寡核苷酸阻断ROCK-1蛋白对人卵巢癌细胞恶性行为的影响

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

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。

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 LipofectamineTM 2000. The expressions of ROCK-I 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 MTI＇ assay. Results The expressions level of ROCK-1 mRNA and protein in the cell lines were decreased significantly after transfection at doses of 10 μmol/L and 20 μmol/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. Condusion 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.