摘要目的 探讨通过RNA干扰技术沉默TIZ基因的表达对卵巢上皮性癌(卵巢癌)细胞生物学行为的影响.方法 设计3对特异性针对TIZ基因的小分子干扰RNA(siRNA),并转染入TIZ基因表达阳性的卵巢癌SKOV3细胞中,采用实时荧光定量PCR(QRT-PCR)技术检测siRNA片段对TIZ基因的沉默率,筛选最佳沉默率的siRNA片段构建pGPU6/GFP/Neo-siRNA-TIZ-573重组干扰质粒,采用脂质体转染法将重组干扰质粒转染入SKOV3细胞(sh-TIZ-573/SKOV3细胞),并设阴性对照细胞sh-NC/SKOV3、阳性对照细胞sh-GA/SKOV3.采用四甲基偶氮唑蓝(MTT)法测定细胞生长曲线,集落形成实验测定细胞生长能力,流式细胞仪检测细胞周期,细胞体外侵袭重建基膜实验、穿膜小室实验和体外黏附实验分别测定细胞体外侵袭、转移及黏附能力.结果 QRT-PCR技术检测显示,TIZ-573 siRNA片段下调TIZ基因表达的作用最明显,沉默率达62％,且成功构建pGPU6/GFP/Neo-siRNA-TIZ-573重组干扰质粒并导入SKOV3细胞中使TIZ基因表达沉默.细胞生长曲线显示,TIZ基因表达沉默后的sh-TIZ-573/SKOV3细胞较对照细胞生长加快.sh-TIZ-573/SKOV3细胞在G0/G1期所占比例(34.9％)比sh-NC/SKOV3、sh-GA/SKOV3细胞少(分别为68.8％、63.4％),分别比较,差异均有统计学意义(P＜0.01).细胞集落形成实验显示,sh-TIZ-573/SKOV3细胞的集落形成率[(89.1±4.6)％]高于sh-GA/SKOV3、sh-NC/SKOV3细胞[(60.8±4.9)％、(57.0±2.6)％],分别比较,差异均有统计学意义(P＜0.01).sh-TIZ-573/SKOV3细胞的侵袭率、转移率和黏附率分别为(48.5±1.7)％、(53.6±3.4)％、(64.9±5.0)％,在TIZ基因沉默前后侵袭、转移和黏附能力无明显改变(P＞0.05).结论 下调TIZ基因的表达对卵巢癌细胞生长有一定的促进作用,其可能发挥抑癌基因的生物学作用.

abstractsObjective To construct TIZ gene RNA interference plasmid,as well as study the biological effects of down-regulating of TIZ gene on ovarian cancer cells.Methods According to the mRNA sequence of TIZ on GenBank,3 pairs of small interference RNA (siRNA) for TIZ gene expression were designed and introduced into SKOV3 cells by liposome transfection reagent; the real-time quantitative PCR (QRT-PCR) was used to detect the interference efficiency and selected the most efficiency siRNA segment;pGPU6/GFP/Neo carrier was used to construct pGPU6/GFP/Neo-siRNA-TIZ-573 restructured interference plasmid,introduced into SKOV3 ccll (sh-TIZ-573/SKOV3).The cell growth curves were made by methyl thiazolyl tetrazolium (MTT) method.The clonogenicity efficiency was observed by clony formation test.The cell cycles were measured by flow cytometry (FCM).The ability of invasion,metastasis and adhesion of ovarian cancer cells were detected by the matrigel invasion assay,transwell migration assay and adhesion assay,respectively.Results QRT-PCR results showed that ovarian cancer SKOV3 cells which transfected by recombinant plasmids pGPU6/GFP/Neo-siRNA-TIZ-573 could down-express TIZ gene.The cell growth curves,FCM and clony formation tests showed that the growth and proliferation of sh-TIZ-573/SKOV3 cells were significantly speeded up compared with the control cells (P ＜ 0.01).There was no significant difference in cell invasion,migration and adhesion between sh-TIZ-573/SKOV3 cells [(48.5 ± 1.7) ％,(53.6 ±3.4)％,(64.9 ±5.0)％,respectively] and the control cells (P ＞0.05).Conclusion The growth of ovarian cancer cells could be speeded up by down-expressing of TIZ gene,which TIZ gene may be play a biological role as a tumor suppressor gene.