Epigenetic profiling of gallbladder cancer and gall stone diseases: Evaluation of role of tumour associated genes

• Methylation frequency of 14-3-3 sigma, MASPIN and THBS1 genes increase from early to advanced GBC grades.
• Higher frequency of methylation and lower expression level of genes characterize the advanced GBC cases.
• SOCS1, HLTF, FLNC and COX2 genes seem not to have any specific epigenetic role in GBC and GSD.
• Epigenetic guided expression analysis may be useful in early diagnosis/prognosis of the disease as well as in therapy.

BackgroundAs 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.Aim of studyTo study the promoter methylation of certain tumour associated genes in the molecular pathogenesis of gallbladder cancer and gall stone diseases.Materials and methodsMethylation 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.ResultsWe 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.ConclusionThe 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.

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