【摘要】 目的探讨Survivin基因影响肾癌侵袭能力的机制。方法利用RNA干扰转染技术靶向沉默肾癌786-O细胞Survivin基因表达,通过RT-PCR及Western blot技术检测Survivin mRNA及蛋白表达水平变化,并检测MMP-2蛋白表达情况。结果成功制备转染Survivin siRNA质粒的低Survivin表达的786-O/low-Survivin细胞系,细胞表达绿色荧光,转染效率超过90%。RT-PCR电泳条带灰度值显示,Survivin siRNA/786-O组的Survivin mRNA表达水平(0.233±0.044)明显低于Control siRNA/786-O组(0.628±0.093)以及Non-siRNA/786-O组(0.657±0.079)(P<0.01)。Control siRNA/786-O组和Non-siRNA/786-O组的Survivin mRNA表达水平差异无统计学意义(P>0.05)。Survivin siRNA转染786-O细胞后,抑制了Survivin基因的转录活性。Western Blot实验显示:Survivin siRNA/786-O组的Survivin蛋白表达水平(2.424±0.303)明显低于Control siRNA/786-O组(15.483±0.343)以及Non-siRNA/786-O组(15.513±0.407)(P<0.01)。Survivin siRNA/786-O组MMP-2蛋白表达水平(0.484±0.111)明显低于Control siRNA/786-O组(10.861±1.347)以及Non-siRNA/786-O组(10.947±1.744)(P<0.01)。Control siRNA/786-O组和Non-siRNA/786-O组的Survivin蛋白及MMP-2蛋白表达水平均差异无统计学意义(P>0.05)。Survivin siRNA转染786-O细胞后,显著下调了Survivin蛋白水平,并下调了MMP-2蛋白表达水平。结论靶向沉默Survivin基因能显著下调肾癌786-O的SurvivinmRNA和蛋白分子表达,并干扰了MMP-2蛋白的表达,揭示出Survivin基因参与调控肾癌侵袭转移的潜在调控机制。更多还原

【Abstract】 Objective To investigate the mechanism of targeted silencing Survivin gene expression in human renal carcinoma cell line-786-O cells to reduce invasion ability of renal carcinoma. Methods The RNA inteference transfection technique was used to silence Survivin gene expression in human renal carcinoma 786-O cells. The expression levels of Survivin mRNA and protein as well as MMP-2 protein were detected by reverse transcription-polymerase chain reaction( RT-PCR) and Western Blot,respectively. Results The 786-O/low-Survivin was established successfully. As compared with control siRNA/786-O and non-siRNA/786-O,and the Survivin siRNA/786-O showed obvious reduction of expression levels of Survivin mRNA and protein( P < 0. 01). Moreover the expression levels of MMP-2 protein were also down-regulated significantly in Survivin siRNA/786-O( P < 0. 01). However there were no significant differences in the expression levels of Survivin mRNA between siRNA/786-O control group and non-siRNA/786-O group( P > 0. 05). After Survivin siRNA was transfected into 786-O cells,which inhibited the transcription activity of Survivin gene. Western Blot assay showed that the expression levels of Survivin protein( 2. 424 ± 0. 303) in Survivin siRNA/786-O group were significantly lower than those( 15. 483 ± 0. 343) in siRNA/786-O control group as well as those( 15. 513 ± 0. 407) in non-siRNA/786-O group( P < 0. 01). Moreover the expression levels( 0. 484 ± 0. 111) of MMP-2 protein in Survivin siRNA/786-O group were significantly lower than those( 10. 861 ± 1. 347) in siRNA/786-O control group as well as those( 10. 947 ± 1. 744) in non-siRNA/786-O group( P <0. 01). However there were no significant differences in the expression levels of Survivin protein and MMP-2 protein between siRNA/786-O control group and non-siRNA/786-O group( P > 0. 05). After Survivin siRNA was transfected into 786-O cells,which obviously down-regulated the expression levels of Survivin protein and MMP-2 protein. Conclusion The targeted silencing of Survivin gene can obviously down-regulate the expression levels of Survivin mRNA and protein in human renal carcinoma cell line-786-O cells,and can interfere the expression of MMP-2 protein,which suggests that Survivin gene may participate in the regulation mechanism of invasion and metastasis of renal cancer.更多还原