利用RNA干扰技术抑制Galectin-3的表达对人大肠癌细胞系LOVO增殖和凋亡的影响

Influences of inhibiting Galectin-3 by RNA interference technique on the proliferation and apoptosis of human colorectal cancer cells LOVO

目的:观察Galectin-3表达与肿瘤细胞增殖和凋亡的关系,探讨利用RNA干扰技术作为肿瘤基因治疗的方法。方法:实验于2005-06/2006-12在南方医院消化研究所完成。从PubMed的序列号NM-002306中按照引物设计软件设计并化学合成4条Galectin-3小干扰片断,从中选出有效的干扰片断,并顺势转染大肠癌细胞系LOVO(为Galectin-3siRNA组),用未转染细胞做为正常对照,以转染阴性干扰片断作为空白对照,免疫印记法验证干扰结果,MTT方法观察干扰后24,48和72h各组细胞增殖吸光度(A值),流式细胞仪检测各组细胞凋亡情况。结果:①用顺势转染的方法成功转染了Gal-3siRNA片断,转染后72h收集细胞并提取细胞浆蛋白,免疫印记法验证结果显示Galectin-3siRNA组灰度值(55.790)与正常对照(97.234)及空白对照的灰度值(90.459)相比,galectin-3表达明显减弱(P<0.05)。②细胞增殖情况:干扰后24,48和72hGalectin-3siRNA组细胞继续生长,但细胞生长减慢,A值低于正常对照和空白对照(24h:1.018±0.002,1.478±0.185,1.169±0.005;48h:2.049±0.008,2.635±0.003,2.532±0.009;72h:2.512±0.506,3.213±0.006,3.060±0.002;P均=0.000)。③细胞凋亡率:各组中凋亡中晚期均多于早期,Galectin-3siRNA组高于正常对照和空白对照[早期:(15.900±2.508)%,(3.813±1.305)%,(5.780±1.56)%;中晚期:(19.750±0.780)%,(5.050±0.120)%,(7.667±0.145)%;P均=0.000]。结论:成功转染并抑制了galectin-3在大肠癌细胞系lovo中的表达,干扰后细胞增殖减慢,凋亡增加。

AIM： To observe the relationship of expression of Galectin-3 with the proliferation and apoptosis of tumor cells and explore the RNA interference technique for anticancer gene therapy. METHODS： The experiment was performed at the Institute of Gastroenterology, Nanfang Hospital from June 2005 to December 2006. Four galectin-3 small interfering RNA （siRNA） was constructed by chemistry synthesis according to primer design software （accession No. NM-002306 in PubMed）. Effective siRNA was collected, and then transfected into colorectal cancer LOVO （as Galectin-3siRNA group）. Non-transfected cells were used as normal control, and negative RNA as blank control. Western-blot was used to verify the interference result. Cell proliferation （absorbance value） was observed by MTT after interference for 24, 48 and 72 hours. Cell apoptosis was measured with flow cytometer. RESULTS： （1）Gal-3siRNA was successfully transfected by direct transfer. After 72 hours, the cytoplasm protein was extracted after collecting cells. Western-blot showed that the expression of Galectin-3 was markedly decreased in Galectin-3siRNA group in gray value（55.790） than normal control（97.234） and blank control group（90.459）（P 〈 0.05）.（2） Cell proliferation： Cells proliferated still in the hGalectin-3siRNA group after 24, 48 and 72 hours, but very slow, and the A value was lower than the normal control group and blank control group （24 hours：1.018±0.002,1.478±0.185,1.169±0.005; 48 hours：2.049±0.008,2.635±0.003,2.532±0.009; 72 hours：2.512±0.506,3.213±0.006,3.060±0.002;P all =0.000）. （3）Cell apoptosis rate： The apoptosis mostly appeared in the middle and advanced stages. It was higher in the Galectin-3siRNA group than the normal control group and blank control group [early stage： （15.900±2.508）%, （3.813±1.305）%, （5.780±1.56） %; middle and advanced stages： （19.750±0.780） %, （5.050±0.120） %, （7.667±0.145） %; P all =0.000]. CONCLUSION： The galectin-3siRNA is successfully transfected in lovo and the expression is significantly decreased. The interference of galectin-3siRNA inhibits the proliferation of the tumor cells, but promotes the apoptosis of tumor cells.