| Objective:Methyl-CpG binding domain protein 1(MBD1),a suppressor of gene transcription,may be involved in inactivation of tumor suppressor genes during tumorigenesis.Over-expression of MBD1 had been detected in human pancreatic carcinomas and correlated with tumor metastasis.The purpose of this study is to investigate the different expression of methyl-related proteins and genes after the silence of MBD1 in pancreatic cancer cell line BxPC-3,observe the effects of MBD1-knockdown on tumor biological characteristics and discuss the role of MBD1 as an impotant transcription regulator in carcinogenesis of pancreatic cancer.Methods:The stable MBD1-knockdown pancreatic cancer cell line(BxPC-3) was established by RNA interference.The differential proteins between control and MBD1-knock-down cells were detected by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The results were confirmed by RT-PCR and western blot.The expression of MBD1-related proteins in tumor tissues of 16 specimens of resected pancreatic cancer and 10 normal pancreatic tissues were determined by immunohistochemical staining.MTT assay and wound healing test were used to detect the changes of BxPC-3 cell growth and cell migratory ability after MBD1 knockdown respectively.In vivo,the pancreatic cancer nude mice model was established to observe the tumor growth and compare the different expression of some related tumor suppressor genes and methyl-related genes between MBD1-knockdown and control group by RT-PCR assay.Results:MBD1-siRNA recombinant plasmid were stably transfected into BxPC-3 cell line,the expression of MBD1 was significantly down-regulated after RNA interference, which was confirmed by RT-PCR and Western blot assay.By two-dimensional gel electrophoresis and mass spectrometry,five up-regulated proteins(including tubulin beta 2,splicing factor arginine/serine- rich isoform 1,ER-60,P4hb,EFHD2) and nine down-regulated proteins(including GRP78,Tollip,HSP A8,Vimentin,Stathmin 1, cofilin 2,hnRNP K,eIF3,ZFP-36) were identified clearly.Most of the identified differential proteins were involved in tumorigenesis,especially related with tumor metastasis,and some were prognostic biomarkers for human malignant tumors.The expressions of Vimentin and Stathmin in pancreatic carcinoma were significantly higher than that in normal pancreas tissues,and the positive expression of Vimentin was closely related with lymph node metastasis.The growth of BxPC-3 cell was suppressed after RNA interference targeting MBD1 while cell migratory ability remained unchanged in vivo and vitro.The mRNA expression of tumor suppressor genes CDH1 and Rb were significantly up-regulated when MBD1,Vimentin and GRP78 were down-regulated in transplanted tumor of nude mice model.Conclusion:The expression of MBD1 in pancreatic cancer cell BxPC-3 could be down-regulated by RNAi.Down-regulation of MBD1 could relieve the inhibition of some tumor suppressor genes and affect the biological features of pancreatic cancer cell BxPC-3,resulting in significant changes of some methyl-related proteins involved in carcinogenesis and metastasis.MBD1,as a transcription regulator,might play an important role in tumorigenesis of pancreatic cancer. |
