SET8 regulates high-phosphorus-induced vascular smooth muscle cell calcification via AKT signaling pathway

Objective To explore the role and mechanism of lysine methyltransferase SET8 in calcification induced by high phosphorus in vascular smooth muscle cells (VSMCs). Methods (1) Male SD rats were selected for in vivo experiments and randomly divided into sham operation group and chronic renal failure vascular calcification group. The thoracic aorta was taken and calcification was detected by von Kossa staining. The expression of SET8 and Caspase-3 was detected by immunohistochemistry. (2) VSMCs were randomly divided into normal group and high phosphorus group (10 mmol/L β-glycerophosphate). Cellular calcification was detected by O-cresol hydrazide complex colorimetric assay and alizarin red staining. Apoptosis was detected by flow cytometry. The expressions of SET8, AKT and Caspase-3 were detected by RT-PCR and Western blotting. (3) In order to further verify the role of SET8 in the apoptosis of VSMCs, liposome transfection was used, and cells were divided into three groups: SET8-shRNA group, empty plasmid group and normal control group. Cellular calcification was detected by O-cresol hydrazide complex colorimetric assay and alizarin red staining. Apoptosis was detected by flow cytometry. The expressions of SET8, AKT and Caspase-3 were detected by RT-PCR and Western blotting. Results (1) In vivo experiments, compared with the sham operation group, vascular calcium deposition in the chronic renal failure vascular calcification group was significantly increased (P<0.05). Immunohistochemistry results showed that SET8 expression was significantly decreased and Caspase-3 was significantly increased in the vascular calcification group (both P<0.05). Correlation analysis showed that SET8 was negatively correlated with vascular calcification and Caspase-3 (r=-0.948, P<0.01; r=-0.961, P<0.01). (2) In vitro, the calcium deposition in the high-phosphorus group was significantly higher than that in the normal group (P<0.05). The results of flow cytometry showed that the number of apoptosis in the high-phosphorus group was significantly higher than that in the normal group (P<0.05). RT-PCR and Western blotting showed that, compared with the normal group, the mRNA and protein expression of SET8 and AKT in the high-phosphorus group decreased, and the mRNA and protein expression of Caspase-3 increased (all P<0.05). (3) After interference of SET8 gene expression, calcification and apoptosis of VSMCs significantly increased, AKT mRNA and protein expression decreased, and Caspase-3 mRNA and protein expression increased (all P<0.05). Conclusions SET8 can inhibit vascular calcification. One of the possible mechanisms is to inhibit the expression of Caspase-3 via promoting AKT activation, thereby inhibiting the apoptosis of VSMCs, and then participating in the regulation of VSMCs calcification induced by high phosphorus. Key words: Myocytes, smooth muscle; Histone-lysine N - methyltransferase; Caspase 3; AKT; β-glycerophosphate