| Human insulin-like growth factor II (IGF- II) is an important fetal growth factor and a cell mitogen. The human gene encoding IGF-II is located on chromosome Ilpl5.5 and contains nine exons and four different promoters(Pl~P4),of which exons 7,8,and the first part of exon 9 code for the IGF- II precursor protein. The 5'part of the gene consists of noncoding leader exons 1~6, of which exons l,4,5,and 6 are each preceded by a distinct promoter (Pl~P4).Under the control of these four promoters, the gene yields five kinds of mRNA species, all of which appear to give rise finally to the same mature protein, a 67-amino acid polypeptide. The four promoters can be activated in a development-dependent and tissue-specific manner. In fetal and neonatal livers promoters P2, P3 and P4 are active, of which P3 is the most active promoter ,and promoter PI is inactive. In adult liver,however,Pl becomes dominant and produces more than 50% of the total IGF- II transcripts, and the activities of P2~P4 decrease significantly or are lost.In recent years it has been found that the overexpression of IGF- II gene was observed and was an early event during hepatocarcinogenesis. It is believed that the mitogenic peptide exerts its autocrine and/or paracrine bioactivity through its interaction with the type I IGF receptor on the hepatocyte surface, which promotes the progression of the cell cycle in G1-"S restriction point, enhances DNA and protein synthesis, and at last results in malignant transformation of hepatocytes.lt has been reported that the overexpression of IGF- II is correlated with the reactivation of P3 andP4 and the loss of PI activity. But the causes and mechanisms responsible for the PI, P3, and P4 activity alterations are not well understood.Thus in the present study we cloned the P1~P4 promoters of IGF- II gene as a normal DNA sequence control from L-02 human fetal hepatocyte line, and the nucleotide sequences and methylation status of the four promoters of the IGF- II gene were detected in the livers of patients with hepatocellular carcinoma and in human hepatoma cells. The correlation of the four promoter nucleotide sequences and methylation status with their transcription control activities was investigated. Structure of the PI ~P4 promoters and their interaction with transcription factors were analyzed in the cell lines using dual luciferase reporter assay system and electromobility shift assay(EMSA)in order to determine the mechanisms by which the P1,P3 and P4 are controlled and their relation to IGF- II overexpression in hepatocellular carcinogenesis.Part I Cloning and sequencing of the human IGF- II gene P1~P4 promotersObjective: To clone the P1~P4 promoters of the human IGF-II gene from L-02 human fetal hepatocyte line and hepatocellular carcinoma tissues, and to analyze their nucleotide sequence differences and the correlation of the four promoter nucleotide sequences with their transcription control activity, which is the basic work for further investigation.Methods: According to the complete nucleotide sequence of the IGF- II gene, a nested primer PCR was performed for amplifying P1~P4 promoter fragments of the gene from L-02 cell line and 8 hepatocellular carcinoma tissues. These PCR products were analyzed by agarose gel electrophoresis, and cloned by using TOPO TA Cloning kit. The positive clones containing P1~P4 fragments were selected and confirmed by sequencing.Results: (1)The P1~P4 promoter fragments of the IGF-II gene that were successfully amplified from the L-02 cell line and 8 hepatocellular carcinoma tissueswere 942bp, 684bp, 1425bp,and 1246bp, respectively. No deletion and mutation were examined in 8 hepatocellular carcinoma tissues as compared with the L-02 cell line and their nucleotide sequences were accordant with GenBank data.Conclusion: The P1~P4 promoters of the IGF-II gene were cloned successfully. (D In this study the four promoter nucleotide sequences from 8 hepatocellular carcinoma tissues are conservative and this may not related to the transcription control mechanism... |
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