Lin Feng, Lin Ping, Liu Xin, Li Dong, Liu Zi-Jun, Zou Hai-Feng, Jiang Ying, Zhao Xue-Fei, Feng Jin-Liang, Yu Xiao-Guang
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Harbin Medical University, Harbin, Heilongjiang 150081, China.
Zhonghua Nan Ke Xue. 2012 Aug;18(8):687-91.
To study the effect of siRNA targeting ADAM17 (ADAM17-siRNA) on the proliferation of prostate cancer PC-3 cells.
After transfecting PC-3 cells with ADAM17-siRNA 1 and ADAM17-siRNA 2, we detected the expressions of ADAM17 mRNA and protein by RT- PCR and Western blotting, respectively. We measured the changes in the proliferation and DNA synthesis of PC-3 cells by MTT and bromodeoxyuridine (BrdU) incorporation assay, examined the cell cycle profile by flow cytometry, and determined the expressions of the genes associated with PC-3 cell proliferation by Western blotting.
Both ADAM17-siRNA 1 and 2 effectively reduced the expressions of ADAM17 mRNA and protein in the PC-3 cells. Knockdown of ADAM17 with the two siRNAs significantly inhibited cell proliferation as compared with the control group (0.43 +/- 0.57 and 0.44 +/- 0.64 vs 0.80 +/- 0.51, P < 0.05) and down-regulated DNA synthesis (0.48 +/- 0.43 and 0.54 +/- 0.59 vs 0.79 +/- 0.72, P < 0.05). The cell cycle profile showed that the cell population of the G1 phase was markedly higher in both the ADAM17-siRNA groups than in the control ([61.83 +/- 2.41]% and [59.78 +/- 1.92]% vs [41.38 +/- 1.53]%, P < 0.05), but that of the S phase remarkably lower in the former two than in the latter ([23.64 +/- 2.56]% and [25.24 +/- 1.86]% vs [33.51 +/- 1.47]%, P < 0.05), with a concomitant decrease in the expression of the cell cycle protein cyclin D1 and increase in the cyclin-dependent kinase inhibitor p21.
ADAM17-siRNA can effectively inhibit the proliferation of PC-3 cells by up-regulating cyclin D1 and down-regulating p21 protein, and ADAM17 has a potential value in the gene therapy of prostate cancer.
研究靶向解聚素和金属蛋白酶17（ADAM17）的小干扰RNA（ADAM17-siRNA）对前列腺癌PC-3细胞增殖的影响。
用ADAM17-siRNA 1和ADAM17-siRNA 2转染PC-3细胞后，分别采用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测ADAM17 mRNA和蛋白的表达。通过噻唑蓝（MTT）法和溴脱氧尿苷（BrdU）掺入法检测PC-3细胞增殖和DNA合成的变化，采用流式细胞术检测细胞周期分布，并通过蛋白质免疫印迹法检测与PC-3细胞增殖相关基因的表达。
ADAM17-siRNA 1和2均能有效降低PC-3细胞中ADAM17 mRNA和蛋白的表达。与对照组相比，两种siRNA敲低ADAM17均显著抑制细胞增殖（0.43±0.57和0.44±0.64比0.80±0.51，P<0.05）并下调DNA合成（0.48±0.43和0.54±0.59比0.79±0.72，P<0.05）。细胞周期分布显示，ADAM17-siRNA组G1期细胞比例均显著高于对照组（[61.83±2.41]%和[59.78±1.92]%比[41.38±1.53]%，P<0.05），而前两者S期细胞比例显著低于后者（[23.64±2.56]%和[
