慢病毒介导CCL5-siRNA对乳腺癌细胞生物学行为的影响

Effects of lentivirus-mediated CC chemokine ligand 5 （ CCL5 ）-RNAi on biological behaviors of human breast cancer cells （MDA-MB-231）

目的探讨CCL5、siRNA对高转移性人乳腺癌细胞生物学行为的影响。方法合成特异性CCL5－siRNA并克隆入慢病毒表达载体pGCSIL—GFP，将重组CCL5－siRNA慢病毒感染高转移性人乳腺癌细胞系MDA—MB-231设为KD组，另设阴性对照组和未感染组；分别采用实时定量聚合酶链反应和Western blot方法检测CCL5－siRNA对CCL5表达的作用；用噻唑蓝比色法、流式细胞术、克隆形成试验和Boyden小室穿透试验观察细胞的增殖、周期、体外聚集和侵袭力的改变。结果CCL5－siRNA慢病毒可显著降低MDA—MB-231细胞CCL5的表达，改变细胞形成克隆和运动侵袭的能力，但对细胞增殖的影响不明显。与阴性对照组（1．00±0．07）和未感染组（0．88±0．15）比较，KD组细胞CCL5mRNA的表达下降为（0．18±0．03），P〈0．01。在上样量相同的条件下，与阴性对照组（1．82±0．18）比较，KD组CCL5蛋白表达降低为（0．33±0．13），P〈0．01。细胞克隆计数：与阴性对照组（0．97±0．09）和未感染组（1．04±0．07）比较，KD组细胞的克隆数降为（0．33±0．10），P〈0．05；穿膜细胞计数：KD组（38±15）明显少于阴性对照组（77±11）和未感染组（69±9），P〈0．05。噻唑蓝比色实验和流式细胞分析提示：KD组细胞的增殖情况与阴性对照组、未感染组相比无明显差别。三组PI值分别为0．48±0．02、0．44±0．05及0．47±0．02（P〉0．05）。结论慢病毒介导的CCL5－siRNA可显著抑制高转移性人乳腺癌细胞的克隆形成和运动侵袭能力，但对其增殖活性无明显影响。

Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB- 231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and Western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting （FACS）. Colony formation and migration ability were determined by colony-forming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 ceils as well as the colony formation and migration ability decreased significantly, but cell＇s proliferation was not affected obviously. Compared with MDA-MB-231 （0. 88 ± 0. 15 ） and MDA-MB-231/CCL5-NC （ 1.00 ± 0. 07 ） cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0. 18 ± 0. 03, P 〈 0. 01. Compared with MDA-MB-23 I/CCL5-NC （ 1. 82 ± 0. 18 ） cells, the expression of CCL5 protein in MDA-MB-231 /CCL5-siRNA decreased to 0. 33 ±0. 13, P 〈0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly （P 〈 0.05）. The clone count in KD group was （0. 33 ± 0. 10）, which was a significant decrease from （0.97 ±0.09） （NC group） and （1.04 ±0.07） （CON group）, P 〈0. 05. The number of cells that migrated through the chamber membrane of KD group （38 ±15） was less than that of NC group （77 ± 11, P 〈0. 05） and CON group （69 ±9, P 〈0. 05）. However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL.5-NC and MDA-MB-231 （P 〉0. 05）, the proliferation index （PI） of group KD, NC and CON were （ 0. 48 ± 0. 02 ）, （ 0.44 ± 0. 05 ） and （ 0. 47 ± 0. 02 ） respectively. The difference was not statistically significant by multiple comparison （P 〉 0. 05 ）. Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.