腺病毒介导的PTEN基因对前列腺癌细胞株PC-3增殖及cyclinD1、p21表达的影响

目的:构建携带抑癌基因PTEN的腺病毒载体,探讨PTEN基因对前列腺癌细胞株PC-3增殖及cyclin D1、p21表达的影响。方法:采用RT-PCR方法从大鼠海马神经元扩增目的基因PTEN,连接入pENTR2A载体,在293A细胞中进行重组腺病毒的包装及扩增。以携带PTEN基因的腺病毒载体感染体外培养的前列腺癌细胞株PC-3。采用间接免疫荧光法检测细胞中PTEN的过表达情况。Western印迹检测PTEN、cyclin D1和p21表达的变化,通过细胞生长实验、平板克隆实验检测PTEN对PC-3细胞增殖能力的影响。结果:成功构建重组腺病毒载体Ad-PTEN;PC-3细胞经Ad-PTEN感染后PTEN蛋白表达水平明显增加。Western印迹实验所得条带进行灰度值分析后与β-actin求比值,PTEN蛋白在Ad-PTEN感染组细胞中表达(0.215±0.065)明显高于对照组[(0.052±0.009),t=4.30,P0.05]和Ad-LacZ组[(0.056±0.008),t=4.21,P0.05]。cyclin D1蛋白在Ad-PTEN感染组细胞中表达(0.256±0.072)明显低于对照组[(0.502±0.087,t=3.77,P0.05)]和Ad-LacZ组[(0.498±0.081,t=3.87,P0.05)]。p21蛋白在Ad-PTEN感染组细胞中表达(0.589±0.076)明显高于对照组[(0.146±0.026,t=9.55,P0.01)]和Ad-LacZ组[(0.163±0.024,t=9.26,P0.01)]。MTT实验显示Ad-PTEN对PC-3细胞的抑制作用自48h后(21.98%)有显著性(t=6.80,P0.01)。平板克隆实验显示Ad-PTEN组克隆形成率[(37.4±4.18)%]明显低于对照组[(54.9±4.81)%,t=4.76,P0.01]及Ad-LacZ组[(56.5±5.42)%,t=4.83,P0.01]。结论:通过腺病毒载体Ad-PTEN使PC-3细胞表达PTEN基因,可以明显抑制细胞的增殖,并可降低cyclin D1表达,同时增加p21的表达。

Effects of adenovirus-mediated PTEN on the proliferation of prostate cancer PC-3 cells and expressions of cyclin D1 and p21

To construct a recombinant adenovirus expression vector containing the anti-oncogene PTEN and to in- vestigate the effects of the PTEN gene on the proliferation of prostate cancer PC-3 cells and the expressions of cyclin D1 and p21 in thePC-3 ceils. Methods ： The PTEN gene was amplified from the rat hippocampus by RT-PCR and cloned into the shuttle plasmid pEN- TR2A. The plasmids were constructed and amplified in 293A cells. Prostate cancer PC-3 cells were cultured in vitro and infected with the adenoviral vector carrying the PTEN gene （Ad-PTEN）. The up-regulation of the PTEN protein was measured by indirect immuno- fluorescence assay; the expressions of PTEN, cyclin D1 and p21 in the cells infected with Ad-PTEN and Ad-LacZ were determined by Western blot; and the effect of PTEN on the cell proliferation was detected by MTY assay and plate colony formation. Results ： The recombinant adenoviral vector Ad-PTEN was successfully constructed. Western blot showed a significantly increased expression of the PTEN protein in the PC-3 ceils infected with Ad-PTEN （0.215 ± 0.065） as compared with that in the control （ [0.052 ＋0.009], t = 4.30, P 〈 0.05） and the Ad-LacZ group （ [ 0. 056 ＋ 0.008 ], t = 4.21, P 〈 0.05）. The expression of cyclin D1 was significantly low- er in the Ad-PTEN-infected PC-3 ceils （0.256 ± 0. 072） than in the control （ [ 0. 502 ± 0. 087 ], t = 3.77, P 〈 0.05 ） and the Ad- LacZ group （[0. 498 ± 0.081 ], t = 3.87, P 〈 0.05 ）, while the expression of p21 remarkably higher in the Ad-PTEN-infected PC-3 cells （0.589 ± 0.076） than in the control （ [0. 146 ~0.026], t = 9.55, P 〈0.01） and the Ad-LacZ group （ [0. 163 ± 0.024], t = 9.26, P 〈0.01 ）. Ad-PTEN significantly inhibited the growth of the PC-3 cells （21.98%） at 48 h （t = 6.80, P 〈0.01 ）. The colo- ny formation rate of the PC-3 cells was （37.4 ± 4.18） % in the Ad-PTEN group, significantly lower than （54.9 ＋4.81 ） % in the con- trol （t =4.76, P〈0.01） and （56.5 ± 5.42）% in the Ad-LacZ group （t =4.83, P〈0.01）. Conclusion： The expression of PTEN induced by Ad-PTEN can significantly inhibit the proliferation of PC-3 cells, down-regulate the expression of cyclin D1, and up- regulate the expression of p21. Natl J Androl, 2014, 20 （3） ： 207 -212