探讨了SEMA3A基因在恶性胶质瘤细胞U251迁移和侵袭能力中的作用, 及其用于胶质瘤临床治疗的可行性. 用特异识别SEMA3A基因的短发卡RNA(short hairpin RNA,shRNA)片段与真核表达载体pFH-GFP-L连接, 再与辅助质粒联用来包装慢病毒. 通过荧光显微镜观察感染胶质瘤细胞U251的感染效率. 通过实时定量PCR技术和Western-blot技术验证了U251细胞中SEMA3A基因的沉默效果. 通过MTT法、PI染色法和Transwell小室分别检测了U251细胞增殖、细胞周期和运动能力的变化. 结果表明, SEMA3A-shRNA慢病毒感染U251能有效下调SEMA3A基因的表达水平(P <0.01), 使U251细胞的增殖能力、迁移和侵袭能力明显降低(P <0.05), 细胞大量阻滞在G2/M期, 并促进了细胞凋亡(P <0.05).
This paper explores the effect of knockdown SEMA3A gene on the migration and invasion of glioma cell line U251. Preliminary discussion of the potential in treatment of glioma was carried out. The highest knockdown efficiency plasmid packaged by a lentivirus packaging vector system was constructed, which contained a pFH-GFP-L vector and a short hairpin RNA (shRNA) sequence targeting the human SEMA3A gene. U251 cells were infected with a modified lentivirus vector. The infection efficiency of shRNA targeting SEMA3A gene was observed by fluorescence microscope. Real-time PCR reaction and
Western-blot were used to investigate the expression of mRNA and protein of SEMA3A. The results show that the lentivirus containing SEMA3A target shRNA are efficient in silencing SEMA3A expression (P < 0.01). Knockdown SEMA3A gene expression could significantly inhibit the proliferation and metastasis of U251 cells (P < 0.05). In addition,a large number of U251 cells are arrested in G2/M phase, promoting apoptosis (P < 0.05).
[1] 陈寿仁, 王占祥, 陈玉英. MicroRNA与胶质瘤侵袭性关系的研究进展[J]. 肿瘤防治研究, 2011,38(1): 106-108.
[2] Maya S. Semaphorin 3A regulates neuronal polarization by suppressing axon formation and promoting dendrite growth [J]. Neuron, 2011, 71(3): 433-446.
[3] Agustin C, Eric L, Chintan P, et al. Neuron-derived semaphorin 3A is an early inducer of vascular permeability in diabetic retinopathy via neuropilin-1 [J]. Cell Metba, 2013, 18: 505-518.
[4] Kei I K, Fumiyasu L. Deletion of SEMA3A or plexinA1/plexinA3 cause defects in sensory afferent projections of statoacoustic ganglion neurons [J]. PLoS One, 2013, 8: e72512.
[5] Mikihito H. Osteoprotection by semaphorin 3A [J]. Nature, 2012, 485: 69-76.
[6] Catalano A. Cross-talk between vascular endothelial growth factor and semaphorin-3A pathway in the regulation of normal and malignant mesothelial cell proliferation [J]. FASEB, 2004, 18: 358-360.
[7] Muller M. Association of axon guidance factor semaphorin 3A with poor outcome in pancreatic cancer [J]. Int J Cancer, 2007, 121: 2421-2433.
[8] Lorena C, Luca T. Semaphorin signaling in cancer cells and in cells of the tumor microenvironment-two sides of a coin [J]. J Cell Sci, 2009, 122: 1723-1736.
[9] Luca T, Paolo M C. Semaphorin signaling in cell migration [J]. Embo Rep, 2004, 5(4): 356-361.
[10] Quenten S, Chenghua G. Vascular endothelial growth factor controls neuronal migration and cooperates with SEMA3A to pattern distinct compartments of the facial nerve [J]. Genes Dev, 2004, 18: 2822-2834.
[11] Lisette M. Semaphorin 3A suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor [J]. Blood, 2008, 111: 2674-2680.
[12] Alfonso C. The neuron immune semaphoring-3A reduces inflammation and progression of experimental autoimmune arthritis [J]. J Immunol, 2010, 185: 6373-6383.
[13] Alfonso C, Paola C. Semaphorin-3A is expressed by tumor cells and alters T-cell signal transduction and function [J]. Blood, 2006, 107: 3321-3329.
[14] Bagnard D, Sainturet N. Differential MAP kinases activation during semaphorin 3A-induced repulsion or apoptosis of neural progenitor cells [J]. Mol Cell Neurosci, 2004, 25: 722-731.
[15] Hanae U, Tomhisa H. Semaphorin-3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer [J]. BBRC, 2011, 44: 60-66.
[16] Bagci T. Autocrine semaphorin 3A signaling promotes glioblastoma dispersal [J]. Oncogene, 2009, 28(40): 3537-3550.
[17] Pan H, Wanami L S. Autocrine semaphorin 3A stimulates alpha2 beta1 integrin expression/ function in breast tumor cells [J]. Breast Cancer Res Treat, 2009, 118: 197-205.
[18] Gang C, Jian S, Ming J. Semaphorin-3A guides radial migration of cortical neurons during development [J]. Nat Neurosci, 2008, 11: 36-44.
[19] Herman J G, Meadows G G. Increased class 3 semaphorin expression modulates the invasive and adhesive properties of prostate cancer cells [J]. Int J Oncol, 2007, 30: 1231-1238.
