摘要
目的:通过RNA干扰(RNA interference,RNAi)抑制人食管鳞癌细胞Eca109Bmi-1表达,探讨其对Eca109细胞生物学特性的影响及可能机制。方法:通过对Bmi-1的小干扰RNA(siRNA)重组腺相关病毒(Bmi-1shRNA)转染Eca109细胞,以转染空病毒(AAV-Null)的Eca109细胞作阴性对照组,以未转染Eca109细胞作空白对照组(PBS),应用蛋白质印迹法分析,绘制细胞生长曲线、流式细胞检测、裸鼠成瘤实验和免疫组化分析等方法观察RNAi对Bmi-1基因的抑制情况及沉默Bmi-1表达在体外及体内对Eca109细胞增殖、侵袭、致瘤和凋亡的影响。结果:转染Bmi-1shRNA后蛋白质印迹法检测结果显示,Bmi-1shRNA组蛋白条带灰度扫描值为681.74±28.89,明显低于AAV-Null组的964.00±41.73(P=0.001)和空白对照组的1 005.51±41.16,P=0.001;AAV-Null组与空白对照组比较,差异无统计学意义,P=0.007。实验组细胞生长曲线较平缓,细胞生长速度较对照组(P=0.009)和空白对照组(P=0.031)明显降低。Eca109细胞转染AAV-Bmi-1shRNA后,Bmi-1shRNA组增殖指数(proliferation index,PI)为0.42±0.13,明显低于AAV-Null组的0.81±0.37(P=0.023)和空白对照组的0.91±0.25(P=0.006),提示RNAi后细胞增殖活性明显降低。裸鼠成瘤实验提示转染Bmi-1shRNA后肿瘤生长减慢,Bmi-1shRNA组肿瘤质量(1.47±0.35)g明显低于AAV-Null组的(1.83±0.28)g(P=0.028)和空白对照组的(1.96±0.40)g,P=0.004;Bmi-1shRNA组细胞凋亡指数(16.9±5.31)%明显高于AAV-Null组的(9.57±3.84)%和空白对照组的(8.13±3.97)%,差异有统计学意义,P值均为0.001。结论:沉默Bmi-1表达能显著抑制Eca109细胞生长,减慢肿瘤细胞的增殖,促进凋亡,抑制肿瘤生长。
OBJECTIVE:To investigate the effect of Bmi-1 gene on the proliferation and apoptosis of human esopha- geal squamous carcinoma cells (ESCC) Ecal09 line, and explore its underlying mechanism. METHODS: Bmi-1 shRNA driven by H1 promoter was constructed to transfect Ecal09 cell line. AAV-Null and normal cell line were utilized in the blank group and negative control group respectively. The expression levels of Bmi-1 protein were analyzed by Western blot and immunohistochemistry respectively. The cell proliferation was determined by CCK-8 assay. The distribution of cell cycle was assessed by flow cytometry. Tumorigenic efficiency was analyzed by nude mice. Cell apoptosis was detected by TUNEL. RESULTS: Western blot result revealed that the erpression level of Bmi in the Bmi-lshRNA transfected group (681.74±28.89) was significantly lower than that in other two groups (vs. AAV-Null 964. 00± 41. 73, P = 0. 001;vs. control 1 005.51±41.16,P=0. 001). Bmi expression levels in the AAV-Null and the control group were not statistically significant (P=0. 007). Extracorporal experiment indicated that the growth rate of Ecal09 cell line in the Bmi-1 shRNA transfected group was significantly lower than those in the AAV-Null and normal cell lines (vs AAV-Null, P= 0. 009;vs control,P= 0. 031). Cell cycle analysis indicated the proliferation index (PI) of Bmi-1 shRNA, AAV-Null and normal transfected Ecal09 cell line were 0. 42± 0. 13,0.81±0.37 and 0. 91± 0. 25 , respectively, which indicated the PI in the Bmi 1 shRNA transfected group was significantly lower than those in the normal and AAV-Null cell lines (vs AAV-Null,P= 0. 023 vs control,P= 0. 006). In vivo experiments exhibited the tumor mass in the Bmi-1 shRNA trans- {ected group (1.47±0.35) g was significantly lower than those in AAV-Nult group (1.83±0.28) g (P=0. 028) and control group (1.96±0.40) g (P=0. 004). The apoplosis index (AI) of Bmi-1 shRNA transfected,AAV-Null,and nor- mal cell lines were (16.9± 5.31 )%, (9. 57±3.84)% and (8. 13 ± 3.97)%, respectively, which manifested that Bmi-1 shRNA transfected cell line possessed a higher AI (vs. AAV-Null, P = 0. 001% vs control, P = 0. 001). CONCLUSION: ShRNA-mediated silengcing of the Bmi-1 gene in Ecal09 cell line can effectively decrease proliferation,growth rate, and promote cell apoptosis.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2013年第22期1703-1708,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81201780)
上海市科委医学引导基金(114119a9300)
关键词
RNA干扰
食管肿瘤
病理学
免疫组织化学
细胞系
肿瘤
转染
细胞增殖
细胞凋亡
RNA interference
esophageal neoplasms/pathology
immunohistochemitry
cell line, tumor
transfection cell proliferation
apoptosis