沉默Y盒结合蛋白-1表达抑制SH-SY5Y细胞增殖促其凋亡的作用与机制

目的:观察短发夹RNA（shRNA）沉默Y盒结合蛋白-1（YB-1）对人神经母细胞瘤SH-SY5Y细胞增殖、凋亡的影响及其调控机制。方法:构建针对YB-1的短发夹RNA真核表达载体，利用LipofectamineTM2000脂质体转染SH-SY5Y细胞，检测蛋白表达水平，鉴别干扰效果；四甲基偶氮唑蓝比色法（MTT）检测干扰与否对SH-SY5Y细胞增殖的影响；流式细胞技术分析干扰与否对SH-SY5Y细胞凋亡的影响；Western blot法检测CyclinD1、Bcl-2及Bax在蛋白水平的变化。结果:Western blot结果显示转染组YB-1表达低于对照组和无效转染组，后两组无显著差异；MTT法检测显示转染组细胞增殖水平低于对照组，无效转染组细胞增殖水平接近对照组；流式细胞术检测结果显示转染组凋亡细胞数占总细胞数的（23.21±1.90）%，高于对照组（5.05±0.83）%（P<0.01），无效转染组（6.24±1.05）%与对照组无显著性差异（P>0.05）；Western blot技术检测转染组CyclinD1、Bcl-2蛋白水平较对照组明显下降，Bax升高，无效转染组同对照组无明显差异。结论:shRNA介导的YB-1表达沉默可抑制SH-SY5Y细胞CyclinD1、Bcl-2表达，促进Bax表达，进而抑制细胞增殖、促进凋亡。

Silencing of Y-box binding protein-1 suppresses neuroblastoma SH-SY5Y cell proliferate and promotes apoptosis

Objective:To investigate the influence of short hairpin RNA (shRNA) mediated silencing YB-1 on neuroblastoma SH-SY5Y cell proliferation and apoptosism then discuss regulatory mechanism.Methods:Establishing the small interfering RNA (siRNA) eukaryotic expression vector targeting YB-1 of human neuroblastoma SH-SY5Y cells，transfection was performed using LipofectamineTM2000 liposome.The expression of YB-1 protein was detected with Western blot after interference.The proliferation of SY5Y cells were detected by MTT assay.The apoptosis of cells was analyzed by flow cytometry.Detected the expression of CyclinD1，Bcl-2 and Bax by Western blot.Results:Expression of YB-1 in transfected group （YB-1 shRNA group） was lower than control group （SH-SY5Y group) (P<0.05).There was no significant difference between control group and invalid transfected group （NC group）.MTT assay showed that cell proliferation level of YB-1 shRNA group was significantly lower than SH-SY5Y group.NC group was similar to SH-SY5Y group.The number of apoptotic cells in YB-1 shRNA group (23.21±1.90)% was higher than SH-SY5Y group (5.05±0.83)% (P<0.01).There was no significant difference between NC group (6.24±1.05)% and SH-SY5Y group (P>0.05) detected by flow cytometry analysis.The level of CyclinD1 and Bcl-2 protein in YB-1 shRNA group was lower than SH-SY5Y group.The level of Bax protein in YB-1 shRNA group was more than SH-SY5Y group.NC group was similar to SH-SY5Y group that detected by Western blot.Conclusion:shRNA mediated silencing YB-1 suppressed neuroblastoma SH-SY5Y cell to proliferate and promoted apoptosis by down regulating CyclinD1 and Bcl-2，as well as up regulating Bax.