微创医学

【摘要】目的探讨Dicer抑制对卵巢癌细胞A2780增殖和迁移能力的影响，以进一步明确Dicer在卵巢癌生物学行为中的作用。方法合成Dicer的小干扰RNA（Dicer siRNA）转染A2780细胞并筛选有效siRNA，实时定量PCR(RTPCR)检测Dicer的mRNA的表达，蛋白印迹法检测Dicer蛋白的表达，MTT检测细胞生长活力，Transwell小室检测细胞迁移能力，流式细胞仪测定细胞周期。结果与未转染组相比，siRNA1658转染组Dicer基因mRNA的表达最低，为(0.194±0.002)，明显低于siRNA1897转染组的(0.535±0.015)，差异有统计学意义（P＜0.001），但与siRNA334组的（0.251±0.014）相比，差异无统计学意义（P＞0.05）。实时定量PCR结果显示，以未转染A2780细胞Dicer mRNA表达水平为100%，siDicerA2780细胞Dicer的mRNA水平相对下降（0.157±0.012），差异有统计学意义（P＜0.001）。以未转染A2780细胞Dicer的蛋白表达水平为100%，siDicerA2780细胞Dicer的蛋白水平相对下降(0.153±0.021)，差异有统计学意义（P＜0.001）。在种板后第3天、第4天、第5天，与对照组细胞比较，siDicer转染的A2780细胞增殖能力增强（P均＜0.05）。与对照组转染siNC的细胞相比，转染siDicer 96 h后，siDicerA2780细胞的增殖活性平均上升了23%。细胞周期分析结果显示siDicerA2780细胞中G0/G1期占(44.26±0.48)%，S期占(25.05±1.20)%，G2/M期占(21.53±0.77)%；对照组siNCA2780细胞中G0/G1期占(52.79±1.11)%，S期占(20.01±1.22)%，G2/M期占(19.80±0.24)%。siDicerA2780细胞S期和G2/M细胞较对照组明显增加(P均＜0.05)，而G0/G1期细胞则减少(P＜0.001)。与对照组siNCA2780细胞的穿膜细胞数相比，抑制Dicer表达的siDicerA2780 细胞的穿膜细胞数增加（3.99±0.29）倍(P＜0.001)。结论Dicer基因具有抑制卵巢癌细胞增殖和侵袭的能力。

【Abstract】 ObjectiveTo investigate the effect of inhibition of Dicer on the proliferation and migration of ovarian carcinoma cell A2780, thus to further specify the role of Dicer in biological behavior of ovarian cancer. MethodsThe synthetic Dicer small interfering(Dicer siRNA) was transfected into A2780 cells and the effective siRNA was selected. The expression of Dicer mRNA and protein were detected by realtime quantitative PCR and Western blot respectively.The growth and viability of cells were measured by MTT. Cellmigration was measured by Transwell chambers.Cell cycle was detected by flow cytometry.ResultsThe expression of Dicer mRNA in the siRNA1658 transfected group (0.194±0.002)was the lowest compared with the nontransfected group, and was significantly lower than that in the siRNA1897 group(0.535±0.015), (P＜0.001).But there was no statistical difference in the expression of Dicer mRNA between the siRNA1658 transfected group and the siRNA334 group（0.251±0.014） (P＞0.05).The result of realtime quantitative PCR showed that the Dicer mRNA level of siDicerA2780 cell relatively decreased （0.157±0.012） when the Dicer mRNA level of nontransfected A2780 cell was defined as 100%(P＜0.001). Dicer protein level of siDicerA2780 cell relatively decreased （0.153±0.021）when the Dicer protein level of nontransfected A2780 cell was defined as 100%(P＜0.001). At the 3rd, 4th and 5th days,the proliferation of A2780 cells transfected by siDicer increased compared with the cells in the control group（all P＜0.05）. After 96 hours of transfection by siDicer, the proliferation activity of siDicerA2780 cells increased by an average of 23% compared with the cells transfected by siNC in the control group.The result of cell cycle analysis showed that siDicerA2780 cells at theG0/G1 phase, S phase and G2/M phase accounted for (44.26±0.48)%，(25.05±1.20)% and (21.53±0.77)% respectively. In the control group, siNCA2780 cells at G0/G1 phase, S phase and G2/M phase accounted for (52.79±1.11)%，(20.01±1.22)% and (19.80±0.24)% respectively. The numbers of siDicerA2780 cells at S phase and G2/M phase were more， but the number of cells at G0/G1 phase was less compared with the cells of the control group(P＜0.001). Dicerinhibited siDicerA2780 cells penetrating membrane achieved （3.99±0.29）folds increase in the number compared to siNCA2780 cells in the control group(P＜0.001). ConclusionDicer gene obtains the ability of inhibiting growth and invasion of ovarian cancer cells.