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

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-231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(MTT)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.MTF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A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组克降数目(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.

Abstract：

Objective To investigate the effect of suppression of CCI5 ligand gene on the proliferation of human breast cancer cells. Methods A lentiviral vector carrying a short interfering RNA (siRNA) targeting CCL5 was transfected 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/CCL5-siRNA and MDA-MB-231/CCL5-siRNA was decreased markedly. The colony number of MCF-7/CCL5-siRNA group was (0. 34 ± 0. 08), significantly lower than 0. 81 ± 0. 12 in the MCF-7/CCL5-N group and 0.92 ± 0.12 in the MCF-7 group (P ＜ 0. 05). The colony number of MDA-MB-231/CCL5-siRNA group was 0. 33 ± 0. 10,significantly lower than 0.97 ±0.09 in the MDA-MB-231/CCL5-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/CCL5-siRNA cells was not significantly different from that of MCF-7/CCL5-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/CCL5-siRNA, MCF-7/CCL5-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/CCL5-siRNA, MDA-MB-231/CCL5-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 in human breast cancer cells may significantly suppress their colony formation ability, rather than affecting their population doubling time to some extent.

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

Objective To investigate the effect of suppression of CCI5 ligand gene on the proliferation of human breast cancer cells. Methods A lentiviral vector carrying a short interfering RNA (siRNA) targeting CCL5 was transfected 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/CCL5-siRNA and MDA-MB-231/CCL5-siRNA was decreased markedly. The colony number of MCF-7/CCL5-siRNA group was (0. 34 ± 0. 08), significantly lower than 0. 81 ± 0. 12 in the MCF-7/CCL5-N group and 0.92 ± 0.12 in the MCF-7 group (P ＜ 0. 05). The colony number of MDA-MB-231/CCL5-siRNA group was 0. 33 ± 0. 10,significantly lower than 0.97 ±0.09 in the MDA-MB-231/CCL5-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/CCL5-siRNA cells was not significantly different from that of MCF-7/CCL5-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/CCL5-siRNA, MCF-7/CCL5-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/CCL5-siRNA, MDA-MB-231/CCL5-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 in human breast cancer cells may significantly suppress their colony formation ability, rather than affecting their population doubling time to some extent.