[Mechanism study of antisense cyclin B1 in tumorigenesis inhibition using proteomic technique].
OBJECTIVE
Previous researches showed that down-regulating the expression of cyclin B1 in tumor cells by RNA interference may inhibit tumorigenesis, but the mechanism remains to be clarified. This study was to reveal the molecular mechanism of antisense cyclin B1 in tumorigenesis inhibition by comparative proteomic technique.


METHODS
A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin B1 was transfected into mouse colon carcinoma cell line CT26. Total proteins of transfected cells and control cells were extracted and separated by two-dimensional gel electrophoresis (2-DE). The differential expression proteins were analyzed with PDQuest software, and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Mascot database searching. The 2 differential proteins with the highest confidence of the peptides were selected and verified by Western blot.


RESULTS
Seven differentially expressed proteins were identified: Axin2, CCTtheta, DR5, and HPCM27 were up-regulated in transfected cells, while RFP17, mKIAA1195, and LOC77035 were down-regulated. The expression abundance differences of Axin2 and DR5, with the highest confidence, were verified by Western blot.


CONCLUSIONS
Several proteins expressed differentially in CT26 cells after transfection of antisense cyclin B1, which take part in some signal pathways in cell proliferation, differentiation, migration, apoptosis, and transcriptional control. The antitumor effect of antisense cyclin B1 may relate to the interplay of the above proteins.
Yang
Tao
T
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, PR. China. ppyangtao@163.com
Zhang
Ling
L
Yan
Fei
F
Tong
Ai-Ping
AP
Liu
Xin-Yu
XY
Liu
Huan-Yi
HY
Huang
Can-Hua
CH
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D060466:Axin Protein; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D019926:Cyclin B / Q000235:genetics*; D056744:Cyclin B1; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D016373:DNA, Antisense / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D060466:Axin Protein; C111729:Axin2 protein, mouse; C534700:Ccnb1 protein, mouse; D019926:Cyclin B; D056744:Cyclin B1; D003598:Cytoskeletal Proteins; D016373:DNA, Antisense; D018076:DNA, Complementary; D020543:Proteome; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011994:Recombinant Proteins