miRNA-27a靶向SPRY2对退变椎间盘血管长入生成作用的影响

MiRNA-27a Targets SPRY2 to Play a Role in Degenerative Disc Vascular Growth

目的:探讨miRNA-27a靶向调控Sprouty同源物2(Sprouty Homolog 2, SPRY2)对人髓核细胞系(nucleus pulposus cells,NPCs)诱导人微血管内皮细胞(Human microvascular vascular endothelial cells, HMEC-1)血管生成作用的影响。方法:收集脊柱侧弯和椎间盘退变(Intervertebral disc degeneration, IDD)患者的椎间盘组织分别作为对照组和IDD组,通过miRNA芯片筛选差异表达的miRNA。RT-qPCR和荧光原位杂交实验验证组织中miR-27a的表达水平。慢病毒转染细胞,细胞分为Control组(未转染);NC组(转染慢病毒空载);sh-miR-27a组(转染miR-27a抑制慢病毒);miR-27a组(转染miR-27a过表达慢病毒);SPRY2组(转染SPRY2过表达慢病毒)及miR-27a+SPRY2组(转染miR-27a和SPRY2过表达慢病毒)。RT-qPCR检测髓核细胞中miR-27a和SPRY2的表达。双荧光素酶报告基因实验验证miR-27a和SPRY2的靶向关系。将经过不同处理的髓核细胞条件培养基与完全培养基混合培养HMEC-1细胞,Transwell和管腔形成实验检测HMEC-1细胞的侵袭和血管生成能力。免疫荧光和ELISA检测髓核细胞和混合培养基中转化生长因子-β1(Transforming growth factor-β1, TGF-β1)含量。结果:与对照组椎间盘组织相比,IDD组miR-27a表达明显增加。与NC组相比,SPRY2在sh-miR-27a组中表达升高(P<0.05),miR-27a组中表达降低(P<0.05)。与NC组相比,miR-27a组HMEC-1细胞侵袭和血管生成能力增强(P<0.05),TGF-β1表达上升(P<0.05);SPRY2组HMEC-1细胞侵袭数减少(P<0.05),管腔样结构未形成。与miR-27a组相比,miR-27a+SPRY2组HMEC-1细胞侵袭和血管生成能力下降(P<0.05),TGF-β1表达下降(P<0.05)。结论:miRNA-27a通过靶向抑制SPRY2的表达促进髓核细胞诱导HMEC-1细胞成血管能力。

Objective: To investigate the angiogenesis of human microvascular endothelial cells(HMEC-1) induced by human nucleus pulposus cells(NPCs) through miRNA-27 a/SPRY2. Methods: Intervertebral disc tissues from scoliosis and intervertebral disc degeneration(IDD) patients were collected as control group and IDD group, and differentially expressed miRNAs were screened by miRNA chip. The expression of miR-27 a in tissues was verified by RT-qPCR and fluorescence in situ hybridization.Lentivirus-transfected cells were divided into Control group(untransfected);NC group(transfected with lv-pNanog);sh-miR-27 a group(transfected with miR-27 a knockdown plasmid);miR-27 a group(transfected with miR-27 a overexpression plasmid);SPRY2 group(transfected with SPRY2 overexpression plasmid) and miR-27 a + SPRY2 group(transfected with miR-27 a and SPRY2 overexpression plasmid). The expression of miR-27 a and SPRY2 in nucleus pulposus cells was detected by RT-qPCR. verified the The targeting relationship between miR-27 a and SPRY2 was verified by dual-luciferase reporter gene assay. HMEC-1 cells were cultured by mixed medium which is mixed by conditioned medium of nucleus pulposus and complete medium. Transwell and lumen formation experiments were used to detect the invasion and angiogenesis ability of HMEC-1 cells. Transforming growth factor-β1(TGF-β1) expreesion in nucleus pulposus cells and mixed medium was detected by immunofluorescence and ELISA. Results: Compared with the control group,the expression of miR-27 a in IDD group was significantly increased. Compared with the NC group, the expression of SPRY2 was increased in the sh-miR-27 a group(P<0.05) and decreased in the miR-27 a group(P <0.05). Compared with the NC group, the invasion and angiogenesis of HMEC-1 cells in the miR-27 a group were enhanced(P<0.05), and the expression of TGF-β1 was increased(P<0.05);Invasion of HMEC-1 cell in the SPRY2 group was reduced(P<0.05), the lumen-like structure was not formed. Compared with the miR-27 a group, the invasion and angiogenesis ability of HMEC-1 cells in the miR-27 a + SPRY2 group reduced(P<0.05), and the expression of TGF-β1 decreased(P<0.05). Conclusion: miRNA-27 a promotes the ability of nucleus pulposus cells to induce angiogenesis of HMEC-1 cells by inhibiting the expression of SPRY2.