沉默Bmi-1表达对人食管鳞癌细胞生长影响观察

Effect of small interfering RNA targeting Bmi-1on the proliferation and invasiveness of human esophageal squamous carcinoma cells

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.