The Investigation Of Function Of5’-untranslated Region Of LINE-1Retrotransposon In HepG2Cells

Aim: To explore the function of5’-UTR (5’-untranslated region) of LINE-1(long interspersed nuclear elements-1) Retrotransposon in HepG2cancer cells. Methods:Different lengths of5’-UTR were prepared and cloned including400bp,680bp and the whole length903bp by using conventional molecular cloning techniques. Sunsequently, the recombinent plasmids were constructed by inserting these gene fragments respectively into report plasmid harbored the luciferase gene. By transfecting these constructs into Human hepatoma (HepG2) cell, the expression of luciferase were detected. The commercial microRNA761kit was prepared by chemical synthesized. The methylation and histone acetylation of Ll-5’UTR were detected by epigenetic approaches in cotransfected HepG2cells. The protein-protein interaction was detected by co-immunoprecipitation.Results:The5’-UTR with different trancated fragments have a promoter activity, in which the680bp of5’-UTR appears the strongest one comparing to the full length of5’UTR. The activity of antisense promoter of5’-UTR is less than sense promoter. The microRNA761hypothesized that may derive from the3’sequence of5’-UTR don’t contribute to the suppression of3terminal in5’UTR. The Ll-ORF1p can suppress5’-UTR activity significantly. The over-expression of L1-ORFlp fail to alter the methylation satues of L1-5’UTR in genome but the alteration of the distribution of histone acetylation needs to be invbestigated furthermore. Notedly, the interaction of ORF-lp with AGO2protein, an important component in RISC, may be responsible for that the L1-ORF1p suppresses5’-UTR activity.Discussion:5’-UTR harbors both sense promoter and antisense promoter and the seqience in its3’terminal beyond680bp suppresses sense promoter activity. The inhibitation of promoter activity of5’-UTR by L1-ORF1may attribute to the alteration of the distribution of histone acetylation and the interaction of Ll-ORF1p with to AGO2protein, which the mechanism needs to be investigated further. To our knowledge, this is first time to report the negative feedback regulation of L1-ORF1p to itself expression. This work shed light on understanding the mechanism of LINE-1in regulation of tumor cells.