| Apolipoprotein B (ApoB) is an essential structural component of chylomicrons (CM) and low density lipoprotein (LDL), and plays a central role in lipoprotein metabolism and cholesterol transport. ApoB exists mainly in two isoforms:apoB-100and apoB-48. ApoB-48is a truncated form of apoB-100generated by mRNA editing via the apoB editosome. ApoB-100is the major protein component of LDL, which transports two-thirds of plasma cholesterol. ApoB-48is responsible for transport dietary lipids chylomicrons, which can also carry cholesterol, but can be cleared quickly. Low levels of plasma apoB-100and LDL-cholesterol can reduce the risk of atherosclerosis and coronary heart disease and other cardiovascular diseases. Study on apoB RNA editing is of great significance on explore the mechanism of RNA editing and the atherosclerosis caused by high cholesterol.Currently, research on apoB RNA editing is on the level of RNA. However, traditional techniques to monitor apoB RNA editing, which are based on RNA extraction and the direct measurement of RNA, are too much time consuming to be used to screen for medicines that target apoB RNA editing. In our previous study, following the characteristic of a CAA(Q) codon is converted into an in-frame translational UAA (stop) codon with a26-nt conservative RNA sequences, we set up a system to monitor apoB RNA editing using a plasmid harboring the fluorescent markers discosoma sp. red fluorescent protein (DsRed) and green fluorescent protein (GFP) upstream and downstream, respectively, of this26-nt sequence. This vector was transfected to Cos-7, Caco-2and CBRH-7919cell lines, we found that only Caco-2and CBRH-7919cells exhibited various colors of fluorescence. These results indicate that this fluorescent protein expressing vector can be used to detect apoB RNA editing and apoB RNA editing is related to the cell cycle.In order to further investigate the effect of cell cycle on apoB RNA editing, we constructed a lentiviral vector, which based on plasmid vector. The stable cell lines, Caco-2and CBRH-7919were obtained. The proportions of different colors at G2/M phase and the phase after the release from G2/M with treatment of colchicine and after removal of it were measured using confocal microscopy and flow cytometry.In confocal microscopy, we observed that the ratio of the red cells to the yellow ones (R2Y) significantly decreased in Caco-2and CBRH-7919stable cells when arrested in G2/M phase with treatment of colchicine. After release from this G2/M phase of arrestment, the R2Y ratio resumed. Quantitative analysis by flow cytometry, showed that the percentage of yellow cells was significantly lower in samples arrested in G2/M phase than that of untreated control samples (P<0.05). The percentage of yellow cells was significantly higher in samples released from G2/M phase arrest (colchicine removed) than that of those arrested ones (P<0.05). These results from both confocal microscopy and flow cytometry analyses suggested that apoB RNA editing is inhibited during G2/M phase and resumes after metaphase. These results at the cellular level were confirmed by sequencing method at the molecular level. Under microscope those bi-nuclear cells released from M phased arrest exhibited yellow fluorescence, not green fluorescence. This phenomenon suggested that apoB RNA editing resumes after cells pass through the spindle checkpoint following the removal of colchicine from the culture media.In this study, we developed an apoB RNA editing detection of lentiviral vector, which overcomes the shortage of plasmid vector low transfection efficiency. This detection system reflects the changing of apoB RNA editing homogeneously and stably in cells, which provides a new approach for study of apoB RNA editing. This study firstly showed that apoB RNA editing was regulated by the cell cycle, and the editing started from anaphase. This research provides a direction for apoB RNA editing on cell cycle. |
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