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English Abstract
Journal Article
Research Support, Non-U.S. Gov't
[Activation of multiple tumor suppressor genes by MBD1 siRNA in pancreatic cancer cell line BxPC-3].
Zhonghua Yi Xue za Zhi [Chinese medical journal] 2008 July 23
OBJECTIVE: 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.
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 CDH1, RASSF1A, TIMP3, P14ARF and Rb in transplanted tumors.
RESULTS: 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).
CONCLUSION: 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.
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 CDH1, RASSF1A, TIMP3, P14ARF and Rb in transplanted tumors.
RESULTS: 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).
CONCLUSION: 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.
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