沉默Survivin基因对子宫内膜癌移植瘤生长及细胞凋亡影响

Effect of Silencing Survivin gene inhibition on growth and apoptosis of endometrial cancer cells derived transplantation tumor

目的 Survivin是凋亡抑制蛋白家族的新成员,具有肿瘤特异性,只表达于肿瘤和胚胎组织,且与肿瘤细胞的分化增殖及浸润转移密切相关。利用慢病毒转染短发夹RNA(short hairpin RNA,shRNA)技术抑制人RL95-2子宫内膜癌细胞Survivin基因表达,探讨沉默Survivin基因对人子宫内膜癌细胞移植瘤生长及肿瘤组织内细胞凋亡影响。方法建立人子宫内膜癌细胞裸鼠移植瘤模型,构建基因靶向Survivin-shRNA序列,利用慢病毒载体介导转染细胞,设置实验组、对照组和空白组,其中实验组原位注射Survivin-shRNA慢病毒至肿瘤组织内,对照组原位注射阴性对照shRNA慢病毒至肿瘤组织内,空白组未予以特殊处理。每隔1周计量注射治疗后移植瘤体积变化并绘制生长曲线。注射治疗4周后,处死全部裸鼠,应用qPCR检测肿瘤组织内Survivin mRNA水平的变化,蛋白质印迹法检测凋亡相关Survivin蛋白和Caspase-3蛋白表达情况,Tunel法检测组织细胞凋亡情况,HE染色观察肿瘤部位组织学变化。结果皮下接种裸鼠RL95-2子宫内膜癌细胞2周即可成瘤,成瘤大小约1.0cm×0.8cm。注射治疗4周后,qPCR检测结果显示,实验组、对照组和空白组Survivin mRNA表达分别为0.243±0.032、0.995±0.027和1.004±0.021,3组间均值差异有统计学意义,F=795.623,P<0.001;两组间比较显示,实验组Survivin mRNA表达水平较其他两组下降,差异均有统计学意义,P<0.05。蛋白质印迹法检测结果显示,实验组、对照组和空白组Survivin蛋白相对表达量分别为0.174±0.064、0.619±0.145和0.628±0.116,3组间均值差异有统计学意义,F=15.691,P=0.004;两组间比较显示,实验组Survivin蛋白水平较其他两组下降,差异均有统计学意义,P<0.05。实验组、对照组和空白组Caspase-3蛋白相对表达量分别为0.407±0.092、0.095±0.015和0.093±0.017,3组间均值差异有统计学意义,F=32.33,P=0.001;两组间比较显示,实验组Caspase-3蛋白较其他两组升高,差异均有统计学意义,P<0.05。Tunel法染色结果表明,实验组细胞凋亡率较对照组和空白组显著升高,P<0.05。连续4周测定各分组肿瘤体积变化,与对照组或空白组相比,Survivin-shRNA慢病毒瘤体注射能使实验组人子宫内膜癌细胞裸鼠移植瘤体积显著缩小,至第4周时平均肿瘤体积为(0.46±0.07)cm3,肿瘤体积减少33%,P<0.05。结论 Survivin-shRNA慢病毒瘤体注射可显著抑制RL95-2子宫内膜癌移植瘤生长,诱导细胞凋亡。Survivin基因可作为抗子宫内膜癌治疗潜在靶点。

OBJECTIVE Survivin is a new member of the apoptosis suppressor protein family. It is tumor-specific and only expressed in tumor and embryo tissues, and is closely related to the differentiation, proliferation and infiltration of tumor cells. Utilizing specific short hairpin RNA（shRNA） to silence the expression of Survivin gene of RL95-2 endometrial cancer cells and to investigate the effect of Survivin gene inhibition on growth and apoptosis of endometrial cancer cells derived transplantation tumor. METHODS The nude mice model of xenografted endometrial cancer and the Survivin gone targeted specific shRNA Lentivirus plasmid were constructed. According to the experiment aim, all tumor-bearing mice were randomly divided into experimental group, control group and blank group. Survivin-shRNA Lentivirus plasmid was intratumorally injected in the experimental group, negative control shRNA Lentivirus plasmid was intratumorally in- jected in the control group, and the blank group was untreated. Changes of tumor volume in each group were monitored once a week and drew the tumor growth curve. After injection therapy for 4 weeks, nude mice were sacrificed and the expression of Survivin mRNA was measured with qPCR, and the expression of apoptosis-related protein, including Survivin protein and Caspas-3 protein, was measured with Western Blot. After iniection therapy for 4 weeks, the ceil apoptosis was tested by using Tunel staining and the histologic changes of tumor sites was detected by HE staining. RESULTS After 2 weeks of RL95-2 endometrial cancer cells transplantation in subcutaneous area of nude mice, tumor size is detected at about 1.0X0.8 cm. After injection therapy for 4 weeks, the results of qPCR showed Survivin mRNA expressions in the experimental group, control group and blank group were 0. 247±0. 035, 0. 997±0. 028 and 1. 007±0. 019, respectively, the mean difference was statistically significant among the three groups （F= 795. 623, P〈0. 001）, and the expression level of Survivin mRNA in the experimental group was lower than that of the other two groups, and the difference was statistically significant （P〈0.05）; the results of Western Blot showed, the Survivin protein expression in experimental group, control group and blank group were 0. 171±0.069, 0. 598±0. 176 and 0. 176±0. 125, respectively, the mean difference was statistically significant among the three groups （F=15. 691, P=0. 004） and the expression level of Survivin protein in the experimental group was lower than that of the other two groups, the difference was statistically significant （P〈0.05） ; the Caspase 3 protein expression in experimental group, control group and blank group were 0. 401±0. 107, 0. 093±0. 018 and 0. 018±0. 026, respectively, the mean difference was statistically significant among the three groups （F= 32.33, P=0. 001）. The expression level of Caspase-3 protein in the experimental group was higher than that of the other two groups, and the difference was statistically significant （P〈0.05）. Compared with the control group and the blank group, the cell apoptosis rate in the experimental group was obviously higher, P〈0.05. Compare the tumor volume of each group, experimental group can significantly narrowed the tumor volume of transplanted endometrial canc or, at the fourth week, the average tumor volume was （0.46±0.07） cm^3 , and the tumor volume decreased by 33%, P〈0.05. CONCLUSIONS Silencing Survivin gone expression by the Survivin-shRNA Lentivirus plasmid injection can inhibit the growth of transplantation tumor and induce apoptosis of endometrial cancer cells. Survivin might be a potential target for anti-endometrial cancer therapy.