siRNA干扰BMI-1基因对膀胱癌EJ细胞增殖的调控作用及其机制

Regulatory effect of siRNA interference targeting BMI-1gene on proliferation of bladder cancer EJ cells

目的:对比观察膀胱癌EJ细胞及人膀胱移行上皮细胞中BMI-1基因及下游p16INK4a、p14ARF基因的mRNA及蛋白表达水平,探讨siRNA干扰BMI-1基因后对EJ细胞增殖的影响及其调控机制。方法:细胞免疫荧光观察BMI-1、p16INK4a和p14ARF蛋白在EJ细胞中表达情况及定位。Real-time RT-PCR检测EJ细胞及膀胱正常移行上皮细胞中3种基因的表达水平,Western blotting检测蛋白水平。构建干扰BMI-1基因siRNA,通过脂质体转染导入EJ细胞中,设立空白对照和阴性干扰对照组,检测BMI-1及下游p16、p14基因表达水平及蛋白表达水平的变化;以CCK-8检测细胞生长观察siRNA干扰BMI-1表达对EJ细胞增殖的抑制作用;用流式细胞术分析细胞凋亡的改变。结果:BMI-1mRNA及蛋白水平在EJ细胞表达高于膀胱移行上皮细胞,而p16INK4a和p14ARFmRNA及蛋白在EJ细胞表达水平稍低于膀胱移行上皮细胞。siRNA干扰BMI-1后可以上调EJ细胞中其下游p16和p14 mRNA及蛋白水平,使EJ细胞增殖能力下降,细胞凋亡增多。结论:siRNA干扰BMI-1基因表达对EJ细胞的体外生长具有明显的抑制作用,其作用机制与上调其下游p16INK4a、p14ARF基因的表达有关。

AIM： To investigate whether siRNA - mediated BMI - 1 gene silencing inhibits the proliferation of EJ ceils by detecting the expression of BMI - 1, pJ6^INK4a and p14^ARF genes at mRNA and protein levels in bladder cancer KJ cells and normal bladder transitional epithelium cells. METHODS： The protein expression and localization of BMI - 1, pJ6^INK4a and p14^ARF in EJ cells were determined by cellular immunofluorescence. An siRNA targeting BMI - 1 gene was synthesized and transfected into bladder carcinoma EJ cells by liposomes. The mRNA expression of BMI - 1, p16INKK4a and p14ARF was detected by real - time PCR and the protein levels were measured by Western blotting in bladder cancer EJ cells and normal bladder transitional epithelium cells. Cell survival was analyzed by CCK -8 assay. Cell apoptosis were examined by flow cytometry. RESULTS： The mRNA and protein expression of BM1-1 in EJ cells was higher than that in bladder transitional epithelium cells. However, the expression of pJ6^INK4a and p14^ARF were opposite. While BMI - 1 gene in EJ cells was silenced by siRNA, the mRNA and protein expression of BMI - 1 were declined whereas the expression of pJ6^INK4a and p14^ARF was increased. The viability of the FJ cells was decreased and the apoptotic cells were increased when BMI - 1 gene was silenced. CONCLUSION： The expression of BMI -1 is inversely correlated with the expression of pJ6^INK4a and p14^ARFin bladder transitional cell carcinoma EJ cells. The siRNA - mediated BMI - 1 gene silencing in bladder cancer EJ cells inhibits the cell growth and up - regulates the expression of pJ6^INK4a and p14^ARF in vitro.