应用RNA干扰技术研究激素非依赖性高转移潜能前列腺癌细胞中survivin的表达及意义

目的研究 survivin 在激素非依赖性高转移潜能前列腺癌中的表达及其与前列腺癌的生物学行为及侵袭和转移潜能的相关性。方法应用 RNA 干扰技术构建 survivin 真核表达载体,转染人激素非依赖性前列腺癌高转移亚系 PC-3M-1E8细胞系。通过细胞生长曲线、肿瘤细胞裸鼠异种接种、软琼脂集落形成实验检测体内、体外细胞生长能力;流式细胞术检测 survivin RNA 干扰质粒对细胞周期及细胞凋亡的影响,Western blot 检测 caspase3活性片段,观察 survivin 抑制细胞凋亡的情况;Matrigel 穿膜实验检测肿瘤细胞体外侵袭能力。结果稳定转染 survivin RNA 干扰质粒的 PC-3M-1E8细胞中 survivin 的 mRNA 及蛋白水平明显降低,蛋白水平与阴性对照组相比,约下降78%～80%,差异有统计学意义(P<0.01);体外培养细胞生长速度及裸鼠体内肿瘤生长速度均明显减慢,锚着不依赖性生长的能力(软琼脂克隆形成数:14.33±3.51)与阴性对照组(52.33±6.81)及空白对照组(54.00±6.00)相比明显降低(P<0.01);凋亡细胞比例明显增加,空白对照组、阴性对照组及干扰阳性组的凋亡比例分别为5.88±0.99、6.97±1.60、16.40±1.95,干扰阳性组的凋亡比例显著高于对照组(P<0.01),并伴有 caspase3活性片段的表达增加;细胞阻滞在 G_0/G_1期(干扰阳性组、阴性对照组及空白对照组的细胞 G_0/G_1期的比例分别为52.71±1.10、43.59±1.83及43.65±3.44,P<0.05),并在细胞形态学上出现多核巨细胞现象;细胞体外侵袭能力明显降低,干扰阳性组的细胞(38.67±6.59)与阴性对照组(46.07±9.97)及空白对照组(47.87±9.58)相比穿膜细胞数明显减少(P<0.05)。结论 survivin 在激素非依赖性高转移潜能前列腺癌中高表达,并与细胞凋亡、细胞生长及肿瘤侵袭有关。抑制 survivin 的表达可能成为临床治疗激素非依赖性前列腺癌的方法之一。

Biological implications of the inhibition of survivin by RNA interference in human androgen-independent prostate carcinoma with highly metastatic potential

OBJECTIVE: To determine the expression level of survivin in androgen-independent prostate carcinoma, and to investigate the biological role of survivin in invasion and metastasis of androgen-independent prostate carcinoma. METHODS: Highly metastatic prostatic cancer cell line PC-3M-1E8 was stably transfected with pSilencer plasmid targeting survivin expression by RNA interference. The biological effects were observed, including anchorage-independent growth, in vitro invasion by soft agar colony formation and Boyden chamber assay, and also in vivo tumorigenesis in nude mice. Cell cycle and apoptosis indices were evaluated by flow cytometry and Western blot analysis of bioactive fragments of caspase 3. RESULTS: The expression of survivin in transfected PC-3M-1E8 cells was markedly depressed at both mRNA and protein levels (about 78% to 80%) as compared with control. The growth of tumor cells was retarded by anchorage-independent growth assay. The survivin transfectants formed smaller and fewer colonies (14.33 +/- 3.51) than the negative (52.33 +/- 6.81) and blank controls (54.00 +/- 6.00). Inhibition of survivin expression was correlated with enhanced apoptosis of tumor cells (percentages of apoptotic cells of the negative control, blank control and experimental groups were 5.88 +/- 0.99, 6.97 +/- 1.60, 16.40 +/- 1.95 respectively), along with an increased expression of activated caspase 3, and cell cycle inhibition at G(0)/G(1) phase (the relative number of cells at G(0)/G(1) phase were 43.65 +/- 3.44, 43.59 +/- 1.83 and 52.71 +/- 1.10, respectively). In addition, multinucleated giant cells were observed along with a marked inhibition of invasion as reflected by fewer penetrating cells by Boyden chamber assay (46.07 +/- 9.97, 47.87 +/- 9.58 and 38.67 +/- 6.59, respectively). CONCLUSIONS: Survivin expression is high in androgen-independent prostate cancer cells and likely may be related to the apoptosis, growth and invasion of the tumor cells. Targeting the survivin pathway by RNA interference appears to be a promising approach for clinical treatment of androgen-independent prostate cancer.