PARPl特异性siRNA对前列腺癌PC3细胞增殖的影响及其机制研究

Effects and underlying mechanism of siRNA targeting PARP1 on the proliferation of PC3 cells

目的研究多聚腺苷二磷酸核糖聚合酶1（PARPl）特异性小分子干扰RNA（siRNA）对雄激素非依赖性前列腺癌PC3细胞株增殖的影响并初步探讨其作用机制。方法设计合成3条PARPl特异性siRNA序列，进行脂质体转染后，通过RT—PCR、蛋白质印迹法检测其对PARPl的干扰效果，筛选出最佳干扰效果的2条siRNA序列；通过单溶液细胞增殖检测法检测干扰PC3细胞内源性PARPl表达对细胞增殖的影响，并检测细胞内Akt及其下游GSK3B活化水平的变化。结果与空白对照组相比，转染阴性对照序列对PARPl的mRNA和蛋白表达水平无明显影响，而转染RNA干扰序列1706、2003和2907均可以明显干扰PARPl的表达，其中mRNA抑制率分别为（52．07±4．65）％、（44．38±9．15）％和（22．05±6．65）％，蛋白抑制率分别为（86．86±4．94）％、（83．30±7．18）％和（63．05±10．19）％。RNA干扰序列1706和2003对PC3细胞的增殖抑制率分别为（38．93±3．87）％和（34．93±1．21）％，并可以下调细胞内Akt、GSK3B的磷酸化水平。结论PARPl特异性siRNA可以明显干扰PC3细胞内源性PARPl的表达并抑制细胞增殖，该抑制作用与Akt的活化抑制以及其下游GSK3B的激活有关。

Objective To observe the effects and study the underlying mechanism of siRNA targe- ting PARP1 on the proliferation of androgen independent prostate cancer PC3 cell line. Methods Three specific siRNA sequences targeting PARP1 were designed and synthesized. And two sequences which had better interfering effect on the expression of PARP1 were evaluated and selected through lipofectamine trans- fection, RT-PCR and Western Blot. The effect of PARP1 silencing on the proliferation of PC3 cells was ob- served with MTS assay and the levels of the phosphorylation of Akt and GSK3[3 were detected by Western Blot. Results Compared to the blank control group, the transfected group with the negative control se- quence had no significant impact on the expression of PARP1, however the transfected group with siRNA- 1706, -2003 or -2907 could significantly suppress the mRNA and protein expression of PARPI. The mRNA inhibition rate reached to （52.07 ±4.65）%, （44.38 ±9.15）% and （22.05±6.65）%, respectively; and the protein inhibition rate reached to （ 86.86 ± 4.94） %, （ 83.30 ± 7.18 ） % and （63.05 ± 10, 19 ） %, respectively. The siRNA-1706 and -2003 could significantly inhibit the proliferation of PC3 ceils; the inhibi- tion rate was （38.93 ± 3.87）% and （34.93 ± 1.21 ）%. And they also could down-regulate the intracellu- lar levels of phosphorylated Akt and GSK313 in PC3 cells. Conclusion PARPl-targeted siRNA can sig- nificantly suppress the expression of endogenous PARP1 and inhibit the proliferation of PC3 cells, which is related to the inhibition of Akt activity and the activation of GSK3β.