摘要
目的观察阻断MBD1基因对胰腺癌抑癌基因表达的影响，探讨MBD1基因在胰腺癌发生发展中的作用。方法设计并合成2条MBD1小干扰RNA（siRNA）序列，克隆进入pGCsi—U6／Neo/绿色荧光蛋白（GFP）构建重组质粒。重组质粒和空质粒转染人胰腺癌细胞系BxPC-3，将3组细胞（转染MBD1-siRNA重组质粒组、转染空质粒组和未转染组）接种于裸鼠，8周后取移植瘤行逆转录聚合酶链反应（RT-PCR）检测MBD1基因和甲基化相关抑癌基因的表达情况。结果成功构建MBD1-siRNA重组质粒并稳定转染胰腺癌细胞系BxPC-3，转染组中MBD1基因表达明显低于空质粒组和未转染组，而CDH1、RASSF1A、TIMP3、P14ARF、Rb等甲基化相关抑癌基因的mRNA水平转染组（179．7±8．1、155．6±10．0、256．7±15．7、199．0±7．9、210．1±9．4）要高于空质粒组（21．0±6．8、22．0±2．7、99．4±10．3、86．0±5．4、15．0±4．9）和未转染组（11．4±6．0、15．3±1．9、110．7±14．1、74．3±4．8、17．4±7．8，均P〈0．05）。结论通过RNA干扰技术，可降低MBD1在胰腺癌中的表达，上调抑癌基因的转录，MBD1介导的转录抑制作用可能在胰腺癌的发生发展中起重要作用。
Objective To observe the effect of MBDI-siRNA on the expressions of multiple tumor suppressor genes in pancreatic cancer cells, and to explore the role of MBD1 in carcinogenesis of pancreatic carcinoma. Methods Two siRNA sequences targeting MBD1 were designed by software and cloned into the expression plasmid pGCsi-U6/Neo/GFP. DNA sequencing was used to confirm if the recombinant plasmid was constructed correctly. The constructed plasmid and the blank plasmid pGCsi-U6/Neo/GFP were stably transfected into human pancreatic cancer cells of the line BxPC-3. Nude mice underwent subcutaneous injection of the BxPC-3 cells transfected with the plasmid pGCsi-U6/Neo/GFP containing MBD1 or the blank plasmid pGCsi-U6/Neo/GFP to establish tumor models and were observed for 8 weeks. Mice without inoculation were used as controls. Eight weeks later the mice were killed with the tumors taken out. RT-PCR was used to detect the mRNA expression of MBD1 and the tumor suppressor genes related with DNA methylation, including CDHI, RASSF1A, TIMP3, P14ARF and Rb in transplanted tumors. Results MBDI-siRNA recombinant plasmids were constructed successfully and stably transfected into the BxPC-3 cells. MBD1 was not detected in the tumors of the MBDI-siRNA group and could be found in the other 2 groups. However, the mRNA expression levels of CDH1, RASSF1A, TIMP3, P14ARF, and Rb of the MBD1-siRNAgroup（179.7 ±8.1,155.6 ± 10.0,256.7 ± 15.7,199.0 ±7.9,210.1 ±9.4） were all significantly higher than those of the other 2 groups （21.0 ± 6.8,22. 0 ± 2. 7,99.4 ± 10. 3,86.0 ± 5.4, 15.0±4.9,11.4±6.0,15.3±1.9,110.7 ±14.1,74.3±4.8, 17.4±7.8,ali P〈0.05）.Condusion RNA interference decreases the expression of MBD1 in pancreatic cancer and up-regulate the transcription of tumor suppressor genes. MBD1 mediated transcriptional repression may play an important role in the development of pancreatic cancer.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2008年第28期1948-1951,共4页
National Medical Journal of China
基金
国家自然科学基金资助项151（30500490）
关键词
胰腺肿瘤
RNA干扰
基因
抑癌基因
Pancreatic neoplasms
RNA interference
Gene
Tumor suppressor gene
