Survivin-shRNA 对A431 皮肤鳞状细胞癌 抑制作用的实验研究

Inhibitory effect of survivin-shRNA on A431 skin squamous cell carcinoma

摘要:

目的 观察Survivin-shRNA 对A431 皮肤鳞状细胞癌的作用效果，探讨核因子κB（NF-κB）在 Survivin-shRNA 对皮肤鳞状细胞癌（SCC）抑制作用中的分子生物学机制。方法 构建Survivin-shRNA 腺病 毒载体，筛选最佳干扰序列。将培养好的A431 细胞混悬液皮下接种于裸鼠，复制A431 裸鼠移植瘤模型，随机 分为空白对照组、阴性对照组、Survivin-shRNA 转染组和Res 阳性对照组，每组5 只，瘤内注射相应试剂，第 20 天处死所有裸鼠，取瘤组织分别测量瘤体积、瘤重，绘制肿瘤生长曲线，计算抑瘤率；HE 染色观察细胞形 态；TUNEL 检测细胞凋亡情况；Western blot 法测定Survivin、IκB、P65、P53、Caspase-3 蛋白的表达。结果 Survivin-shRNA 转染组或Res 阳性对照组的裸鼠移植瘤，瘤体积及瘤重降低，与空白对照组及阴性对照组比 较差异有统计学意义（P <0.05）；TUNEL 检测结果示Survivin-shRNA 转染组、Res 阳性对照组的裸鼠移植瘤， 细胞凋亡率增高，与空白对照组及阴性对照组比较差异有统计学意义（P <0.05）；Survivin-shRNA 转染组及 Res 阳性对照组可见肿瘤细胞较少，分布稀疏，有变性的液化坏死灶，癌细胞皱缩变圆，胞核固缩；SurvivinshRNA 转染组或Res 阳性对照组裸鼠移植瘤，Survivin、P65 蛋白的表达均降低，与空白组对照组及阴性对照组 比较，差异有统计学意义（P <0.05），IκB、P53、Caspase-3 蛋白表达增高，与空白对照组及阴性对照组比较， 差异有统计学意义（P <0.05）。结论 Survivin-shRNA 可以抑制皮肤鳞状细胞癌裸鼠移植瘤的生长，其机制 之一可能是通过抑制NF-κB 信号通路，活化抑癌基因P 53，进而促进肿瘤细胞凋亡，抑制SCC 移植瘤的生长。

Abstract:

Objective To observe the effect of survivin-shRNA on skin squamous cell carcinoma (SCC) A431 cells, to and explore the molecular biological mechanism of NF-κB in the inhibition of survivin-shRNA on skin SCC. Methods Survivin-shRNA adenovirus vector was constructed to screen the best interference sequence. The cultured A431 cell suspension was subcutaneously inoculated in nude mice to construct the transplanted tumor model of A431. The nude mice were randomly divided into a blank group, a rAd-EGFP negative control group, a rAdsurvivin- shRNA transfection group and a Res positive control group, with 5 rats in each group. The corresponding reagents were injected into the tumors. All the mice were sacrificed on the 20th day. The tumors were isolated, the volume and weight of the tumors were measured, the tumor growth curves were drawn, the tumor inhibition rate was calculated. HE staining was used to observe the morphology of the tumor cells. TUNEL was used to detect cell apoptosis. Western blot was applied to test the protein expression levels of survivin, IκB, p65, p53 and caspase3. ANOVA was used to analyze the results. Results After survivin-shRNA or Res treatment in the nude mice, the tumor volume and weight decreased significantly, there were significant differences compared with the blank group and the rAd-EGFP negative group (P < 0.05). The results of TUNEL showed that the apoptosis rates were significantly increased in the rAd-survivin-shRNA transfection group and the Res positive control group, the two groups had significant differences from the blank group and the rAd-EGFP negative group (P < 0.05). In the survivin-shRNA transfection group and the Res positive control group, microscopic observation showed that decreased and sparse tumor cells, degenerative liquefaction necrotic foci, cancer cell shrinkage and becoming round, and karyopycnosis. After survivin-shRNA or Res treatment of nude mice, the expressions of survivin and p65 protein were decreased, the differences were statistically significant compared with the blank and rAd-EGFP negative groups (P < 0.05), while the expressions of IκB, p53 and caspase3 proteins were significantly increased compared with the blank control group and the rAd-EGFP negative control group (P < 0.05). Conclusions Survivin-shRNA can inhibit the growth of skin SCC xenografts in nude mice, one of the mechanisms may be through inhibition of the NF-κB signaling pathway, activation of tumor suppressor gene P53, and promoting apoptosis of tumor cells, eventually leading to inhibited growth of SCC xenografts.