The Effects Of RNA Interference Target CDC25A Gene On Proliferation Of Human Liver Cancer HEPG2 Cells And Subcutaneous Tumur Of Human Liver Cancer HEPG2 Cells In Nude Mice

AIM:To observe the change to the CDC25a gene on the expression of mRNA and protein after the RNA interference (RNAi) through the recombinant lentiviral vector, and then research the change to the proliferation of HepG2 cell and the growth of tumor in nude mice. Meanwhile, this study investigates its mechanism of actions.Methods 1. Real-time fluorescence quantitative PCR was applied to detect the expression of CDC25a mRNA levels in HepG2. SMMC-7721 and Sk-hep-l,then screen experiments using cells group.2. Constructed five siRNA targeting CDC25a gene vectors (CDC25a-RNAi vector) through RNA interference technology,and then transfect stably to human liver cancer cell lines HepG2.Screened the most effective targets.3. Divided the cells into three groups:the experimental group is CDC25a gene silencing groups,through chronic diseases particles (LV-CDC25a-RNAi1,2,3,4,5) infected HepG2 cells (KD group); negative control group is the group that use the comparison Lentiviral Particles (LV -RNAi-NC) infected HepG2 cells(NC group); the normal control with no treatment(CON groug). Interference effects and select the most effective target through RT-PCR、Real-time fluorescence quantitative PCR and Western blot to detect the expression of CDC25a mRNA and protein.4. RT-PCR and Real-time fluorescence quantitative PCR was applied to detect the expression of Cyclin E and CDK2 mRNA levels in the HepG2 cells.5. MTT assay、Giemsa stain method were applied to measure the proliferation of human hepatoma cell HepG2, flow cytometry was applied to measure the cell cycles.6. Divided 30 female nude mice into three groups, each group included 10 nude mice. Vaccination of three groups of cells in the left armpit of female nude mice subcutaneous respectively, formed tumors (that is, the successful construction of nude mouse transplantation tumor model).Observed the tumors formation and growth in nude mice for 4 weeks.The tumors volume and weight were measured after 4 weeks. Transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction(qPCR) were used to detect the expression of CDC25a mRNA levels in the subcutaneous tumors, Western blot was used to detect the expression of CDC25a protein levels in the subcutaneous tumors.RESULTS:1. According to the results of qPCR, the CDC25a’ mRNA expression in the human liver cancer cells HepG2 was the higher than in SMMC-7721 and Sk-hep-1 cells. So we choose the HepG2 cells as the experiments using cell.2. Used the Successful builded CDC25a-RNAi vectors to infected HepG2 cells,we can find that the cell fluorescent display rate> 95%.Western blot and RT-CR results showed that:compared to the negative and blank control,five kinds of targets in the CDC25a silence group the CDC25a mRNA and protein expression levels decline significantly,among them with LV-CDC25A-RNAi(9928-1R)(KDl) interference target on the highest efficiency, choosed as the experimental group, there was no statistically significant difference between the negative and blank control group (P>0.05).3. The PCR showed that:the cyclin E mRNA and Cdk2 mRNA expression of silent group cell is below negative control drapes and normal control subjects (P<0.05).4. MTT assay showed that:the cells of experimental group after lentiviral transfection at 490 nm absorption luminosity values in 48,72,96 and 120 hour was lower than the negative control group and the normal control group, the difference was significant (P< 0.05);the time at 24 hour although lower than negative control and normal control group, but the difference was not significant (P=0.065). Giemsa staining results showed that:the number of clones in the experimental group was 24, significantly lower than the negative control group (clone number 98), the normal control group (99 clones), the difference was significant (P<0.05). The results of flow cytometry reveal that the cell of silence group block in G1 phase.5. Tumors growth rate、volume and weight in the experimental group were significantly lower than the control and negative control group(P<0.05). CDC25a mRNA and protein expression in experimental group’s tumor tissue was lower than the negative control and blank control group (P<0.05).CONCLUSION:1.The proliferation of human hepatoma cells can be inhibited by using technology of RNAi.2. The expression of CDC25a gene could significantly be silenced by using technology of RNAi, the growth of subcutaneous tumor could be inhibited also.3. It suggests that CDC25a gene can be considered as a alternative target for the treatment of liver cancer.