Role of CMKLR1 on mouse vascular smooth muscle cells proliferation and related mechanism

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To explore the proliferation property of vascular smooth muscle cells (VSMCs) in the stable CMKLR1 gene knock-down mouse VSMCs line and explore related mechanism. The short hairpin RNA sequence targeting to knockdown the coding regions of mouse CMKLR1 mRNA was synthesized and subsequently employed to construct recombinant lentivirus vector.Mouse VSMCs were cultured and infected with the recombinant lentivirus (knockdown VSMCs). mRNA and protein CMKLR1 expression in Knockdown VSMCs was measured by real-time PCR and Western blot and compared with those in normal VSMCs (vehicle VSMCs) and lentivirus control VSMCs (control VSMCs). The proliferation of normal, knockdown and control VSMCs was induced by platelet-derived growth factor-BB (PDGF VSMCs) and measured by cell number counting and BrdU.The phosphorylated c-Jun N-terminal kinase (p-JNK) protein was investigated by Western blot. The relative level of CMKLR1 mRNA in knockdown VSMCs (0.23±0.04) was significantly downregulated compared with which in vehicle VSMCs (1.05±0.05) as well as control VSMCs (0.99±0.04) (P<0.01). The relative level of CMKLR1 protein in knockdown VSMCs (0.29±0.04) was also significantly decreased, compared with which in vehicle VSMCs (1.06±0.04) as well as control VSMCs (0.95±0.02) (P<0.01). The VSMCs number ((50.33±1.20)×10(3)/cm(2)) and BrdU A450 nm value (1.80±0.05) in PDGF VSMCs were significantly increased in vehicle VSMCs ((42.02±1.53)×10(3)/cm(2,) 1.55±0.04) (both P<0.05). Compared with those in vehicle VSMCs, the VSMCs number ((23.33±2.03)×10(3)/cm(2)) and BrdU A450 nm value (1.32±0.02) in knockdown VSMCs were significantly decreased.The proliferation property between PDGF VSMCs and control VSMCs was similar(P>0.05). Compared with the relative level of p-JNK protein (1.03±0.03) in vehicle VSMCs, the p-JNK protein level was significantly increased in PDGF VSMCs (1.36±0.02, P<0.05) and significantly downregulated in knockdown VSMCs (0.79±0.05, P<0.05). Knockdowning CMKLR1 gene can reduce the proliferation of mouse vascular smooth muscle cells, which was related with the down-regulation of p-JNK expression.