| Survivin基因的shRNA 干扰对结肠癌SW480 细胞增殖和凋亡的影响* |
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| 引用本文： | 王学虎, 傅仲学, 赵 渝, 谌 伟, 吴星烨, 王春毅. Survivin基因的shRNA 干扰对结肠癌SW480 细胞增殖和凋亡的影响*[J]. 中国肿瘤临床, 2010, 37(9): 485-489. DOI: 10.3969/j.issn.1000-8179.2010.09.002 |
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| 作者姓名： | 王学虎 傅仲学 赵渝 谌伟 吴星烨 王春毅 |
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| 作者单位： | 作者单位:重庆医科大学附属第一医院普通外科(重庆市400016) |
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| 基金项目： | 国家自然科学基金，教育部高等学校博士学科点专项科研基金 |
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| 摘 要： | 目的：以携带shRNA 片段的腺病毒为载体，对结肠癌细胞SW480 中Survivin基因的表达进行干扰，研究其对肿瘤细胞中Survivin基因沉默效果及对细胞周期、凋亡和增殖的影响。方法：将构建好的携带Survivin-shRNA 片段腺病毒，体外转染结肠癌细胞株SW480。以EGFP为报告基因，采用流式细胞计数测定不同感染复数（MOI）下的转染效率，选取适当病毒浓度进行下一步实验。通过RT-PCR 和Western blot检测基因沉默后结肠癌细胞内Survivin mRNA和蛋白的表达水平；在Annexin V-FITC 和PI染色后通过流式细胞术检测并分析Survivin基因沉默后细胞周期和凋亡的变化；同时采用噻唑蓝（MTT）法、克隆增殖实验对细胞不同时期增殖活性进行观察，明确对细胞增殖的抑制时效性。结果：腺病毒转染后MOI 值在0～50时，剂量与转染效率成正比，确定最佳MOI 值为50并进行后续实验；shRNA 干扰后细胞内Survivin mRNA和蛋白表达水平降低，较对照细胞组差异有统计学意义（P<0.01）。 流式细胞仪检测结果显示基因沉默后细胞凋亡率升高，与对照组相比差异有统计学意义（P<0.01）。 同时基因沉默后，细胞周期也有明显变化，表现为G1/S 期细胞增多和G2/M期细胞减少，与对照细胞组相比差异有统计学意义（P<0.05）。 MTT 和单克隆平板实验均显示Survivin基因的沉默，对细胞的增殖和生长均具有明显抑制作用（P<0.05）。 结论：采用腺病毒对结肠癌进行靶向Survivin基因的shRNA 干扰，能有效降低目的基因的表达。其介导的Survivin基因的沉默，可以有效的诱导结肠癌细胞凋亡，同时Survivin基因可以通过抑制细胞G1/S 期转化来阻止细胞分裂，抑制细胞生长。
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| 关 键 词： | Survivin RNA干扰 增殖 凋亡 结肠癌 |
| 收稿时间： | 2009-04-21 |
| 修稿时间： | 2009-09-01 |
Influence of the Survivin Gene on the Proliferation and Apoptosis of Colon Carcinoma Cells |
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| WANG Xuehu, FU Zhongxue, ZHAO Yu, SHEN Wei, WU Xingye, WANG Chunyi. Influence of the Survivin Gene on the Proliferation and Apoptosis of Colon Carcinoma Cells[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2010, 37(9): 485-489. DOI: 10.3969/j.issn.1000-8179.2010.09.002 |
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| Authors: | WANG Xuehu FU Zhongxue ZHAO Yu SHEN Wei WU Xingye WANG Chunyi |
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| Affiliation: | Department of General Surgery, First Affiliated Hospital of Chongqing Medical University, Chongqing400016, China |
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| Abstract: | Objective:To evaluate the interference effects of using a replication-defective adenovirus vector on the Sur -vivin gene in the colon carcinoma cell line SW480 and to investigate the proliferation and apoptosis of SW480 cells after Survivin gene silencing. Methods:To study the biological function of Survivin in colon cancer, RNA interference was applied to knock down survivin expression in the colon cancer cell line SW 480 by recombinant replication-defective adenovirus pro -ducing Survivin small hairpin RNA (Survivin-shRNA). The infection rates were detected at different multiplicity of infection (MOI) at 48h after transfection and the optimized one was chosen for the subsequent experiments. After Survivin gene si-lencing, expression of Survivin mRNA and protein was detected by RT-PCR and Western blot, respectively. The cell cycle and the degree of cell apoptosis were analyzed by flow cytometry (FCM) at 48h after transfection. To analyze the effect of shRNA-Survivin on the proliferation of tumor cells, we performed MTT and colony formation assays. Results: The infection rate was significantly different when the MOI was at 10-50, and the rate was dose-dependent. There was no difference be -tween MOI= 50and MOI=100 . The optimized MOI was 50and was used at this level for the following experiments. RT-PCR and Wertern blot results demonstrated that expression of Survivin protein and the Survivin gene was down-regulated in co-lon cancer cells after Survivin gene silencing. The rate of apoptosis was obviously increased after Survivin gene silencing (P<0.05). After Survivin gene silencing, the number of cells in the G1/S phase was increased and that in G2/M phase was decreased, with a significant difference (P<0.05). MTT and colony-formation assays showed that shRNA-Survivin had an obvious inhibitory effect on the growth of colon cancer cells in the experimental group (P<0.05). Conclusion:Survivin was over expressed in colon cancer SW480 cells and the replication-defective adenovirus vector carrying Survivin-shRNA can suppress expression of Survivin mRNA and protein and thus can induce cell apoptosis and block cell division by blocking cells in G1/S phase. |
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| Keywords: | Survivin |
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