The Effect Of Cell Cycle And Expression Of Cyclin B1and Cyclin C Protein In Hepatocellular Carcinoma Cell Line Hepg2and SMMC-7721after Silencing β-catenin Gene

Cancer is a global disaster event, and primary hepatic carcinoma is one ofthe ten big malignant tumors, its dath rate rank the third in digestive system. Itstarts hidely, scarce of typical symptom in the beginning, once found it hasbeen to belonging to late stage, and operation therapeutic efficacy is not ideal,so it has been researched by foundation and clinical researches.RNAi is a geneslincing technique at the post transcriptional level caused by introduction of adouble-stranded RNA which induces the degration of mRNA containingspecific homologous sequences, It was first reported by FIRE et al in1998. It isone of the most important technological research findings in the last century,mainly used in signaling pathway and some related gene function. Now, massdata indicate: tumori-genesis are reported to be strongly associated withabnormalities in cell cycle regulation, The cell cycle is a complex process withmyriad genes involved and elaborate and complex signaling mechanisms toallow for this critical cellular process. The main factors taking part in theregulation of cell cycle are cyclins,CDKs and CKIs. The normal functions ofthese regulatory factors keep dynamic equilibrium of cell cycle and ensure thenormal progress of cell cycle. Dysfunction of cell cycle often causes abnormalcell growth and eventually tumor. The cytoplasmic protein β-catenin is acentral molecule in wnt/β-catenin signaling pathway and plays a key roleduring the genesis and development of tumors. Current researches indicated:cyclin A activated in S stage, cyclin E,cyclin D mainly produced a markedeffect at G1/S checkpoint, and the expression of cyclin A,E,D1were affectedafter silencing β-catenin gene in HCC, but it is unclear if other cyclins were affected.Our experiments are just the results of RNAi technology in practicalapplication. small interference RNA (siRNA) against β-catenin was transfectedinto HCC cell line HepG2and SMMC-7721, viewing the expression of theβ-catenin in the different cell line,and detect the cell cycle,cell growth and therelated cyclins in the different cell line at different times.Small interference RNA was transfected into HCC cell line HepG2andSMMC-7721, useing western blotting to detect the expression of β-cateninprotein, in the experiment, we found β-catenin protein expression wasdecreased at72,96h.Analysis of cell cycle by flow cytomery. The cell cycle was analyzed byflowctomery at48h,72h and96h after the transfection. In our experiments,the cell cycle was arrested in G0/G1phase after knockdown of β-catenin bysiRNA at72h in two cell lines. With the time passing, the cell cycle proceededto G2/M phase at96h. The reasons probably were that the inhibition ofβ-catenin affected cell cycle and or some factors compensated with thedecrease of β-catenin. These factors may explain cell cycle was arrested inG0/G1phase at72h and reverted to some extent at96h. The results of MTTalso indicated: the cell growth of the two cell lines were both inhibited at72h.As above, β-catenin may regulate cell cyle, sequentially affect cell growth. Ourresults is alike to those of Hu's studies showing that cell cycle is arrested inG0/G1phase after inhibiting β-catenin gene.In our experiments, cyclin C protein expression increased at72h andreverted at96h, cyclin B1protein expression decreased at72h and reverted at96h. These findings have important implications: silencing β-catenin gene mayinduce the changes of cell cycle and the expression of cyclin C and cyclin B1,they are targets for developmental signals to regulate gene expression. So wethink Wnt/β-catenin signaling pathway may affect cell cycle through regulating the expression of cyclin C and cyclin B1.In addition, because the cell cycle wasarrested in the G0/G1phase at72h, the increase of cyclin C expressionpromoted the progress of cell cycle from to S phase and G2phase or promotedthe progress of the cell cycle from G0phase to G1phase. The decrease of cyclinB1inhibited the progress from G2to M phase or inhibited the progress of thecell cycle from G1to S. The relation that the change of cyclin C and cyclin B1in our experiments with cyclin A, E, D1needs to be further studied.To sum up, the results of our experiments still require validation in othercell lines and in animal studies. But the change of cell cycle,cyclin B1,cyclinC protein expression after silencing β-catenin in two cell lines may bring ussome new idears: irregular activity of cyclins is frequently involved in cancer,targeting Wnt/β-catenin signaling pathway, and its downstream target genecyclin C and cyclin B1for cell cycle arrest in a particular phase may be potentanti cancer agent in the future tumor therapies.