DNMTl siRNA对人胰腺癌PaTu8988细胞周期的调控机制研究

Mechanism of DNMT1 siRNA on cell cycle regulation of pancreatic carcinoma cell line PaTu8988

目的研究沉默DNA甲基转移酶1(DNMTl)基因对人胰腺癌PaTu8988细胞增殖和细胞周期的影响及可能机制。方法培养的人胰腺癌PaTu8988细胞分为空白对照组、阴性siRNA(15nmol/L)组、阴性siRNA(30nmol/L)组、DNMT1 siRNA(15nmol/L)组和DNMT1 siRNA(30nmol/L)组。转染48h后,采用real-time PCR和Western blotting评价沉默效率和细胞周期调控基因p21的表达情况;WST-8法检测细胞增殖情况;流式细胞技术检测细胞周期变化。结果转染48h后,15nmol/L和30nmol/L浓度的DN-MT1 siRNA均明显抑制了DNMT1 mRNA和蛋白的表达;DNMT1 siRNA转染后人胰腺癌PaTu8988细胞增殖受抑制,各组细胞增殖抑制率依次为0%±12.0%、13.7%±8.2%、23.4%±7.3%、17.1%±5.8%和36.9%±14.2%,其中DNMT1 siRNA(30nmol/L)组的细胞增殖抑制率明显高于其他各组(P<0.01);DNMT1 siRNA(15nmol/L)组和DNMT1 siRNA(30nmol/L)组的G2/M期细胞百分率均显著低于其他各组,S期细胞百分率明显高于其他各组(P<0.05);DNMT1 siRNA(30nmol/L)组细胞周期调控基因p21 mRNA相对表达量明显高于其他各组(P<0.01)。结论DNMTl siRNA能特异性抑制胰腺癌细胞DNMTl的表达,并抑制细胞增殖、促使细胞出现S期阻滞,其机制可能与上调细胞周期调控基因p21的表达有关。

Objective To investigate the effects and the mechanism of DNA methyhransferase 1 （DNMT1） gene silencing by small interfering RNA （siRNA） on cell cycle and proliferation of pancreatic carcinoma cell line PaTu8988. Methods PaTu8988 cells were divided into five groups： blank control group, negative control siRNA （15nmol/L） group, negative control siRNA （30nmol/L） group, DNMT1 siRNA （15nmol/L） group and DNMT1 siRNA （30nmol/L） group. Rcal-time PCR and Western blotting were used to assess the efficiency of DNMT1 gene silencing, and the expression levels of cell cycle regulation gene p21 after transfection for 48h. Cell proliferation was then analyzed by WST-8 assay and cell cycle was evaluated by flow cytometry. Results The expressions of DNMT1 mRNA and protein were inhibited in both DNMT1 siRNA （15nmol/L） group and DNMT1 siRNA （30nmol/L） group after DNMT1 siRNA transfection for 48h. The proliferation of PaTu8988 calls was inhibited by DNMT1 siRNA. The inhibition rates were 0%±12. 0%, 13. 7%±8. 2%, 23. 4%±7.3%, 17. 1%±5.8% and 36. 9%±14. 2%, respectively, in the five groups listed above. The inhibition rate of DNMT1 siRNA （30nmol/L） group was significantly higher than those in other four groups （P〈0. 01）. The percentages of G2/M phase cells were significantly lower, while the percentages of S phase cells were significantly higher in cells infected with DNMT1 siRNA compared with those in other groups （P〈0. 05）. The level of p21 mRNA in DNMT1 siRNA （30nmol/L） group was significantly higher than those in other four groups （P〈0. 01）. Conclusions DNMT1 siRNA can specifically knockdown the expression of DNMT1 gene and inhibit the proliferation of PaTu8988 cells, and induce S phase cell cycle arrest, which may occur through up-regulate the expression of p21 mRNA.