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Loss of Imprinting of IGF2 and H19, Promoter Usage of IGF2, Loss of Heterozigosity of IGF2R and Helicobacter pylori Infection in Laryngeal Squamous Cell Carcinoma (LSCC)

The imprinted gene IGF2 codes for the mitogenic peptide, comprising 67 amino acids and contributing to tumor growth through its autocrine or endocrine effects. Its expression, directed from four different promoters, is regulated on the level of DNA methylation, since it is reciprocally imprinted with H19. IGF2 biallelic expression is known as loss of imprinting (LOI), which physiologically occurs in prenatal liver and chondrocytes, from the P1 promoter. IGF2/H19 LOI, IGF2-R loss of heterozygosity (LOH) analysis and presence of H. pylori were tested on 36 squamous laryngeal carcinomas (LSCC), in addition to IGF2 promoter usage analysis in all non-tumorous and tumorous laryngeal tissue that expressed IGF2. For imprinting analyses a RFLP based method was used, IGF2-R LOH was detected by PCR followed by polyacrilamide gel electrophoresis/silver staining. H. pylori presence was detected by nested PCR and promoter usage was analysed by RT PCR with promoter specific primers. The informativity for loss of imprinting (LOI) analyses was: 44% (16/36) for the IGF2 and 53% (19/36) for the H19. Biallelic expression of IGF2 was observed in 27% (4/15) of non-tumorous laryngeal tissues and in 40% (6/15) of LSCC tumors. H19 LOI was observed in 24% (4/17) of LSCC tumors. One tumor sample showed LOH of both IGF2 and H19 coupled with monoallelic expression of these genes. For IGF2R, 69% (25/36) of samples were heterozygous and LOH was detected in 12% (3/25) tumors. H. pylori was found in 16% (13/82) of carcinoma tissues. P1 promoter was used in 4/15 (26%) non-tumors (two with IGF2 LOI) and in 8/15 (53%) LSCC tumors (four with IGF2 LOI). We conclude that IGF2 and H19 imprinting is disturbed in LSCC and IGF2 promoter usage is highly variable in malignant and adjacent normal laryngeal tissue. The presence of H. pylori had no effect on IGF2/H19 imprinting. In addition, the P1 promoter usage is not responsible for IGF2 LOI.

loss of imprinting; IGF2; H19; loss of heterozigosity; IGF2R; Helicobacter pylori

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