Effects Of Pcsk9siRNA On THP-1 Derived Macrophages Apoptosis Induced By OxLDL

AIM: To study the expression of pcsk9 and apoptosis in THP-1 derived macrophages induced by oxLDL, to investigate inhibitory effect of siRNAs(small interfering RNA,siRNA) on pcsk9 gene expression and the effect of pcsk9 siRNA on THP-1 derived macrophages apoptosis induced by oxLDL .METHODS: THP-1 derived macrophages were induced to differentiation into macrophages by PMA treatment for 24h. The experiments were designed as follows: cells were incubated with oxLDL with a concentration of 0μg/ml, 25μg/ml, 50μg/ml, 75μg/ml,100μg/ml for 48h separately. The apoptosis of THP-1 derived macrophages was observed by staining with Hoechst33258. The expression of pcsk9 was analyzed by reverse transcription polymerase chain reaction and Western blot. siRNAs for pcsk9 gene was designed and synthesized, then be transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000.Transfect efficiency was assessed by fluorescence microscope assay. RT-PCR(reverse transcription polymerase chain reaction) and Western blot were conducted to detect the expression of pcsk9 after 24h,48h separately. The most efficient siRNA was selected to transfect into THP-1 derived macrophages.24h later,oxLDL was used to treat cells for 48h,and Hoechst 33258 staining and flow cytometer were conducted to determine apoptosis.RESULTS: As shown in Hoechst33258 staining assay,the number of cells with nuclear condensation induced by 75μg/ml oxLDL increased significantly. RT-PCR and Western blot showed that, in THP-1 derived macrophages, pcsk9 was upregulated with increasing concentration of oxLDL, while 75μg/ml oxLDL increased significantly. 6h after FAM-siRNA was transfected into THP-1 derived macrophages, we collected cells and visualized green fluorescence under the fluorescence microscope whereas the normal cells non-transfected siRNA could not be seen. The result showed that siRNA was successfully transfected into cells.RT-PCR and Western blot results showed that when transfected the most effective siRNA with different concentration such as 30nmol/L,50 nmol/L,80nmol/L into cells,the effect of suppression cut down obviously at 80nmol/L. Then we selected the mostly efficient siRNA-2 transfect into THP-1 derived macrophages,after 24h add 75μg/ml oxLDL for 48h, compared with control,the apoptosis of THP-1 cell transfected siRNA was suppressed by staining with Hoechst 33258 and flow cytometer.CONCLUSIONS: oxLDL could induce THP-1 derived macrophages apoptosis.The expression of pcsk9mRNA and protein were increased by oxLDL in a concentration-dependent manner, 75μg/ml oxLDL increased significantly.Expression of pcsk9 gene could be effectively suppressed by siRNA. THP-1 derived macrophages apoptosis induced by oxLDL could be effectively suppressed by pcsk9 siRNA.