Proteomic Research Of Antisense CyclinB1 On The Mechanism Of Anti-tumor Effect

Background and Objective: Cyclin B1, a cell cycle protein in mitosis phase, connects with cyclin-dependent kinase 1 (CDK1) to form mitosis promoting factor (MPF) and plays an important role in the regulation of cell cycle. It is over-expressed in a variety of tumors and is thought to be an potential malignant indicator of tumor, which is related to the genesis, differentiation, metastasis and prognosis of tumor. Our previous researches showed that applying antisense cyclin Bl specially targeted in the CT26 colon carcinoma cell lines to down-regulate its expression might induce G1 arrest and cell apoptosis , thus inhibit the proliferation activity of tumor cells in vitro and in vivo, but the mechanism of tumor proliferation inhibition remained to be clarified. And other researches on this mechanism are still lack and most of them are limited to nucleinic acid or individual protein level. While using proteomic methods, we can investigate the molecular constructions and interaction rules in large scale and high throughput from a whole, dynamic aspect. It may provides a powerful means to explore the genesis, development and pathopoiesis mechanism of tumor. This study is to reveal the molecular mechanism on tumor proliferation inhibition of mouse full-length cyclin B1 antisense cDNA using comparative proteomic methods.Methods: Firstly, a recombinant plasmid containing the full-length antisense cDNA of mouse cyclin Bl (AS-mCLB1) and pcDNA3.1 empty vector were introduced into CT26 colon carcinoma cell lines to establish AS-mCLBl(Ac) and pcDNA3.1 (Pc) stable transfectants respectively. The activity of cell proliferation of Ac, Pc and untransfected (Nc) groups were measured by MTT and cell growth assay. And the cell cycles and apoptosis were determined using flow cytometry. Then we used two-dimensional gelelectrophoresis (2-DE) and matrix-assisted laser desorption / ionization time of flight (MALDI-TOF-MS) mass spectrometry to extract and identify the differential expression proteins between Ac and Pc or Nc groups. And we employ RT-PCR and western blot to verify part of the differential proteins on the mRNA and protein levels. Finally, tumorigenicity and survival were observed in the BALB/c mice which were implanted with Ac, Pc and Nc cells respectively. And the difference of angiogenesis and apoptosis was detected with HE staining and in situ end-labeling (TUNEL).Result: Obvious G1 arrest, cell apoptosis and inhibition of cell growthwere observed in the Ac cells. And 25 differential expression proteins were identified by comparative proteomics methods, among which 10 proteins were up-regulated and 15 proteins were down-regulated in the experiment group. These differential proteins are related to cell apoptosis, angiogenesis, oncogene inactivation, transcriptional control and signal transduction,etc. RT-PCR and western blot were employed to verify 6 differential proteins on the mRNA and protein levels. In the animal experiment, tumors in the Ac group developed on day 12 versus day5, 6 in the two control groups after inoculation respectively. And the survival time of Ac group is longer than the two control groups markedly. The HE staining and TUNEL assay showed less angiogenesis and more apoptosis in the experiment group.Conclusion: This study confirmed mouse full-length cyclin B1 antisense cDNA could induce G1 arrest and apoptosis of tumor cells and thus inhibit its proliferation by reducing the expression of cyclin B1 gene. Multiple differential expression proteins are revealed through proteomic methods between Ac and Pc or Nc groups, which are related to cell apoptosis, angiogenesis, oncogene inactivation, transcriptional control and signal transduction,etc. Accordingly, it suggested that the tumor proliferation inhibition of mouse full-length cyclin B1 antisense cDNA might involve in cell cycle arrest, induction of apoptosis, inactivation of oncogene, reduction of angiogenesis, impact of transcription molecular activity and expression changes of correlated signal molecules on cyclin B1 or other signal pathways,etc.