Inhibitory Effects Of CDCA5-siRNA On Proliferation And Apoptosis Of Human Hepatoma Cell Line HepG2

BACKGROUNDHepatocarcinoma is a frequently maliganant tumor in the world,55%of the newincidence occurred in China. Every year the incidence rate is26-32/100,000in Chinese,even70-80/100,000in some high prevalent area. Hepatocarcinoma is one of the mostlethal disease, ranking the third highest mortality rate in all malignancies. Most of thepatients are clinically asymptomatic metastasis progress occurs in the early stage.Hepatocarcinoma is not sensitive to radiotherapy or chemotherapy. Therefore, it is vitalimportant to seek for new treatment beside operation, radiotherapy and chemotherapy.Currently gene therapy becomes a new strategy in tumor treatment. Cell division cycleassociated5(CDCA5) belongs to quantitative trait locus (QTL) gene area. Sororin isrequired for stable binding of cohesin to Chromatin and is essential for the maintenance ofgenome integrity. Recent research shows that upregulation of CDCA5is closely related tolung cancer, cervical cancer and prostrate cancer. Our previous study found that CDCA5ishigh expressed in human hepatocarcinoma tissue and human hepatoma cell line. Targetedtreatment of CDCA5gene is expected to become a new direction in the therapy targetinghepatocarcinoma. We used small interfering RNA (siRNA) technology to inhibit theexpression of CDCA5in human hepatocarcinoma cell line in vitor, then observe the effectof CDCA5gene inhibition on cell proliferation and apoptosis.OBJECTIVE:To investigate influence of CDCA5small interfering RNA(CDCA5-siRNA) onproliferation and apoptosis of human hepatocarcinoma cell line HepG2. METHODS:1. Screening of human hepatocarcinoma cell line with high expression of CDCA5.Western blot is used to detected the expression of CDCA5protein in humanhepatocarcinoma cell line HepG2,Hep3B,Huh7and HL-7702.2. Designing of siRNA and optimizing of transfection efficiency.Design and fabricate for the CDCA5small interfering RNA (siRNA) sequences (1,2and3). The FAM-siRNA was use transfected into human hepatocarcinoma cell line byLipofectAmineTM RNAiMAX transfection reagent. Transfection efficiency was assessedby inverted fluorescence microscope.3. Inhibitory effects of CDCA5-siRNA on proliferation and apoptosis of humanhepatoma cell line HepG2.The human hepatocarcinoma cell lines HepG2were divided into siRNA group (A, Band C groups), universal scrambled negative control siRNA group (NC group) and Blankgroup. Three CDCA5-siRNAs with different sequences were transfected into HepG2respectively. Western blot method was used to measure expressions of CDCA5proteinlevels at72h after transfection． The cell proliferation was assessed by CCK-8at24,48,72,96,120h after transfection. The Annexin V-FITC/PI double-labeled flow cytometrywas employed to measure the apoptosis at48h after transfection.RESULTS:1. A cell line with high expression of CDCA5gene, Hep G2was screened.2. Transfection efficiency was above90%when the concentration siRNA was10nM, andLipofectAmineTM RNAiMAX1.75μl/ml.3. The expression of CDCA5protein significantly decreased in the CDCA5-siRNA groups(A,B and C groups) compared with NC group and Blank group（P<0.05). Group B had thelowest expression rate and highest inhibition rate (89.3%).4. The relative growth rate of group B was significantly lower than those of NC group andBlank group since48hours after transfection （P<0.05), and the lowest (66.58±2.58%) was at72h in group B. The early and late apoptosis rate were (17.43±2.31)%and(22.37±2.21)%in group B, higher than those of other two group（P<0.05).CONCLUSION:1. siRNA down-regulats the expression of CDCA5gene in human hepatoma cell lineHepG2.2. Inhibition of the CDCA5gene can suppressed the cell proliferation and increase cellapoptosis.