The Effect Of Cyclin E RNAi On The Growth And Cell Cycle Of Hepatocellular Carcinoma Cells HepG2

Background and aimsUncontrolled proliferation induced by deregulation of cell cycle is an important mechanism for tumorigenesis. cyclin E which is a kind of cyclins, combined with CDK2, plays a great role in G1/S transition. Overexpression of cyclin E leads to existing of cyclin E during the whole cell cycle and subsequently activates CDK2and promotes phosphorylation of pRb continuously and then induces tumorigenesis as a result of increased G1/S transition. Additionally, intracellular accumulation of cyclin E leads to chromosome instability, which is an alternative mechanism for the tumorigenesis induced by overexpression of cyclin E. It is reported that cyclin E overexpression was related to the centrosome amplification and structural abnormalities, indicating that overexpression of cyclin E resulted in centrosome abnormalities and then induced tumorigenesis.Cyclin E is probably related to hepatic tumorigenesis. Hepatocellular carcinoma (HCC) is the most common tumor in the world. Our country is prevalent for HCC and occupies over50%populations who were died of HCC in the world. Clinical studies and animal experiments showed that overexpression of cyclin E was observed in patients with HCC and was probably related to differentiation, invasion and clinical stages of HCC.Based on the effect of cyclin E on cell cycle and the relationship between cyclin E and HCC, we investigate the effect of decreased expression of cyclin E on the growth and cell cycle of hepatocellular carcinoma cells by using siRNA vector for cyclin E to inhibit the expression of cyclin E in hepatocellular carcinoma cells HepG2.MethodsAccording to the principle of selection of siRNA sequences, two single-stranded DNAs targeting to cyclin E siRNA were designed and obtained and then were formed into double-stranded hairpin DNA by annealing. Two recombinants of pGFP-V-RS-siCE951and pGFP-V-RS-siCE1122were obtained by inertion of hairpin DNA into siRNA vector, pGFP-V-RS. The inserted sequences were analyzed and compared with designed sequences. Constructed cyclin E siRNA expression vectors were transfected into HepG2using LipofectAmineTM2000. All the cells were divided into four groups:silencing group1(transfected with pGFP-V-RS-siCE951), silencing group1(transfected with pGFP-V-RS-siCE1122), unrelated siRNA control group (transfected with pGFP-V-Con) and blank control group (added by transfection reagent without any transfection). RT-PCR and Western-Blot were used to study the expression of cyclin E in the four groups. The cell cycles and proliferation of HepG2cells were tested by flow cytometry and CCK-8kit respectively. Soft agar clone experiment was used to test clone formation of HepG2.Results1. GCAAAAGG TTTCAGGGTAT and GGACAAAGCCCGAGCAAAG were selected as the siRNA targeting sequences by homologous analysis.2. Two recombinants of pGFP-V-RS-siCE951and pGFP-V-RS-siCE1122which were consistent with our designs were obtained.3. Silencing group1and2showed decreased cyclin E mRNA compared with unrelated siRNA and blank control groups (0.215±0.021and0.178±0.019VS0.406±0.037and0.372±0.042respectively, p<0.05). There was no significant difference between the two control groups (p>0.05).4. Cyclin E expresion was observed in all groups in western blot experiments. It is showed that cyclin E expression was higher in unrelated siRNA and blank control groups compared to silencing group1and2.5. In proliferation test, At3d,4d and5d, the absorbance in silencing group1and2decreased significantly compared with the two control groups (p<0.05).6. Compared with the two control groups, the number of cells in silencing group1and2showed decrease in S and G2/M and increase in G0/G1(p<0.05). Statistical significance was not observed between the two control groups (p>0.05).7. In soft agar clone formation experiment, the number of clones in silencing group1and2decreased significantly compared with unrelated siRNA and blank control groups (124.6±16.3and141.2±15.2VS209.2±25.8and226.7±34.2respectively, p<0.05). There was no significant difference between the two control groups (p>0.05).Conclusions1. Constructed cyclin E siRNA vectors, pGFP-V-RS-siCE951and pGFP-V-RS-siCE1122, in which GCAAAAGGTTTCAGGGTAT and GGACAAAGCCCGAGCAAAG were selected as target sequences, dramatically decreased expression of cyclin E in HepG2. Therefore, the selected target sequences were optimal for cyclin E siRNA.2. Inhibition of cyclin E expression decreased the number of cells entering S and G2/M and increased the number of cells in G0/G1. Additionally, Inhibition of cyclin E expression inhibited the growth of HepG2.