摘要
目的:研究下调HDAC1的表达对大肠癌细胞增殖、凋亡和细胞周期的影响.方法:设计合成HDAC1的特异性shRNA,将其插入至pSilencer载体中,并将重组后的pSilencer质粒载体经脂质体包裹转染SW480细胞株.用RT-PCR和Westernblot检测shRNA对细胞内HDAC1基因表达的影响,同时采用Westernblot对细胞周期相关基因进行检测.采用MTT法检测生长抑制作用.运用流式细胞术检测细胞的凋亡情况和细胞周期分布.结果:成功构建和筛选出HDAC1特异性的shRNA质粒载体,与空白对照组相比,转染HDAC1-shRNA的大肠癌细胞HDAC1mRNA蛋白质表达水平明显下降(30.4%±4.5%vs64.6%±4.4%,P<0.01;27.4%±4.5%vs58.1%±3.3%,P<0.01),p21/WAF-1/CIP-1表达增加(97.4%±2.6%vs62.6%±3.4%,P<0.01),CdK2和CyclinE蛋白下降(CdK2:27.7%±6.0%vs42.6%±4.1%,P<0.01;CyclinE:42.0%±8.5%vs82.8%±3.7%,P<0.01),细胞生长抑制率增加(24h:35.9%±4.9%vs1.2%±0.6%,P<0.01;48h:47.5%±7.0%vs1.3%±0.6%,P<0.01;72h:45.7%±6.2%vs1.0%±0.5%,P<0.01;96h:48.2%±4.7%vs1.2%±0.7%,P<0.01),凋亡率明显增加(31.3%±2.8%vs3.9%±0.7%,P<0.01),G0/G1期和G2/M期细胞比例增加(G0/G1:64.5%±0.9%vs57.8%±1.8%,P<0.01;G2/M:17.4%±1.3%vs14.5%±0.6%,P<0.05),S期细胞比例相应下降(17.5%±1.0%vs27.7%±1.5%,P<0.01).结论:HDAC1特异的shRNA能够有效下调HDAC1基因,诱导大肠癌细胞发生凋亡和细胞周期阻滞,从而抑制细胞的增殖.
AIM： To construct the HDAC1 specific recombinant plasmid vector and investigate its effects on the apoptosis, proliferation and cell cycle distribution of colorectal cancer cells. METHODS： HDAC1 specific short hairpin RNA （shRNA） plasmid vector was constructed and then transfected into the cultured SW480 cell line with lipofectamine 2000. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blot were used to detect the expression of HDAC1 mRNA and protein, respectively, and p21^/WAF-1/CIP-1, CdK2, and Cyclin E proteins were detected by Western blot, too. The growth inhibition of SW480 cells was evaluated by MTT assay. Cell apoptosis and cell cycle distribution were determined by flow cytometry. RESULTS： The levels of HDAC1 mRNA andprotein in HDAC1-shRNA group were significantly lower than those in negative control group （30.4% ± 4.5% vs 64.6% ± 4.4%, P 〈 0.01; 27.4% ± 4.5% vs 58.1% ± 3.3%; both P 〈 0.01）. In comparison with negative control group, HDAC1 silence led to a significant increase of p21^/WAF-1/CIP-1 protein （97.4% ± 2.6% vs 62.6% ± 3.4%, P 〈 0.01） and decrease of CdK2 and Cyclin E protein （27.7% ± 6.0% vs 42.6% ± 4.1%, P 〈 0.01; 42.0% ± 8.5 % vs 82.8% ± 3.7%, P 〈 0.01）. MTT assay revealed that transfection of HDAC1-shRNA inhibited the growth of SW480 cells, and the inhibitory rates were markedly higher at 24, 48, 72, and 96 h time points than those in negative control group （24 h： 35.9% ± 4.9% vs 1.2% ± 0.6%, P 〈 0.01; 48 h： 47.5% ± 7.0% vs 1.3% ± 0.6%, P 〈 0.01; 72 h： 45.7% ± 6.2% vs 1.0% ± 0.5%, P 〈 0.01; 96 h： 48.2% ± 4.7% vs 1.2% ± 0.7%, P 〈 0.01）. The percentage of apoptosis cells in HDAC1-shRNA group was significantly higher than that in negative control group （31.3% ± 2.8% vs 3.9% ± 0.7%, P 〈 0.01） and the cells were increased at G0/G1 and G2/M phase （G0/G1： 64.5% ± 0.9% vs 57.8% ± 1.8%, P 〈 0.01; G2/M： 17.4% ± 1.3% vs 14.5% ± 0.6%, P 〈 0.05）, but decreased at S phase （17.5% ± 1.0
出处
《世界华人消化杂志》
CAS
北大核心
2008年第11期1173-1178,共6页
World Chinese Journal of Digestology
关键词
HDAC1基因
短发卡状RNA
大肠癌
凋亡
增殖
逆转录聚合酶链反应
HDAC1
Short hairpin RNA
Colorectal cancer
Apoptosis
Proliferation
Reverse transcription polymerase chain reaction
