DNA聚合酶β靶向siRNA对PC3细胞生物学行为的影响

摘要

Aim:To observe the effect of silencing the overexpression of DNA polp via RNA interference on cellular biological behavior of PC3 cell line. Methods: DNA polβ targeted siRNA eukaryotic expression vector ( pSUPERneoGFP-PS1 ,pSUPERneoGFP-PS2 and pSUPERneoGFP-CS) were transfected into PC3 cell lines. Non transfected and empty pSU-PERneoGFP transfected groups were established. The protein expression of DNA polp was tested by Western blot. The expression of DNA polβ mRNA was tested by RT-PCR. The cellular biological behavior including the changes of cell cycle and cellular spontaneous mutation rate were observed. Results: DNA polp targeted eukaryotic expression vectors ( pSU-PERneoGFP-PSl and pSUPERneoGFP-PS2) were successfully transfected into PC3 cell lines and displayed obvious inhibitory effects on DNA polp protein overexpression. The expressions of DNA polβ mRNA among the five groups had significant difference ( F = 126. 264,P < 0.001 ). In addition, different sites of targeted siRNA presented different degree of inhibitory effect, whereby leading to different changes of cellular biological behavior. S-period frequency of cell cycle and the spontaneous mutation rate of hypoxanthine-guanine phosphonbosyl transferase( HGPRT) gene in cells with overexpession of polβ partly suppressed were significantly decreased compared with control groups, whereas the S-period frequency of cell cycle and the spontaneous mutation rate of HGPRT gene in cells with overexpession of DNA polp nearly completely suppressed were significantly increased compared with control group. Conclusion; It is suggested that PC3 cells were dependent on suitable low level expression of DNA polβ.%目的:观察DNA聚合酶(pol)B靶向siRNA表达载体转染人前列腺癌PC3细胞对细胞生物学行为的影响.方法:采用已构建的DNA polβ靶向siRNA真核表达载体(pSUPERneoGFP-PS1、pSUPERneoGFP-PS2和pSU-PERneoGFP-CS),利用脂质体包裹转染PC3细胞,并设未转染组和转染空psuPERneoGFP组.观察各组DNA polβmRNA和蛋白的表达及相应的细胞周期和自发突变率等细胞生物学行为的变化.结果:转染psuPERneoGFP-PS1和pSUPERneoGFP-PS2可沉默PC3细胞DNA polβ蛋白的表达.5组间DNA polβ mRNA表达的比较,差异有统计学意义(F=126.264,P<0.001);转染pSUPERneoGFP-PS1和pSUPERneoGFP-PS2组细胞较未转染、转染空pSU-PERneoGFP和转染pSUPERneoGFP-CS组显著降低(P<0.05).5组间S期细胞比例和自发突变率比较,差异有统计学意义(F=72.501和6.860,P<0.05);转染pSuPERneoGFP-PS2组细胞S期比例及自发突变率较对照组显著降低,而转染pSUPERneoGFP-PS1细胞S期比例及自发突变率显著高于对照组(P<0.05).结论:适宜的DNA polβ低水平表达是PC3细胞遗传稳定性维持所依赖的.