RNA干扰DNA甲基转移酶1对人胰腺癌PaTu8988细胞增殖和凋亡的影响

RNA interfering on proliferation and apoptosis of pancreatic cancer cell line PaTu8988 by DNMT1 siRNA

目的观察以DNA甲基转移酶1（DNA methy transferas 1，DNMT1）基因为靶基因的RNA干扰对人胰腺癌PaTu8988细胞增殖及凋亡的影响。方法设计、合成针对DNMT1基因的siRNA和阴性对照siRNA（N—siRNA）。实验分为空白对照组、脂质体组（仅予脂质体）、N—siRNA组（转染30 nmol N-siRNA）、siRNA组（转染30 nmol siRNA）。siRNA转染48h后，实时PCR法检测DNMT1 mRNA水平；WST-8法检测细胞增殖；流式细胞技术检测细胞凋亡。结果siRNA组DNMT1mRNA的抑制率为（86．0±4．3）％，明显高于N—siRNA组的（40．1±2．2）％和空白对照组的0（P〈0．01）；细胞存活率为（47．6±5．6）％，明显低于N—siRNA组的（68．1±4．1）％和空白对照组的100％（P〈0．01）；细胞凋亡率为（14．94±2．89）％，明显高于空白对照组的（7．51±1．12）％、脂质体组的（7．06±0．39）％、N—siRNA组的（8．84±1．44）％（均P〈0．01）。结论siRNA能特异、有效地抑制人胰腺癌PaTu8988细胞DNMT1 mRNA的表达，同时抑制细胞增殖、促进细胞凋亡。

Objective DNA methytransferase 1 （DNMT1） is highly expressed in many cancers and lowly expressed in normal adult cells. This study was to assess effects of DNMT1 gene silencing on proliferation and apoptosis of pancreatic cancer cell line PaTu8988. Methods It is divided into four groups： Control group, Lipofectamine 2000 group, Negative control siRNA （ N-siRNA ） group （ 30 nM ） and siRNA group （ 30 nM）. The expression levels of DNMTI mRNA were detected by real-time PCR to assess the efficiency of DNMT1 gene silencing. Cell proliferation was analyzed by WST-8 assay; Cell apoptosis was evaluated by flow cytometry. Results Relative to Control group, 48h after transfection of DNMTI siRNA, The inhibitory rates of DNMT1 mRNA levels in PaTu8988 cells was （ 86.0 ± 4.3 ） %. Cell survival rate was declined to 47.6 ± 5.6% from Control group（ 100. 7 ± 3.0% ）, Lipofectamine 2000 group（64.5 ± 6.8% ）, N-siRNA group （68. 1 ± 4.1% ） （ P 〈 0.05 ） ; Cell apoptosis rate was increased from Control group （7.51 ± 1.12 ） % to siRNA group （14.94±2.89）% （P〈0.05）, respectively, 48h after transfection of DNMT1 siRNA. Conclusions DNMTI siRNA can efficiently and specifically knockdown the expression of DNMT1 mRNA and inhibit the proliferation of PaTu8988 cells, and induce cell apoptosis. It provides evidence for gene therapy of pancreatic cancer.