survivin反义RNA对卵巢癌细胞株SKOV3生长的抑制作用

目的观察survivin反义RNA对卵巢癌细胞株SKOV3的生长抑制作用及其对裸鼠皮下种植瘤成瘤能力的影响。方法构建能在真核细胞中稳定表达survivin反义RNA的载体pcDNA3-SVVas,应用基因重组技术,将pcDNA3-SVVas经脂质体lipofectam ine 2000介导转染卵巢癌细胞株SKOV3(SKOV3/SVVas),同时以空载体pcDNA3转染SKOV3细胞(SKOV3/neo)作为对照;绘制细胞生长曲线,采用免疫组化、RT-PCR和流式细胞仪检测SKOV3/SVVas、SKOV3/neo和SKOV3细胞survivin mRNA及蛋白的表达和细胞凋亡率。将24只裸鼠随机分为3组,每组8只,分别将SKOV3/SVVas、SKOV3/neo、SKOV3细胞接种于裸鼠皮下,观察成瘤情况及肿瘤体积变化。结果与SKOV3细胞比较,SKOV3/SVVas细胞增殖能力明显降低;SKOV3/SVVas细胞survivin蛋白阳性细胞率及survivin mRNA表达量分别为(37.5±1.0)%、0.407±0.022,与SKOV3细胞的(81.2±0.4)%、0.793±0.042和SKOV3/neo细胞的(80.4±0.8)%、0.734±0.039比较,差异均有统计学意义(P<0.01);SKOV3/SVVas细胞凋亡率显著增加,为(27.4±9.6)%,与SKOV3和SKOV3/neo细胞比较,差异有统计学意义(P<0.05)。SKOV3/SVVas组裸鼠成瘤率降低,出现目检可见肿瘤的平均时间延长为(14.0±1.0)d,与SKOV3/neo组的(6.1±0.8)d和SKOV3组的(5.8±0.9)d比较,差异有统计学意义(P<0.01);与SKOV3、SKOV3/neo组比较,SKOV3/SVVas组裸鼠肿瘤生长速度缓慢,肿瘤体积的差异均有统计学意义(P<0.01)。结论稳定表达的survivin反义RNA能够有效抑制SKOV3细胞生长,降低survivin的表达,诱导SKOV3细胞凋亡;转染survivin反义RNA的SKOV3/SVVas细胞在裸鼠皮下的成瘤能力降低。

Effect of antisense survivin RNA transfection on the growth of ovarian carcinoma SKOV3 cells in vitro and in vivo

OBJECTIVE: To study the inhibition effect of survivin antisense RNA on the growth of ovarian carcinoma SKOV3 cells, and the tumorigenic ability of the transfected SKOV3 cells implanted subcutaneously in nude mice. METHODS: The recombinant vector pcDNA3-SVVas was constructed by directed cloning of fragments of survivin cDNA into the vector. The ovarian carcinoma SKOV3 cells were transfected with pcDNA3-SVVas by lipofectamine 2000 (SKOV3/SVVas cells), and blank vector pcDNA3 as control (SKOV3/neo cells). The effect of survivin antisense RNA on cell growth was assessed by growth curve. The inhibition of expression of endogenous survivin protein and mRNA was evaluated by immunohistochemical stain and RT-PCR. The apoptosis of cells was assessed by flow cytometry. Twenty-four nude mice were divided into three groups, then SKOV3/SVVas, SKOV3/neo and SKOV3 cells were implanted subcutaneously. The growth of tumor was observed and the tumor volume calculated. RESULTS: The expression of survivin significantly decreased in SKOV3/SVVas cells in comparison to that in SKOV3 and SKOV3/neo cells. The growth of the SKOV3/SVVas cells became significantly slower than those of SKOV3 cells and SKOV3/neo cells (P < 0.01). The expression of survivin protein and mRNA in the SKOV3/SVVas cells were (37.5 +/- 1.0)% and 0.407 +/- 0.022 respectively, compared with SKOV3 [(81.2 +/- 0.4)%, 0.793 +/- 0.042] and SKOV3/neo [(80.4 +/- 0.8)%, 0.734 +/- 0.039]. The difference was significant (P < 0.01). After transfected with pcDNA3-SVVas, cells were found apoptosis in early stage by flow cytometry. The apoptosis rate was (27.4 +/- 9.6)%. There was significant difference between SKOV3/SVVas cells and SKOV3 cells, as well as between SKOV3/SVVas and SKOV3/neo cells (P < 0.05). The tumorigenic ability of SKOV3/SVVas cells was reduced. The first time that tumor could be detected in SKOV3/SVVas group, (14.0 +/- 1.0) days was significantly prolonged compared to SKOV3/neo, (6.1 +/- 0.8) days and SKOV3, (5.8 +/- 0.9) days (P < 0.01). In SKOV3/SVVas group, 5 of 8 nude mice were found tumor, the growth rate of tumor was slower compared with the other two groups. When compared in volume, the difference was significant (P < 0.01). CONCLUSIONS: Stable expression of survivin antisense RNA can effectively inhibit the growth of SKOV3 cells and reduce the expression of endogenous survivin proteins and mRNA, induce the apoptosis of cells. Survivin antisense RNA can inhibit the tumorigenesis of SKOV3 cells in nude mice.