Study On The Heterogeneity Of APOB100-MRNA Editing In Cells

Apolipoprotein B (ApoB) is an essential structural component of low density lipoprotein (LDL) and chylomicrons (CM) responsible for triglyceride and choelsterol tranport. LDL functions as the major carrier of plasma cholesterol in human. ApoB protein exists mainly in two isoforms:apoB-48and apoB-100. They both are encoded by the same gene. ApoB mRNA editing is a process that the cytidine (C6666) is deaminated to uridine, leading to an in-frame translational stop codon---UAA. Thus, the truncated form---apoB-48is produced. ApoB-48-containing lipoprotein is cleared more rapidly than LDL. Therefore, the risk suffering from LDL-cholesterol related diseases declines as the level of LDL in plasma drops down. This study on apoB mRNA editing is of a great significance on the quest for the mechanism of RNA editing and/or the atheroslerosis because of high cholesterol.Research on ApoB mRNA editing now is on the level of RNA in some cell lines. Traditional research methods are not convenient for high-throughput detection of apoB mRNA editing for their high background and complexity of RNA extraction and its instability. As a stop codon was produced within the conserved sequence in some cell lines, the apoB-expressing vector within DsRed was inserted with both GFP and the conserved apoB mRNA editing sequence on this convenience in this experiment. Cell lines, Caco-2and CBRH-7919, transfected with the above expression vector were used. These transfected cell lines were either present in green or yellow. It suggests that there exit heterogeneity of apoB editing among the same cell line.Cells, present on two colors---green and yellow, were cultured separately after sorting with FCM. Yellow cells took on green after cultureing for48h, and vice versa. This suggests that the heterogeneity of apoB RNA editing is of dynamic factors in cells instead of the property of each cell.Transfection with constructed vectors were done in both cell lines arrested by colchicine in M phase. Both cell lines were mainly present in green under a LSCM, and a lot of yellow cells arose in the green ones after30h cultured without colchicine. Quantitive analysis of cells treated with colchincine by FCM, showed that proportion of yellow ones to all cells with color in Caco-2or CBRH-7919cells declined14.0%(p<0.01) or6.0%(p<0.01) compared with their respective controls. After colchincine removal, the proportion increased8.9%(p<0.01) or16.8%(p<0.01) respectively, compared with their respective colchicine treatments. Data of FCM confirmed the observation under LSCM. Both methods together confirmed that the heterogeneity of apoB mRNA editing was because of cell cycle. That is to say, apoB mRNA editing shows difference during cell cycle process. This preliminarily suggests that those characteristics in developmental stages of apoB mRNA editing be related with the cell cycle phases.High-throughput apoB mRNA editing detection was constructed in this experiment. With the aid of this design, apoB RNA editing could be observed in cells under microscope directly. The level of editing could be measured by FCM, too. This experiment preliminarily showed that apoB mRNA editing was a dynamic process dependent on cell cycle. The study provides a new sight for apoB mRNA editing.