Association of the promoter methylation and protein expression of Fragile Histidine Triad (FHIT) gene with the progression of differentiated thyroid carcinoma

Abstract

The role of aberrant methylation of fragile histidine triad (FHIT) promoters in the differentiated thyroid carcinoma (DTC) is not yet clear. Therefore, we investigated the association of the status of FHIT promoter methylation and FHIT protein expression with the clinicopathological progression of DTC, using PCR-based methylation assay and immunohistochemical technique. While no FHIT gene promoter methylation was observed in the matched non-cancerous epithelium (NCE) specimens, 24.6% of DTC samples demonstrated methylation in the FHIT promoter region. The protein expression of FHIT in NCE and DTC was 100.0% and 41.5% (P<0.01), respectively. There was a negative correlation between promoter methylation and protein expression of FHIT gene (P<0.05). Additionally, the methylation status appeared to be significantly associated with the pathological grade, tumor TNM stage, and lymph node metastasis (P<0.05), and FHIT proteins were weakly expressed in only about 20% of DTC with grade II pathological changes, TNM stage III/IV, or lymph node metastasis. Finally, the gender and tumor classification but not age marginally affected the promoter methylation and protein expression of FHIT. Our results suggest that methylation of the promoter region may play a key role in inactivation of FHIT - possibly leading to subsequent carcinogenesis and progression of DTC.

Keywords: Fragile histidine triad; carcinogenesis; differentiated thyroid carcinoma; methylation; tumor progression.

Figures

Figure 1

Representative results from PCR based Hpall restriction enzyme assay. A 282 bp PCR product is seen following incubation with Hpall in a methylated DTC (B: H), but not in an unmethylated DTC (A: H), as the methylated restriction site (CCGG) is resistant to digestion by the enzyme. All 65 NCE were unmethylated in the FHIT promoter region, while 16 of 65 DTC were methylated. MW: molecular weight ladder; W, water; U, undigested; H, incubated with Hpall; M, incubated with Mspl.

Figure 2

Representative micrographs of IHC staining intensity of FHIT protein in the thyroid tissue A, Normal thyrocyte shows strong cytoplasmic staining intensity (score 3; × 200). B, Normal thyrocyte shows moderate cytoplasmic IHC staining intensity (score 2; × 400). C, Follicular thyroid carcinoma shows weak cytoplasmic IHC staining intensity (score 1; × 400). D, Papillary thyroid carcinoma shows no FHIT-specific IHC staining (score 0; × 400).

Figure 3

FHIT gene promoter methylation and its protein expression in DTC and NEC. FHIT gene promoter methylation and its protein expression were shown in DTC (n=65) and NEC (n=65). **, p

Figure 4

Association of FHIT gene promoter methylation and its protein expression with pathological and clinical manifestations in DTC. FHIT gene promoter methylation and its protein expression in DTC with grand I (n = 43) and grade II (n = 22) pathological changes (A), TNM stage I/II (n = 48) and TNM stages III/IV (n = 17) (B), and metastasis (n = 26) and non-metastasis (n = 39) (C) were determined by PCR-based methylation analysis and IHC staining, respectively. *, p

Figure 5

Association of FHIT gene promoter methylation and its protein expression with gender, age and tumor classification in DTC. FHIT gene promoter methylation and its protein expression in DTC of (A) male (n = 19) and female (n = 46), (B) the patients at