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目的探讨反义Snail转录因子抑制卵巢癌转移的机理。方法采用RTPCR技术分别检测卵巢透明细胞癌细胞株ES2、卵巢腺癌细胞株OVCAR3、卵巢浆液性囊腺癌细胞株HO8910和高转移卵巢浆液性囊腺癌细胞株HO8910PM细胞中Snail、E钙黏蛋白mRNA的表达水平。将HO8910细胞分为两组,分别转染反义Snail质粒(反义组)和空载体质粒(对照组),观察Snail对E钙黏蛋白mRNA表达的逆转作用,及对卵巢癌细胞体外侵袭、运动的抑制作用。结果PCR产物凝胶电泳结果显示,SnailmRNA在ES2、HO8910、HO8910PM细胞中有表达,而E钙黏蛋白mRNA仅在OVCAR3细胞中有表达。HO8910细胞瞬时转染0、24、48h时,SnailmRNA的表达水平分别为0.897±0.005、0.865±0.010、0.338±0.014,0与24h时相比,差异有统计学意义(P<0.05);24h与48h时相比,差异有统计学意义(P<0.01);同时,E钙黏蛋白mRNA开始有表达,并随转染时间的增加而增加,0、24、48h时,E钙黏蛋白mRNA表达水平分别为0、0.130±0.001、0.217±0.005,各时间点间分别比较,差异均有统计学意义(P<0.05)。HO8910细胞稳定转染后,反义组、对照组SnailmRNA表达水平分别为0.126±0.010、0.355±0.020,两组比较,差异有统计学意义(P<0.05);反义组E钙黏蛋白mRNA开始有表达,表达水平为0.467±0.040,而对照组为0,两组比较,差异有统计学意义(P<0.01);同时,检测间质细胞标志物纤维连接蛋白mRNA的表达水平,反义组为0.191±0.010,显著低于对照组的0.582±0.010(P<0.01)。体外细胞运动实验表明,反义组、对照组跨膜细胞数[分别为(48±2)、(396±29)个/高倍镜(×200)]比较,差异有统计学意义(P<0.01)。重组细胞基底膜(Matrigel)侵袭实验显示,两组穿透基底膜细胞数[分别为(35±10)、(219±72)个/高倍镜(×200)]比较,差异有统计学意义(P<0.05)。结论卵巢癌细胞中存在SnailmRNA与E钙黏蛋白mRNA表达的逆转关系,抑制SnailmRNA的表达对卵巢癌细胞的侵袭和运动有抑制作用,可能为卵巢癌转移的基因治疗提供新的靶点。
Objective To investigate the mechanism of antisense Snail transcription factor inhibiting ovarian cancer metastasis. Methods RTPCR was used to detect the expression of Snail, E-cadherin in ovarian clear cell carcinoma cell line ES2, ovarian adenocarcinoma cell line OVCAR3, ovarian serous cystadenocarcinoma cell line HO8910 and highly metastatic ovarian serous cystadenocarcinoma cell line HO8910PM respectively mRNA expression level. The HO8910 cells were divided into two groups and transfected with antisense Snail plasmid (antisense group) and empty vector plasmid (control group) respectively. The reversal effect of Snail on E-cadherin mRNA expression and invasion in vitro of ovarian cancer cells were observed, Inhibition of exercise. Results The PCR products of gel electrophoresis showed that Snail mRNA was expressed in ES2, HO8910 and HO8910PM cells, while E-cadherin mRNA was only expressed in OVCAR3 cells. The expression levels of Snail mRNA in HO8910 cells at 0, 24 and 48h were 0.897 ± 0.005, 0.865 ± 0.010 and 0.338 ± 0.014, respectively. Compared with 24h, the difference was statistically significant (P <0.05) (P <0.01). At the same time, E-cadherin mRNA began to be expressed and increased with the increase of transfection time. At 0, 24 and 48 hours, E-cadherin mRNA expression The levels were 0, 0.130 ± 0.001 and 0.217 ± 0.005, respectively, with statistical significance (P <0.05). After stable transfection of HO8910 cells, the expressions of Snail mRNA in antisense group and control group were 0.126 ± 0.010,0.355 ± 0.020, respectively. The difference was statistically significant (P <0.05). The mRNA expression of E-cadherin The expression level was 0.467 ± 0.040, while the control group was 0, the difference between the two groups was statistically significant (P <0.01); at the same time, the level of interstitial cell marker fibronectin mRNA expression, antisense group 0.191 ± 0.010, significantly lower than the control group 0.582 ± 0.010 (P <0.01). In vitro cell motility experiments showed that the number of transmembrane cells in the antisense and control groups were (48 ± 2) and (396 ± 29) / high magnification lenses (× 200), respectively, with significant difference (P <0.01 ). Matrigel invasion assay showed that there were significant differences in the numbers of cells penetrating the basal lamina [(35 ± 10) and (219 ± 72) / high magnification lenses (× 200), respectively] P <0.05). Conclusion There is a reverse relationship between Snail mRNA and E-cadherin mRNA expression in ovarian cancer cells. Inhibition of Snail mRNA expression may inhibit the invasion and motility of ovarian cancer cells, which may provide new targets for the gene therapy of ovarian cancer metastasis.