[使用单一短发夹RNA载体同时抑制LNCaP细胞中的人端粒酶逆转录酶和雄激素受体基因表达]

[Simultaneous inhibition of both hTERT and androgen receptor gene expression in LNCaP cells using single shRNA vector].

作者信息

Li Gong-hui, Cheng Sheng, Zhou You-feng, Chen Zhao-dian

机构信息

Department of Urology, Sir Run RUN Shaw Hospital, Zhejiang University Medical College, Hangzhou, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2009 Nov 3;89(40):2853-7.

PMID:20137668

Abstract

OBJECTIVE

To explore the feasibility of inhibition of hTERT and androgen receptor (AR) gene expression simultaneously in LNCaP cells by single shRNA vector.

METHODS

Templates DNA of both hTERT and AR siRNA were inserted into Pgenesil vector to construct a new vector Pgenesil-hTERT-AR-shRNA by RNAi-DNA vector technology. Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA vectors were transfected into prostate cancer LNCaP cells respectively. The levels of AR mRNA, apoptosis and proliferation of each cell group were determined by FQ-PCR, Annexin V method and MTT.

RESULTS

The level of hTERT mRNA of control group cells and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.51 +/- 0.08) x 10(8), (7.32 +/- 0.43) x 10(7), (2.94 +/- 0.15) x 10(6), (4.45 +/- 0.25) x 10(7) and (3.17 +/- 0.18) x 10(6) (copies/ml) respectively. The level of AR mRNA of control cell groups and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.92 +/- 0.11) x 10(5), (6.47 +/- 0.32) x 10(5), (3.70 +/- 0.24) x 10(4), (1.22 +/- 0.06) x 10(4) and (7.21 +/- 0.41) x 10(3) (copies/ml) respectively. These data indicate that the expression of hTERT or AR gene could be significantly inhibited by Pgenesil-hTERT-shRNA or Pgenesil-AR-shRNA while Pgenesil-hTERT-AR-shRNA could simultaneously inhibit both hTERT and AR gene expression. The apoptosis rate and the inhibition rate of cell growth of Pgenesil-hTERT-AR-shRNA group were significantly higher than those of Pgenesil-hTERT-shRNA group or Pgenesil-AR-shRNA group (P < 0.05).

CONCLUSION

It is feasible to inhibit both hTERT and AR gene expression simultaneously by single shRNA vector. It will be a new research strategy of gene therapy for prostate cancer.

摘要

目的

探讨利用单一短发夹RNA（shRNA）载体同时抑制LNCaP细胞中人端粒酶逆转录酶（hTERT）和雄激素受体（AR）基因表达的可行性。

方法

采用RNA干扰- DNA载体技术，将hTERT和AR siRNA的模板DNA插入Pgenesil载体，构建新型载体Pgenesil - hTERT - AR - shRNA。分别将Pgenesil - HK - shRNA、Pgenesil - hTERT - shRNA、Pgenesil - AR - shRNA和Pgenesil - hTERT - AR - shRNA载体转染至前列腺癌LNCaP细胞。采用荧光定量聚合酶链反应（FQ - PCR）、膜联蛋白V法和MTT法分别检测各细胞组中AR mRNA水平、细胞凋亡及增殖情况。

结果

对照组细胞以及转染Pgenesil - HK - shRNA、Pgenesil - hTERT - shRNA、Pgenesil - AR - shRNA和Pgenesil - hTERT - AR - shRNA的细胞中hTERT mRNA水平分别为（1.51±0.08）×10⁸、（7.32±0.43）×10⁷、（2.94±0.15）×10⁶、（4.45±0.25）×10⁷和（3.17±0.18）×10⁶（拷贝/毫升）。对照组细胞以及转染Pgenesil - HK - shRNA、Pgenesil - hTERT - shRNA、Pgenesil - AR - shRNA和Pgenesil - hTERT - AR - shRNA的细胞中AR mRNA水平分别为（1.92±0.11）×10⁵、（6.47±0.32）×10⁵、（3.70±0.24）×10⁴、（1.22±0.06）×10⁴和（7.21±0.41）×10³（拷贝/毫升）。这些数据表明，Pgenesil - hTERT - shRNA或Pgenesil - AR - shRNA可显著抑制hTERT或AR基因表达，而Pgenesil - hTERT - AR - shRNA可同时抑制hTERT和AR基因表达。Pgenesil - hTERT - AR - shRNA组的细胞凋亡率和细胞生长抑制率显著高于Pgenesil - hTERT - shRNA组或Pgenesil - AR - shRNA组（P < 0.05）。