohistochemistry further confirmed the down-regulation of SOCS1 in GBC.
  
  
  CONCLUSIONS
  The present study infers that accumulation of epigenetic alterations increases poor prognosis of GBC patients. Out of seven genes, MASPIN and THBS1 play key epigenetic role in GBC, but not in GSD. The reason for downregulation of SOCS1 only in GBC, and unaltered expression of 14-3-3 sigma protein in all the GBC and GSD tissue samples is not clear. Further investigation on the expression pattern of these genes in GBC cell lines may elucidate their likely functional role in in association with gallbladder cancer.
authors.affiliation: Centre for Genomics, Molecular and Human Genetics, Jiwaji University, Gwalior 474 011 MP, India.
authors.forename: Tekcham Dinesh
authors.initials: TD
authors.lastname: Singh
authors.affiliation: Department of Pathology, Cancer Hospital and Research Institute, Gwalior 474 007 MP, India.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Department of Surgical Oncology, Cancer Hospital and Research Institute, Gwalior 474 007 MP, India.
authors.forename: Braj Raj
authors.initials: BR
authors.lastname: Shrivastav
authors.affiliation: Centre for Genomics, Molecular and Human Genetics, Jiwaji University, Gwalior 474 011 MP, India. Electronic address: pktiwari.ju@gmail.com.
authors.forename: Pramod Kumar
authors.initials: PK
authors.lastname: Tiwari
mesh_terms: D000293:Adolescent; D000328:Adult; D019175:DNA Methylation / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003720:Densitometry; D044127:Epigenesis, Genetic; D005260:Female; D005706:Gallbladder Neoplasms / Q000235:genetics*; D042882:Gallstones / Q000235:genetics*; D052138:Genes, Neoplasm; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D016622:Silver Staining; D055815:Young Adult
chemical_list: D018076:DNA, Complementary
keywords: Biomarker; Epigenetic; Gall stone disease; Gallbladder cancer; Methylation & prognosis