| ObjectiveThe one of main mechanism of malignancy tumor is that the cell cycle regulation out of order induce cell unlimited proliferation .Skp2 is one of the essential human F-box protein family for DNA duplication, which enduce specificitly recognice substrate and participate the cell cycle regulation.RNAi(RNA interference)can efficiently target specific interference in the expression of target genes, and then act as the simply and validly implement to take the place of gene knock-out. The prophase experiment had confirmed that Skp2-siRNA can active suppress the expression level of mRNA and the proliferation condition of Eca-109 cells by realtime-PCR and CCK-8. In this study, we detect continuously the expression of Skp2 and p21WAF1/CIP1 protein and the change of cell cycle after Skp2-siRNA transfection on Eca-109 cells , then analyse the relationship between Skp2 and p21WAF1/CIP1 and their effects on cell cycle, and provid theoretical basis for targeted therapy.MethodsThe Skp2-siRNA was designed and synthesized and then transfected Eca-109 esophageal carcinoma cell line by RNAi-Mate vector.The cell line was divided into five groups, including the blank control group, the siRNA transfection group, the mock transfection group, the negative control group and the positive control.The expression of Skp2 and p21WAF1/CIP1 protein were detected with immunocytochemistry and observed with HIPAS-2000 computer image acquisition system, the result was analyzed with IPP system. Two-three ten thousand cell counts were performanced by ModFit analyse with flow cytometry and its results were analyzed statistically by ANOVA. Results1.The transfection effection is exceed eighty percentag after fluorescent labelled siRNA transfection 6 hours by the fluorescence microscope count.2.According to cell morphology after Skp2-siRNA transfection 24 hours, the cells begun to become round and shrink, the growth became slower than control group at the same time. The part of cell begun to fall off after 48 hours, and the cells fall off more after 72 hours.3.The result of immunocytochemistry:After transfection with Skp2- siRNA, the Skp2 protein expression of Eca-109 esophageal carcinoma cell line began to decrease 24h later,the IOD value is 5.74±0.21, decreased maximum at 48h, the IOD value is 2.81±0.19, and began to increase 72h later, the IOD value is 8.12±0.26. Compared with blank control group,the difference has statistics significance(p<0.01). There were statistics significance among 24h, 48h, 72h in siRNA transfection group(p<0.01).The IOD value of Skp2 protein among 24h,48h,72h in the mock transfection group was respectively 13.35±0.17,13.10±0.21,14.02±0.23, and had no statistics difference from blank control group(p>0.05).The IOD value of Skp2 protein among 24h,48h,72h in the negative control group was respectively 13.65±0.38,13.05±0.10,13.79±0.24, and had no statistics difference from blank control group(p>0.05).4. The result of immunocytochemistry: After transfection with Skp2-siRNA, the p21 WAF1/CIP1 protein expression of Eca-109 esophageal carcinoma cell line began to increase 24h later, the IOD value is 8.21±0.34, increase maximum at 48h, the IOD value is 14.03±0.18, and began to decrease 72h later, the IOD value is 5.55±0.37. Compared with blank control group,the difference has statistics significanc(ep<0.01).There were statistics significance among 24h, 48h, 72h in siRNA transfection group(p<0.01).The IOD value of p21WAF1/CIP1protein among 24h,48h,72h in the mock transfection group was respectively 2.47±0.20,2.82±0.17,2.05±0.20, and had no statistics difference from blank control group(p>0.05).The IOD value of p21WAF1/CIP1protein among 24h,48h,72h in the negative group was respectively 2.67±0.11,2.94±0.17,2.27±0.12. and had no statistics difference from blank control group(p>0.05).5. The result of flow cytometry:After transfection with Skp2-siRNA, the percent of G1 phase of 24h, 48h, 72h was respectively 49.00±1.72%, 64.22±1.62%, 55.44±1.26%, which were higher than in blank group. Compared with blank control group, the difference has statistics significance (p<0.01).There were statistics significance among 24h, 48h, 72h in siRNA transfection group(p<0.05).The percent of G1 phase among 24h,48h,72h in the mock group was respectively 39.46±1.03%,36.42±0.52%,37.58±1.34% and had no statistics difference from blank control group(p>0.05).The percent of G1 phase among 24h,48h,72h in the negative group was respectively 36.26±1.07%,36.97±1.13%,35.07±1.11% and had no statistics difference from blank control group(p>0.05).After transfection with Skp2-siRNA, the percent of S phase of 24h,48h,72h was respectively 42.85±0.89%, 32.88±1.30%, 36.15±1.51%, which were lower than in blank group.Compared with blank control group,the difference of 24h from 48h or 72h had statistics significance (p<0.01),and the difference of 48h had no statistics difference from 72h in control group(p>0.05).The percent of S phase among 24h,48h,72h in the mock transfection group was respectively 45.18±1.47%,44.31±0.79%,44.70±1.34%, and had no statistics difference from blank control group(p>0.05).The percent of S phase among 24h,48h,72h in the negative group was respectively43.10±0.85%,44.47±1.04%,46.34±1.41%, and had no statistics difference from blank control group(p>0.05).ConclusionsSkp2-siRNA can inhibit the growth and proliferation of Eca-109 esophageal carcinoma cell line, which is mediated by effectively down- regulate Skp2 protein expression and upregulate p21WAF1/CIP1 protein expression,and then block the cell cycle at the G1 stage. RNAi technique may become important means to inhibit the growth of tumour cell...
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