Effect Of Lentivirus Mediated Cdks Interference On Apob RNA Editing

Apolipoprotein B (ApoB) is an essential structural component of lipoprotein rich in triglyceride (CM, VLDL) and cholesterol (LDL),and plays a central role in lipoprotein metabolism and transport. ApoB exists mainly in two isoforms:apoB-100 and apoB-48. ApoB-48 is a truncated form of apoB-100 generated by RNA editing via the apoB editosome. During ApoB RNA editing, a conservative sequence of 26 nt is enough to ensure the transition from CAA to UAA. ApoB-100 is the only receptor of LDL, which transports plasma cholesterol to all tissues in LDL form. ApoB-48 is responsible for synthesis and secretion of dietary lipids chylomicrons,with a very short half-life, while it can be recognized and cleared quickly. As low levels of plasma apoB-100 and hypercholesterolemia can reduce the risk of atherosclerosis andand other cardiovascular diseases, study on apoB RNA editing is of great significance on exploration of the mechanism of RNA editing and the atherosclerosis caused by high cholesterol.In previous study, a plasmid vector with the 26 bp conservative sequence was constructed, harboring the fluorescent markers red fluorescent protein (DsRed) and green fluorescent protein (GFP) upstream and downstream, respectively.Two cell lines with ApoB RNA editing function,Caco-2 and CBRH-7919 were transiently transfected with this vector, exhibiting multi-color of fluorescence in constrast that nonfunctional cell line of ApoB RNA editing exhibited monocolor of green fluorescence. It suggested that RNA editing be indicated by fluorescent proteins beside the RNA editing site. The green fluorescence of cells changed into multi color after several days culture of the mixed cells sorted by cytometer. Further arresting of the cycled transformed cell with this plasmid with cholchicine, showed that color change was synchronized with cycling phases. This previous study in our lab then suggested that the apoB RNA editing is related to cell cycling.In order to further confirm the effect on cell cycle of ApoB RNA editing, lentiviral vectors for stable transfection were constructed on the basis of the transient expression vector in the experiment. The stably-transfected cell lines, Caco-2 and CBRH-7919, were then got. Both stable lines, were then transfected with RNAi lentiviral vectors silencing CDK1 or CDK2 gene that arrests cells in M or S phase during cell cycle. Cells silenced with CDK1 or CDK2 were checked by confocal microscopy and flow cytometry. Results under confocal microscope showed that red and yellow fluorescence appeared in multinucleated cells instead of the mononulceated cells. Also the proportion multinucleated cells vs. monoculeated ones increased obviously in contrast with the control of no CDK1 silence. While there was no significant change as for CDK2 silence. Flow cytometric analysis data are otherwise showed similar trendency to that under microscopy, the red and yellow cell proportion increasing obviously in comparision to its control (P<0.01). On the other hand, cells of CDK2 silencing had no significant change in contrast with its nonsilenced controls (P>0.05). Cycling phase was analyzed with flow cytometry. Data showed that those CDK1-silenced cells were arrested in late G2/M. These similar results were further confirmed by sequencing method at the molecular level. Those above experiments suggested that ApoB RNA editing start from anaphase in cell cycling.In this study, an ApoB RNA lentivirus mediated editing detection system was constructed, which was very homogeneous and stable in reporting change ApoB RNA editing in cells and had higher transfection efficiency than that of the transient transfection with plasmid. It provides a new method for apoB RNA editing. This study firstly proved that apoB RNA editing be regulated in the cell cycle, and the editing started from anaphase. This research provides a solid support for apoB RNA editing on cell cycle.