Effect Of Core-binding Factor α1siRNA Targeted Suppression On Vascular Smooth Muscle Cells Calcification Induced By High Phosphorus

【Objective】To investigate the effect of Cbfα-1small interfering RNA (siRNA) targetedsuppression on osteogenic differentiation and calcification of vascular smooth musclecells (VSMC) induced by high phosphate in vitro, and to explore the role of Cbfα-1inVSMC calcification induced by high phosphate, in order to discuss the mechanisms ofvascular calcification of chronic kidney disease. It may provide the treatment of targetfor vascular calcification of chronic kidney disease.【Methods】1. The explants derived from thoracic aorta were used for primary culture ofvascular smooth muscle cells and identification of cells was carried on bymorphological identification and direct immunohistochemical staining of α-SMA.Passage3to8of cells were used for experiments.2. Four of the Cbfα-1siRNA were designed and synthesized. VSMC weretransfected by using cationic lipid vetors. Determined transfection efficiency by theFAM fluorescent labeling-siRNA. Screened effective siRNA sequences by RT-PCR.3. VSMC were transfected with effective siRNA sequences. VSMC were dividedinto four groups:(1) normal phosphate group (Pi1.3mmol/L);(2) high phosphategroup (Pi2.6mmol/L);(3) siRNA transfection group: high phosphate (2.6mmol/L)+transfection reagent+Cbfα-1-siRNA(effective siRNA sequences);(4) negativetransfection control group: high phosphate (2.6mmol/L)+transfection reagent+negative control siRNA. Cbfα-1and osteopontin(OPN) mRNA expression wasdetected by RT-PCR24and48h after transfection. Cbfα-1and OPN proteinexpression was determined by Western Blot48and72h after transfection. Calciumdeposition was visualized by Alizarin stain method6d after transfection. All of the experiments were repeated for3times at least.【Results】1. Under inverted phase contrast microscope we found VSMC werecharacterized by the ribbon spindle shape and showed “hill and valley” morphologicalappearance. Direct immunohistochemical staining of α-SMA showed positive stainingin the cultured VSMC, and there were more than95%VSMC in the cell cultures.2. The experiment showed that the transfection efficiency was about55%at theconditions of Cbfα-1siRNA with concentration of100nmol/L and Lipo according to8μL/well. The results of RT-PCR showed that Cbfα-1mRNA level was inhibitedmost significantly by Cbfα-1siRNA-1952with suppression ratio which was up to81.77%after transfection for24h. Cbfα-1siRNA-1952was finally chosed as effectivesequences.3.24h after transfection, compared with normal phosphate group, the Cbfα-1andOPN mRNA expression was significantly increased in high phosphate group (P<0.01).The expression of Cbfα-1mRNA in siRNA transfection group was significantly lowerthan that in high phosphate group and negative transfection control group(P<0.01),but the expression of OPN mRNA was no significant difference among three groups（P＞0.05）.48h after transfection, the expression of Cbfα-1and OPN mRNA insiRNA transfection group was lower than that in high phosphate group and negativetransfection control group (P<0.05), but whith was higher than that in normalphosphate group(P<0.05).4.48h after transfection, compared with normal phosphate group, the Cbfα-1andOPN protein expression was significantly increased in high phosphate group (P<0.01).The expression of Cbfα-1protein in siRNA transfection group was significantly lowerthan that in high phosphate group and negative transfection control group(P<0.01),but the expression of OPN protein was no significant difference among three groups（P＞0.05）.72h after transfection, the expression of Cbfα-1and OPN protein insiRNA transfection group was lower than that in high phosphate group and negativetransfection control group (P<0.05), but whith was higher than that in normalphosphate group(P<0.05). 5. Alizarin stain results showed that the calcium deposition in cell layers was notobvious in normal phosphate group, and the calcium deposition was obvious in highphosphate group and negative transfection control group respectively, but the calciumdeposition was significantly reduced in siRNA transfection group.【Conclusion】1. High phosphate induces VSMC calcification in vitro. Specific siRNA caneffectively inhibit the expression of the Cbfα-1mRNA and protein of VSMCtransfected by using cationic lipid vetors.2. Cbfα-1gene silencing can significantly inhibit the transformation of vascularsmooth muscle cells into osteoblast-like cells and calcification induced by highphosphate in vitro. Cbfα-1may be a potential therapeutic target in vascularcalcification of chronic kidney disease.