趋化性细胞因子受体CXCR4基因沉默对大肠癌细胞体外侵袭及增殖能力的影响

Effect of specific suppression of chemokine receptor CXCR4 by short interference RNA on the invasion capability and proliferation of colorectal cancer cells

目的:研究趋化性细胞因子受体CXCR4短干扰RNA(siRNA)对大肠癌细胞系体外侵袭及增殖能力的影响.方法:利用T7 RNA聚合酶体外合成以CXCR4为靶基因的siRNA,用脂质体转染大肠癌SW480细胞,同时设立空白对照组和无关对照组.于转染后48h采用RT-PCR方法检测CXCR4 mRNA水平,免疫印迹方法检测CXCR4和MT1-MMP的蛋白质水平,Boyden小室模型检测体外侵袭能力的变化,流式细胞术检测细胞周期的分布情况,MTT法测定细胞增殖状况.结果:SW480细胞转染CXCR4 siRNA 48h后,与空白对照和无关对照相比,CXCR4 mRNA水平明显下调(51.53%±6.1% vs 78.4%±3.3%.P<0.01:51.53%±6.1% vs 87.4%±5.3%,P<0.01),CXCR4的蛋白质水平明显降低(47.3%±3.7% vs 107.2%±3.6%,P<0.01;47.3%±3.7% vs 114.7%±4.8%,P<0.01),MT1-MMP蛋白表达水平也明显下降(43.8%±2.5% vs 64.4%±4.4%,P<0.01;43.8%±2.5% vs 67.0%±2.9%,P<0.01),细胞的体外侵袭能力减弱(26.5%±6.1% vs 73.7%±3.4%,P<0.01;26.5%±6.1% vs 64.5%±5.7%,P<0.01),细胞周期的分布无明显差异.在无SDF-1存在的情况下,各组细胞的增殖无明显改变;经SDF-1刺激后,各组细胞增殖增加,但CXCR4 siRNA转染组细胞的增殖显著低于空白对照组和无关对照组(24h:0.55±0.03 vs 0.68±0.06,0.71±0.04,P<0.05;48h:0.67±0.04 vs 0.89±0.03,0.94±0.07,P<0.05;72 h:0.72±0.06 vs 1.36±0.08,1.53±0.07,P<0.01).以上各指标在空白对照和无关对照之间无显著性差异(P>0.05).结论:以CXCR4为靶向的siRNA能够有效下调CXCR4基因,降低SDF-1诱导的大肠癌细胞系体外侵袭能力及增殖海性.

AIM： To study the inhibitory effect of chemokine receptor CXCR4 short interference RNA （siRNA） on the invasion capability and proliferation of colorectal cancer cell line SW480. METHODS： SiRNA specifically targeting CXC chemokine receptor CXCR4 was designed in vitro using T7 RNA polymerase. Colorectal cancer SW480 cells were cultured under standard condition and the siRNA was transfected into SW480 cells with Lipofectamine 2000. Negative control and mock control were used at the same time. The levels of CXCR4 mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting 48 h after transfection respectively. The changes of invasion capability and MT1-MMP protein in SW480 cells were evaluated using Boyden Chamber invasion assay and Western blotting respectively. The cell cycle distribution and proliferation status were detected by flow cytometry and MTT assay respectively. RESULTS： Forty-eight hours after transfection with CXCR4 siRNA, in comparison with those in negative and mock control group, the levels of CXCR4 mRNA （51.53% ± 6.1% vs 78.4% ± 3.3%, P 〈 0.01; 51.53% ± 6.1% vs 87.4% ± 5.3%, P 〈 0.01） and protein （47.3% ±3.7% vs 107.2% ±3.6%, P 〈 0.01; 47.3% ± 3.7% vs 114.7% ±4.8%, P 〈 0.01） decreased significantly （47.3% ±3.7% vs 107.2% ±3.6%, P 〈 0.01; 47.3% ±3.7% vs 114.7% ±4.8%, P 〈 0.01）; the invasion capability （26.5% ±6.1% vs 73.7% ±3.4%, P 〈 0.01; 26.5% ±6.1% vs 64.5% ± 5.7%, P 〈 0.01） and the MT1-MMP protein expression （43.8% ± 2.5% vs 64.4% ±4.4%, P 〈 0.01; 43.8% ± 2.5% vs 67.0% ±2.9%, P 〈 0.01） in SW480 cells decreased significantly, but the cell cycle distribution didn＇t change remarkably. At the absence of SDF-1, cell proliferation status was similar to that in control and mock control group. After stimulation by SDF-1, the prolifera- tion of SW480 cells increased in all the groups, but it was much less active in the CXCR4 siRNA group than in the other two control groups. CONCLUSION： CXCR4 siRNA can inhibit the expression of CXCR4 and decrease the invasion capability and proliferation of SW480 cells.