MBD1 RNA干扰对胰腺癌细胞株BxPC-3生长影响的实验研究

Effect of MBDI siRNA on growth of pancreatic cancer cell line BxPC-3

目的观察阻断MBD1表达后对胰腺痛细胞牛长增殖的影响，探讨MBD1在胰腺癌发生发展中的作用。方法采用RNA下扰技术，构建MBD1siRNA重组质粒，脂质体介导转染人胰腺癌细胞株BxPC3，RT-PCR和Western印迹检测转染前后MBD1 mRNA与蛋白的表达变化，MTT法检测转染前后BxPC-3细胞的生长曲线，将细胞接种于裸鼠，观察转染前后胰腺癌细胞体内移植瘤的生长情况。结果转染siRNA-MBD1质粒后胰腺癌细胞株BxPC～3MBD1的mRNA与蛋白表达水平明显降低。MTT法检测细胞的生长曲线，发现转染组较转染卒质粒组和未转染组生长明显缓慢（P〈C0．01）；体内实验显示，将转染前后的细胞接种于裸鼠，转染组移植榴生长速度明显较其他两组减慢（P〈0．01），RT-PCR检测移植瘤MBD1mRNA表达的变化，转染组中未能测到明显的MBD1表达。而转染组中CDH1、Rb等抑癌基因mRNA的表达明显高于转染空质粒组和未转染组。结论通过RNA干扰技术，可在转录和翻译水平降低MBD1在胰腺癌细胞株BxPC-3中的表达，并能抑制肿瘤细胞的生长，但其作用机制尚不清楚，MBD1介导的转录抑制作用可能在胰腺癌的发生发展过程中起到重要的作用。

Objective To observe the effect of MBD1 siRNA on the growth of pancreatic cancer cell line BxPC 3, and explore the role of MBD1 in the carcinogenesis of the pancreatic carcinoma. Methods One siRNA sequence targeting MBD1 was designed by software and cloned into the expression plasmid pGCsi-U6/Neo/GFP. DNA sequencing was used to confirm that the recombinant plasmid was constructed correctly. The constructed plasmid and the control plasmid were stably transfected into human pancreatic cancer cell line BxPC-3. RT-PCR and Western blot were used to detect the MBD1 mRNA and protein expression after RNA interference. The cell growth curve was measured by MTT assay. The three kind of cells （BxPC 3, BxPC-3/vector, BxPC 3/MBD1-siRNA） were injected subcu- taneously to nude mice, which were observed for 8 weeks for tumor formation respectively. Resnlts RT-PCR and Western blot showed that the MBD1 mRNA and protein expression were significantly lower in the BxPC-3/MBD1 siRNA group than in BxPC 3 and BxPC-3/vector group （P〈0. 01）. In vivo and vitro, the growth of BxPC-3 cell was suppressed after transfected with MBD1 siRNA. The MBD1 mRNA in tumor of BxPC 3/MBD1-siRNA group could not be detected by RT PCR. The mRNA levels of some tumor suppressor genes including CDH1 and RB were up regulated significantly after RNA interference targeting MBD1. Conclusion The stable MBDI-siRNA recombinant plasmid can decrease MBD1 expression in human pancreatic cancer cell BxPC 3 and inhihit the growth of the cell notably in vivo and vitro, the mechanism is still unclear. MBD1 mediating transcriptional repres- sion may play an important part in the development of pancreatic cancer.