Methylated Status Of Insulin-like Growth Factor-Ⅱ Gene Promoter Cpg Island Associated With Development Of Hepatocellular Carcinoma

Objective:The formation and development of hepatocellular carcinoma (HCC) is associated with the abnormal expression of many genes. Genetic and epigenetic changes regulate the expression of cancer-related genes, which is the core biological process in HCC. It was reported that insulin-like growth factor-Ⅱ(IGF-Ⅱ) was highly expressed in HCC tissue, while the methylation rates of IGF-Ⅱpromoter was low, which suggested that IGF-Ⅱgene expression was closely related to its promoter methylation status. In this study, the relationship between the changes of IGF-Ⅱpromoter methylation status and the expression of IGF-Ⅱwas investigated during rat hepatocarcinogenesis. Meanwhile, the correlations between the methylation status of IGF-Ⅱpromoter and the clinicopathological features in human HCC liver tissues were analyzed. The role of IGF-Ⅱpromoter methylation status in the course of HCC was explored.Methods: Hepatoma models were induced with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley (SD) rats. Morphological changes of livers were observed by pathological examinations (H&E staining). The CpG island methylation status of rat hepatic IGF-ⅡP2 was observed by methylation-specific polymerase chain reaction (MSP). Dynamic changes of IGF-Ⅱin rat livers and serum were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). Simultaneously, the methylation status of human IGF-Ⅱgene P3 CpG island was investigated by MSP in the self-control human HCC and normal control tissues.Results: H&E staining confirmed the rat hepatocytes from granule-like degeneration to atypical hyperplasia and HCC development after rats induced with 2-FAA. In the course of HCC development, an dynamic-increasing tendency of hepatic and serum IGF-Ⅱexpressions was found from normal liver tissues to degeneration to precancerous to cancerous ones. Positive relationship was found between hepatic and serum IGF-Ⅱexpressions (r = 0.97, t = 5.97, P =0.000). The methylation rates of rat IGF-ⅡP2 was 100% in control, 83.3% in degeneration, 11.1% in precancerous, and 0% in cancerous tissues, respectively. Significant differences were found between the precancerous, cancerous ones and the control ones and also between the cancerous and degeneration ones. The negative relationships were found between protein expression and methylation status. The methylation rates of IGF-ⅡP3 were 0% in human HCC, 47.5% in their adjacent, and 100% in normal tissues, respectively, with significant differences among them. No significant correlation was found between the methylation rates of IGF-ⅡP3 in HCC tissues and clinical parameters. However, in the adjacent tissues, the methylation rates of IGF-ⅡP3 in poor-differentiated HCC were lower than that in well-differentiated ones (P = 0.000 ). Meanwhile, the methylation rates of IGF-ⅡP3 in HBsAg-positive HCC patients was significantly lower than those with HBsAg-negative (P = 0.040 ). But no difference was found between the methylation rates and sex, age, tumor size and AFP.Conclusions:The expression of IGF-Ⅱgene is closely associated with the methylation status alteration of its promoter, suggesting that the demethylation modification contribute to the formation of HCC by up-regulating gene expression. It is helpful to explore the mechanism of methylation status alteration in the carcinogenesis of HCC.