寡核苷酸阻断ROCK-1蛋白对人卵巢癌细胞恶性行为的影响

目的通过反义寡核苷酸(ASODN)对ROCK-1的特异性阻断,探讨ROCK-1蛋白在卵巢癌转移中的作用。方法将ROCK-1反义寡核苷酸(ASODN)以脂质体介导,转染人卵巢癌细胞系SW626和Caov-3细胞,采用逆转录聚合酶链反应(RT-PCR)与Western blot印迹法,检测转染前后ROCK-1蛋白的表达,Boyden小室观察ROCK-1 ASODN对SW626和Caov-3细胞侵袭及迁移能力的影响,采用二苯基溴化四氮唑蓝(MTT)比色法,测定转染前后细胞增殖及黏附能力的变化。结果转染ROCK-1 ASODN后,2株细胞内ROCK-1蛋白的表达明显减少,最大抑制率可达49.0%;细胞的侵袭能力受到明显抑制,10μmol/L组和20μmol/L组SW626细胞的侵袭能力分别为对照组的75.6%±3.8%和54.7%±2.9%,Caov-3细胞为68.8%±4.7%和50.0%±4.5%;转染2种浓度ASODN的SW626和Caov-3细胞的随机运动能力分别为对照组的80.0%±1.3%、63.7%±1.9%、72.0%±1.3%和55.9%±2.5%;定向运动能力分别为对照组的83.9%±1.4%、64.1%±1.3%、72.5%±3.4%和54.5%±1.9%。转染后2株细胞的体外黏附能力和增殖能力,均未显示出明显的变化。结论ROCK-1蛋白的表达与人卵巢癌细胞的体外侵袭和迁移密切相关,阻断ROCK-1的表达,可有效地抑制卵巢癌肿瘤细胞的转移。

Effect of blocking of ROCK-1, an effector of small G protein Rho, on the malignant behavior of ovarian tumor cells in vitro

OBJECTIVE: To investigate the possible role of ROCK-1 in ovarian cancer invasion and metastasis. METHODS: ROCK-1 ASODN was transfected into SW626 and Caov-3 cell lines mediated by Lipofectamine 2000. The expressions of ROCK-1 mRNA and protein were detected by RT-PCR and Western-blot assay. Boyden chamber was used to assess the effect of ROCK-1 ASODN on the invasion and migration of the cell lines. The changes in the adhesion and proliferation of the transfected cells were detected by MTT assay. RESULTS: The expressions level of ROCK-1 mRNA and protein in the cell lines were decreased significantly after transfection at doses of 10 micromol/L and 20 micromol/L ROCK-1 ASODN. When compared with the control group, the invasion capability of transfected cells was inhibited to an extent of 75.6% +/- 3.8% and 54.7% +/- 2.9%, respectively, for SW626 cell line, and 68.8% +/- 4.7% and 50.0% +/- 4.5% for Caov-3 cell line, respectively. The random migratory activity of these two cell lines was inhibited by 80.0% +/- 1.3%, 63.7% +/- 1.9%, 72.5% +/- 3.4% and 55.9% +/- 2.5%, respectively, and the inhibition of chemotaxis activity of the two cell lines was 83.9% +/- 1.4%, 64.1% +/- 1.3%, 72.5% +/- 3.4% and 54.5% +/- 1.9%, respectively. No significant difference was found in the adhesion and proliferation of the cells transfected with ROCK-1 ASODN and control cells. CONCLUSION: The expression of ROCK-1 was closely related to the invasion capability and migratory activity of ovarian cancer cells. ROCK-1 may play a crucial role in invasion and metastasis of ovarian cancer.