The Effect Of RNA Interference Against C-myc Oncogene On Cell Cycle

C-myc is a eternal oncogene and plays a critical role in regulating cell proliferation, growth, apoptosis and differentiation. Overexpression of c-myc is one of the most common alterations in human cancers and is mostly responsible for the maglinant transformation. In our study ,the pSilencer-c-myc plasmid expressing SiRNA against c-myc mRNA was transfected into A549 cells by positive ion method. 72h after transfection, cells were harvested and the levels of c-myc mRNA were detected. RT-PCR results showed that c-myc mRNA of transfected cells was decreased to 40% compared with the untransfected cells. At the same time, the c-Myc protein levels were estimated by WesternBlotting and displayed a decrease of 70% .So it can be concluded that the RNA interference against c-myc was successful.The Flowcytometry was employed to analyze the effect of c-myc RNAi on the cell cycle of A549 cells 72h after transfection.Our results showed that the RNA interference decreased the proportion of G0/G1 phase cells by 16%,from 51.5±1.7% to 35.1±1.1%, and the percentage of S phase cells was increased by 14%,from 41.3±1.4% to 55.1 ±1.6%. But no change of G2/M cell numbers was observed between control cells and transfected cells(p>0.05). These data declared that c-myc RNAi induced a S-arrest of cell cycle.To investigate how the S-arrest happened, analyses of the mRNA levels of cell cycle-relative gene were performed, including cyclin,CDK and CKI .Our experiments revealed that after RNA interference: 1) The mRNA levels of cylcin D3,E,A were markedly downregulated in different extents (p<0.05) , by 22±8%,30±2%and 45±7%, respectively.However, no difference of cyclin Dl mRNA was observed (p>0.05). 2) CDK2 and CDK4 mRNA levels were decreased (p<0.05),by 44±4%, 39±4%,respectively. 3) In opposition to the formers, p21 and p27 mRNA were elevated (p<0.05) , by 36±6% and 44±8% (p<0.05).To be conscious of whether the changes of mRNA induced alterations of protein expression, protein levels of Cyclin and CDK were detected. Our data showed that 72h after transfection,the protein levels of cyclin D3,E,A and CDK2,4 were decreased (p<0.05) ,by 42 + 5%, 41+2%, 57 + 5% and 56±9 %, 36±5% respectively.This confirmed that the downregulations of cyclin D3, E, A and CDK2,4 mRNA were responsible for decreases of their protein.All these results explained that after c-myc RNA interference and the decreased expression of c-Myc, the activations on cyclins and CDKs, as well as the repressions on CKIs were impaired.This directly induced the S-arrest.In conclusion,as a gene therapy method,RNA interference against c-myc indicates an excellent future.