The Investigation Upon Mechanism Of LINE-1Response Genes In HepG2Cells

In the eukaryotic genomes, many repetitive DNA sequences were identified in the introns of genes. Some of them are non-coding sequences, these sequences were characterized to participate in regulation of gene transcription and translation. The activation of these elements can change the expression of adjacent genes. It can also cause mutation, expression alteration and genom remodelling if it inserts in or near the coding sequence. Among these repetitive sequences, a retrotransposon called LINE-1which is widely distributed in the genome is found to play an important role in cell proliferation, differentiation and tumorigenesis. LINE-1is inactivated in normal differentiated cells by promoter hypermethylation. But however, it can be activated during the development of germ cell, embryonic and tumor cell. To date, LINE-1is identified be activated in almost all tumor cells, suggesting that the activation of LINE-1may involve in the common mechanism of tumorigenesis. So clarify the regulatory mechanism of LINE-1apparently facilitate to understand the tumorigenesis, molecular mechanisms of development and regulation. Apparently, LINE-1is a promising target for tumor drugs.The full-length LINE-1gene encoded two proteins, termed ORF1p and ORF2p, which are required for its retrotransposition. The mechanism termed Target-primed reverse transcription (TPRT) is considered to be responsible for this process. Among two proteins encoded by LINE-1, ORF2p encompasses nucleic acid enzymes and exonuclease activity, while ORF1p has the activity of nucleic acid binding with its own RNA to form RNP. Comparating to ORF2p, the function of ORF1p is still keep obscrue. To identify and characterize ORF1p response genes, Genechip was employed and some interesting genes were validated and characterized. With transfected HepG2cells, the screened ORF1p response genes were confirmed by real time RT-PCR. The condidate genes franked by LINE-1homology sequence in their upstream and downstream respectively were analyzed furthermore. To categorize the contribution of different transcripts in LINE-1,5'UTR with sense and antisense string as well as ORF1regions were investigated by recombinating construction. The some genes identified above were investigated by their expressions, alteration of promoter methylation and mRNA degradation in trasnfected HepG2cells.