抑制CC类趋化因子配体5基因表达对乳腺癌细胞增殖能力的影响

Effects of down-regulation of CC chemokine ligand 5 （ CCL5 ） on proliferation of human breast cancer cells in vitro

目的探讨抑制CC类趋化因子配体5（CCL5）基因表达对人乳腺癌细胞增殖能力的影响。方法用特异性CCL5 RNA干扰（RNAi）序列慢病毒载体感染人乳腺癌细胞MCF-7和MDA-MB-231，分别为KD1组和KD2组；另在MCF-7和MDA-MB-251细胞中分设阴性病毒载体感染的阴性对照组（NC1组和NC2组）和未感染组（CON1组和CON2组）。采用实时定量逆转录聚合酶链反应（RT—PCR）检测转染病毒后乳腺癌细胞中CCL5的表达，四甲基偶氮唑蓝（MTF）比色法和流式细胞术（FACS）分析细胞的增殖情况，平板克隆形成实验观察细胞的克隆形成能力。结果CCL5 RNAi慢病毒可显著降低MCF-7和MDA-MB-231细胞中CCL5基因的表达。MTT法检测结果显示，在不同的培养时间，MCF-7和MDA-MB-231细胞的KD组、NC组与CON组细胞培养上清一值差异均无统计学意义（均P〉0．05）。FACS分析结果显示，KD1组、NC1组和CON1组的增殖指数（PI）值分别为0．48±0．03、0．43±0．01和0．45±0．02；KD2组、NC2组和CON2组的PI值分别为0．48±0．02、0．44±0．05和0．47±0．02（两两比较，均P〉0．05）。荧光显微镜下观察显示，KD组的克隆体积及每克隆的细胞数明显小于NC组和CON组。KD1组和KD2组克隆数目（0．34±0．08和0．33±0．10）明显少于NC1组（0．81±0．12）、NC2组（0．97±0．09）、CON1组（0．92±0．12）和CON2组（1．04±0．07），差异有统计学意义（P〈0．05）。结论CCL5基因的表达下调对乳腺癌MCF-7和MDA-MB-231细胞群体倍增时间无明显影响，但可显著降低细胞的克隆形成能力，从而使肿瘤细胞的恶性增殖受到抑制。

Objective To investigate the effect of suppression of CCL5 ligand gene on the proliferation of human breast cancer cells. Methods A lentiviral vector carrying a short interfering RNA （siRNA） targeting CCL5 was transfeeted into human breast cancer cell line MCF-7 and MDA-MB-231 cells. The expression of CCL5 mRNA in the cells was detected by real-time PCR. The proliferation of MCF-7 and MDA-MB-231 cells was assessed by MTT assay and FACS assay, and the colony formation ability of both cell lines were measured, respectively. Results Real time PCR showed a good knockdown effect of CCL5 in both cell-lines. Colony-forming assay showed that the ability of colony formation of MCF-7/CCLS-siRNA and MDA-MB-231/CCLS-siRNA was decreased markedly. The colony number of MCF-7/CCLS-siRNA group was （0.34 ±0.08）, significantly lower than 0.81 ±0.12 in the MCF-7/CCIS-N group and 0.92 ±0.12 in the MCF-7 group （P 〈 0.05）. The colony number of MDA-MB-231/CCLS-siRNA group was 0.33 ± 0. 10, significantly lower than 0.97 ±0.09 in the MDA-MB-231/CCLS-N group and 1.04 ±0.07 in the MDA-MB- 231 group （P 〈0.05）. However, MTT assay revealed that the proliferation of MCF-7/CCLS-siRNA cells was not significantly different from that of MCF-7/CCLS-N or MCF-7 cells, respectively （ P 〉 0.05 ）, and the same result was found in MDA-MB-231 cells. FACS assay showed that the proliferation index （PI） of groups MCF-7/CCLS-siRNA, MCF-7/CCLS-N and MCF-7 were 0.48 ± 0.03, 0.43 ± 0.01 and 0.45 ± 0.02. The PI of groups MDA-MB-231/CCLS-siRNA, MDA-MB-231/CCLS-N and MDA-MB-231 cells were 0.48 ± 0.02, 0.44 ±0.05 and 0.47 ± 0.02. There was no statistical difference among them （ P 〉 0.05 ）.Conclusion The down-regulation of CCL5 gene Jin human breast cancer cells may significantly suppress their colony formation ability, rather than affecting their population doubling time to some extent.