Activation of multiple tumor suppressor genes by MBD1 siRNA in pancreatic cancer cell line BxPC-3

Abstract

To observe the effect of MBD1-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. 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 CDH1, RASSF1A, TIMP3, P14ARF and Rb in transplanted tumors. MBD1-siRNA recombinant plasmids were constructed successfully and stably transfected into the BxPC-3 cells. MBD1 was not detected in the tumors of the MBD1-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-siRNA group (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, all P < 0.05). 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.