长链非编码RNA-ANCR对小鼠血管平滑肌细胞成骨样分化的影响

Effects of long non-coding RNA-ANCR on the osteoblastic differentiation of vascular smooth muscle cells

目的观察长链非编码RNA-抗分化非编码RNA(anti-differentiation non-coding RNA,ANCR)对动脉钙化细胞模型的影响,探讨其可能的作用机制。方法以10 mmol/Lβ-甘油磷酸钠(beta-glycerophosphate,β-GP)诱导小鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)成骨样分化以建立动脉钙化细胞模型,构建ANCR过表达慢病毒载体并感染小鼠VSMCs,q PCR检测Runt相关转录因子2(Runt related transcription factor 2,Runx2),骨形成蛋白-2(bone morphology protein-2,BMP-2)和ANCR的表达,Western blot检测胞质蛋白Runx2、BMP-2及核内核转录因子-Kappa B(nuclear factor kappa B,NF-κB)p65的表达,茜素红染色观察矿化结节的形成。结果 (1)β-GP刺激后小鼠VSMCs中Runx2和BMP-2的表达显著升高,差异有统计学意义(P<0.05),茜素红染色可见明显的矿化结节。(2)β-GP刺激后小鼠VSMCs中ANCR的表达显著降低,Runx2和BMP-2的mRNA表达显著增加,差异有统计学意义(P<0.05)。(3)ANCR过表达慢病毒感染后与对照组相比,Runx2和BMP-2的mRNA水平和蛋白水平均显著下降,NF-κBp65的蛋白表达显著降低,钙化结节形成显著减少,差异有统计学意义(P<0.05)。结论 ANCR能抑制Runx2和BMP-2的表达,且可能通过削弱NF-κB信号通路抑制β-GP诱导的小鼠VSMCs成骨样分化。

Objective To investigate the effects of the long non-coding RNA-anti-differentiation non-coding RNA （ANCR） on the osteoblastic differentiation of vascular smooth muscle cells （VSMCs） and explore its possible mechanism. Methods 10 mmol/L of beta-glycerophosphate （β-GP） was used to induce osteoblastic differentiation of mouse VSMCs, which was infected by lentivirus vectors over-expressing ANCR. The mRNA expression of Runt related transcription factor 2 （ Runx2）, bone morphology protein- 2 （ BMP- 2） and long non-coding RNA-ANCR were detected by quantitative polymerase chain reaction （qPCR）. The protein expression of Runx2, BMP-2 and nuclear factor kappa B （NF-KB） p65 subunit were determined by Western blot. The formation of mineralized nodules was detected by Alizarin Red Staining. Results （ 1 ） The expression of Runx2 and BMP-2 significantly increased in mouse VSMCs stimulated by β-GP and the formation of mineralized nodules stained by Alizarin Red was also significant. （2） The expression of ANCR significantly decreased in the mouse VSMCs stimulated by 13-GP, the expression of Runx2 and BMP-2 was also significantly increased. （3） Over-expression of ANCR significantly reduced the mRNA and protein expression of Runx2 and BMP-2 in β-GP-stimulated VSMCs, decreased the protein expression of NF-KB and attenuated the formation of mineralized nodules. Conclusion ANCR may decrease the expression of Runx2 and BMP-2. ANCR may also attenuate the osteoblastic differentiation of VSMCs through inhibiting the NF-KB signaling pathway.