RNA干扰趋化因子受体4基因表达对神经母细胞瘤体外侵袭能力的影响

Knockdown of chemokine receptor type 4 （CXCR4） expression blocks neuroblastoma metastasis by RNA interference

目的构建趋化因子受体4（Q（cR4）小分子干扰RNA（siRNA）表达载体，研究其对体外神经母细胞瘤侵袭能力的影响。方法选择CXCR4高表达的神经母细胞瘤SH-SY5Y细胞系，设计合成人CXCR4基因不同靶点的能编码siRNA的3条双链DNA序列，克隆到真核表达载体pSilencer^TMneo中构建siRNA表达载体，体外脂质体介导转染SH-SY5Y细胞，用半定量RT-TCR分析CXCR4基因mRNA的变化，用免疫组织化学和Westernblot分析CXCR4蛋白表达，Transwell小室检测细胞的侵袭能力。结果成功构建了CXCR4-siRNA表达载体，转染后半定量RT-PCR检测神经母细胞瘤细胞CXCR4mRNA丰度分别为siR1转染组0．32±0．09、siR2转染组0．35±0．13和siR3转染组0．33±0．11，相对于对照组0．58±0．13表达下降（P〈0．05）；转染后免疫组化检测神经母细胞瘤细胞CXCR4的蛋白表达分别为siR1转染组75．98±4．81、siR2转染组75．52±3．95和siR3转染组76．35±6．51，相对于对照组92．196±3．89表达下降（P〈0．01）；转染后Western blot检测神经母细胞瘤细胞CXCR4的蛋白表达分别为siRl转染组0．1103±0．0023、siR2转染组0．1203±0．0015和siR3转染组0．1308±0．0018，相对于对照组0．4832±0．0012表达下降（P〈0．01）；且转染后神经母细胞瘤细胞侵袭能力较对照组53．11±3．72降低（P〈0．05），siR1转染组为25．48±2．81、siR2转染组为30．89±2．77、siR3转染组为18．83±1．79。结论CXCR4-siRNA表达载体通过降低CXCR4基因的蛋白表达能显著抑制神经母细胞瘤细胞的体外侵袭能力，有望为神经母细胞瘤的基因治疗开辟新途径。

Objective To explore the effect of silencing chemokine receptor type 4 （CXCR4） by siRNA on the invasion capability of neuroblastoma cell line SH-SYSY in vitro. Methods Three siRNAs targeting CXCR4 were chemically synthesized and transfected into SH-SY5Y cells. The transfection efficiency was observed under fluorescence microscope. CXCR4 expression at mRNA and protein levels were detected by semi-quantitative RT-PCR and Western blotting. The invasion capability of the cells was evaluated by Boyden Chamber in vitro. Results Compared with control groups, after the SH-SY5Y cells being transfected with the three CXCR4 targeting siRNAs, CXCR4 mRNA in transfected cells significantly decreased （0. 32 ± 0. 09, 0. 35 ± 0. 13 and 0.33 ± 0. 11 vs 0. 58 ±0. 13, P〈 0. 05）, CXCR4 protein detected by immunohistochemistry was decreased （75.98 ± 4. 81, 75. 52 ± 3. 95 and 76. 35 ± 6. 51 vs 92. 196 ± 3.89, P〈0. 01 ）, CNCR4 protein detected by Western blotting was also decreased （0. 1103 ±0. 0023, 0. 1203± 0. 0015 and 0. 1308 ± 0. 0018 vs 0. 4832 ± 0. 0012, P〈0. 01）. The invasion capability of the SH-SY5Y cells was decreased 48 hours after the cells were transfeeted （25.48±2.81, 30.89±2.77 and 18.83±1.79 vs 53.11±3.72, P〈0.05）. Conclusions Silencing CXCR4 by siRNA decreases the invasion capability of SH-SY5Y cells.