Smo siRNA对裸鼠食管癌移植瘤细胞凋亡的影响

SiRNA-mediated Smoothened gene silencing induces tumor cell apoptosis in a nude mouse model bearing human esophageal carcinoma

目的:探讨小干扰RNA(small interfering RNA,siRNA)对人食管癌裸鼠移植瘤细胞凋亡影响.方法:使用Smo siRNA转染人食管癌EC9706细胞,设无关序列组和空白对照组为对照,将转染好的细胞注射到裸鼠肩胛旁区,4wk后取瘤组织.采用免疫组织化学SP法、Western blot方法检测裸鼠移植瘤组织中Smo、Gli1蛋白的表达;应用原位杂交、半定量RT-PCR方法检测裸鼠移植瘤组织中Smo、Gli1 mRNA的表达;运用TUNEL法和透射电镜等方法观察裸鼠移植瘤细胞的凋亡情况.结果:转染siRNA后,免疫组织化学和原位杂交结果显示,3组组织中siRNA干扰组Smo蛋白(8.37±1.73)及mRNA(2.32±0.63)、Gli1蛋白(3.53±0.37)及mRNA(3.35±0.87)阳性表达细胞数均低于对照组(无关对照组Smo蛋白及mRNA分别为8.42±1.49、9.61±0.85,空白对照组Smo蛋白及mRNA8.37±1.73、9.82±0.63;无关对照组Gli1蛋白及mRNA分别为0.89±0.06、8.41±1.64,空白对照组Gli1蛋白及mRNA0.91±0.05、8.53±1.38),且差异均具有统计学意义(P<0.05);Westernblot及RT-PCR结果显示,与对照组相比(无关对照组Smo蛋白及mRNA分别为9.61±0.85、0.89±0.06;空白对照组Smo蛋白及mRNA为0.91±0.05、0.96±0.07;无关对照组Gli1蛋白及mRNA分别为0.87±0.08、0.89±0.07,空白对照组Gli1蛋白及mRNA分别为0.84±0.06、0.87±0.06;siRNA干扰组中的Smo(0.33±0.06)及mRNA(0.35±0.07)、Gli1(0.29±0.05)及mRNA(0.29±0.05)的表达量明显下调,组间两两相比差异具有统计学意义(P<0.05);TUNEL结果显示,siRNA干扰组凋亡率(apoptosis rate,AI)明显升高,组间两两比较AI差异具有统计学意义(P<0.05);透射电镜检测结果显示,实验组与对照组相比凋亡细胞数明显增多.细胞胞质固缩、电子致密度增高,可见凋亡早期细胞,染色质固缩并凝结成形态不同的块状,凋亡晚期细胞可见,细胞核裂解为碎块状,产生凋亡小体.两对照组超微结构无明显差异,细胞膜完整,线粒体等细胞器正常,细胞核基本正常.结论:Smo siRNA可通过下调裸鼠食管癌细胞中Smo基因的表达,进而体内诱导裸鼠食管癌移植瘤细胞的凋亡.

AIM： To investigate the inhibitory effect of small interfering RNA （siRNA）-mediated silencing of the Smoothened （Smo） gene on tumor cell apoptosis in a nude mouse model bearing human esophageal carcinoma. METHODS： An Smo siRNA or a non-relevant siRNA was used to transfect human esophageal carcinoma EC9706 cells. Non-transfected cells were used as a blank control. The transfectedcells were injected into the parascapular region of nude mice. After 4 wk, tumor tissue samples were taken to detect the expression of Smo and Gli1 proteins and mRNAs by immunohisto-chemistry, in situ hybridization, Western blot, semi quantitative RT-PCR, and to determine cell apoptosis by TUNEL assay and transmission electron microscopy. RESULTS： Immunohistochemistry and in situ hybridization analyses showed that the expression levels of Smo protein （8.37 ± 1.73 vs 8.42 ± 1.49, 8.37 ± 1.73） and mRNA （2.32 ± 0.63 vs 9.61 ± 0.85, 9.82 ± 0.63） and Gli1 protein （3.53 ± 0.37 vs 0.89 ± 0.06, 0.91 ± 0.05） and mRNA （3.35 ± 0.87 vs 8.41 ± 1.64, 8.53 ± 1.38） were significantly lower in the Smo siRNA group than in the non-relevant group and blank control group （all P 0.05）. Western blot and RT-PCR results also revealed that the expression levels of Smo protein （0.33 ± 0.06 vs 9.61 ± 0.85, 0.91± 0.05） and mRNA （0.35 ± 0.07 vs 0.89 ± 0.06, 0.96 ± 0.07） and Gli1 protein （0.29 ± 0.05 vs 0.87 ± 0.08, 0.84 ± 0.06） and mRNA （0.29 ± 0.05 vs 0.89 ± 0.07, 0.87 ± 0.06） were significantly lower in the Smo siRNA group than in the non-relevant group and blank control group （all P 0.05）. TUNEL results showed that the apoptosis rate increased significantly in the Smo siRNA group compared with two control groups （both P 0.05）. Transmission electron microscopy analysis revealed cytoplasmic pyknosis, increased electron density, chromatin condensation and condensation, nuclear fragmentation, and presence of apoptotic bodies in the Smo siRNA group. Two control groups had no such histopathological changes. The number of apoptotic cells increased significantly in the Smo siRNA group compared to control groups. CONCLUSION： SiRNA-mediated Smo gene silencing induces tumor cell apoptosis in a nude mouse model bearing human esophageal carcinoma.