The Effect And Significance Of Methylation At The Promoters Of Human Insulin Like Growth Factor Ⅱ Gene In Hepatocellular Carcinoma

Objective To investigate the status of methylation at promoters of Insulin like growth factor â…¡ (IGF-â…¡) in human hepatocellular carcinoma (HCC) tissues, assess the role of IGF-II promoters methylation in the process of occurrence and development of HCC.Methods Twelve HCC and pair-matching adjacent nonneoplastic tissues, 10 liver tissues of HBV, 7 normal liver tissues specimens, taken directly from patients and confirmed by pathology section, and the cell lines BEL7402, SMMC7721, HepG2, L02 were subjected to analysis. According to the templat, genomic DNA extracted from HCC tissue specimens, the promoters of IGF-â…¡ (P1-P4) were amplified by means of nested polymerase chain reaction (nested PCR) and then sequencing. Restriction enzyme Hpa â…¡ and Msp I were utilized to detect the methylation pattern of the IGF2 promoter. The genomic DNA extracted from tissue specimens and cell lines were modified by bisulfite sodium and determined by methylation specific PCR ( MSP ).Results The IGF-â…¡ promoters of HCC have the coincidence with the normal sequence published by MEDLINE (NT₀09308) .The IGF-II promoter P1 methylation was detected in 9 of 12 (75% ) HCC tissues, 8 of 12 (67%) adjacent nonneoplastic tissues and all cell lines, which contrasted sharply with HBV ( 20%, 2/10) and normal liver tissues (14%,1/7). All HCC, adjacent nonneoplastic tissues and cell lines have demethylation at IGF-â…¡ promoter P3, whereas only 40% (4/10) HBV and 29% (2/7) normal liver tissues stand on the same status. The demethylation at IGF-â…¡ promoter P4 was observed in all HCC, adjacent nonneoplastic tissues and cell lines, far different to HBV (10%, 1/10) and normal liver tissues (14%, 1/7). There is no different methylation status at IGF-â…¡ promoter P2 identified in all groups. No distinction of methylation was found at promoter P1-P4 between HBV and normal liver tissues. Four promoters of IGF-â…¡ display the similar methylation pattern in HCC between different degrees of tumor cell differentiation or sizes of primary focus.Conclusions The abnormal activity of IGF-â…¡ in HCC may be not correlated with gene deletion and mutation. The methylated P1 and demethylated P3, P4 could result in the inactive adult promoter P1 and active fetal promoter P3, P4 which led to aberrant expression of IGF-â…¡. The methylation status of IGF-â…¡ promoter P1-P4 is not correlated with HBV infected, the degree of tumor cell differentiation and the size of primary focus in HCC. The methylation of IGF-â…¡ promoters may play an capital role in the occurrence and development of HCC.