| Non-coding DNA thinked as junk DNA ever was approved to have an important role in cell regulation recently and given rised to more attention for its special function. L1(long interspersed repeated element 1,LINE-1)with broad distribution in genome, which composed about 17% in human genome for average. Most of L1 sequences are 5'truncted and only full-length sequence with 50-100 copies is considered to have a competence for retrotransposon. As an active retrotransposon, the activity of L1 are suppressed by epigenetics modification (hypermethylation in its promotor regions) in differentiated cells and, however, are actived in immortalized cells and early stage of embryonic development with a manner of TPRT(target-primed reverse transcription). L1 is thinked as"Dancer"in genome and contributed to genome-shapping greatly. Recent studies show that its activation is closely linked to tumor formation and progression. So, apparently, to study L1 will shade light on understanding of tumorigenesis progression and benefiting target-based drug development.In human tumors, the promotors of L1s are hypomethylated, which bonded to activate for its retrotransposition. To identify the alteration of methylation of CG islands in L1 promoter, we employed the approach of digestion with methylation- sensitivity restrictive enzyme prior for CG targeted site and subjected to PCR amplification with the primers franked up-stream and down-sream of this CG site. The priciples is as follows: the designed size of PCR product will appear if the CG site failed to be cut by methylation- sensitivity restrictive enzyme, and otherwise, the designed size of PCR product will not appear if the CG site is cut by methylation- sensitivity restrictive enzyme. To conduct this experiment, we used two isoschizomers, MSPI and Hpaâ…¡, which have same target sequence 5'-CCGG-3'. The DNAs propared from three tumors cells and normal blood cells were digested with HepG2 and MSPI respectively and followed by PCR amplification. As a result, compared to normal blood cells the CG sites of L1 promoter in tumor cells are hypomethylated.It is known that L1 encoded two proteins, which are ORF-1p and ORF-2p, and the features of ORF-1p are almost unknown except that its amount is excessive extremly than ORP-2p. Noteworthly, there is not any homology of ORF-1p with known proteins checked upto date. To study the function of L1 encoded ORF-1p in cancers cells, RNAi technology was employed, in which the siRNA sequences were designed based on its sequence by software in Ambion company website. By transfecting into different tumor cells, the down-regulation of L1 encoded ORF-1p was confirmed by the approaches of real time RT-PCR and Western blot respectively. Subsequently, the features of transfected tumor cells were characterized by the approaches of MTT,flow cytometry and soft-agar anchored colony formation. The experimental results shown that with significant down-regulation of L1 by siRNA the characters of transfected tumor cells were appeared to change as follows: Cell proliferation was significantly reduced, cell cycle was arrested in S phase, colony formation was significantly attenuated.To explore the mechanisms of cell cycle arrest, the cell cycle related protein, P21 and P15, were selected to detect their alteration upon expression. With recombinant constructs by reporter gene system, the expression of report gene were identified in transfected cells with down-regulation of L1 encoded ORF-1p. The result shown that up-regulation of p21 and p15 in transfected cells was significant. To screen ORF-1p response genes further, the gene chip was employed and as a result an amount of genes were identified, which were categorized into different signalling pathways after analyzing by bioinformation.Taken togather, the activation of L1 in different tumor cells was identified by hypomethylation in its promoter region. Subsequently, the transfected cells were characterizing by cell proliferation, cell cycle and colony formation. The mechanism concerning cell cycle arrest elicited by down-regulation of L1 encoded ORF-1was investigated further. Moreover, ORF-1p response genes were also identified by gene chip and subsequently were categorized into different signalling pathways.
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