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目的:探讨miR200c对卵巢癌细胞SKOV3和HO-8910转移潜能的影响及其调控机制。方法:人工合成miR200cmimic(实验组)和对照无关序列(阴性对照组),用脂质体将其转入SKOV3和HO-8910细胞,以SKOV3和HO-8910为空白对照,培养48h后,应用RT-PCR技术检测miR200c的相对表达;采用Transwell小室装置检测其对细胞侵袭和迁移能力的影响;蛋白质印迹法检测细胞中上皮向间质转化相关蛋白ZEB1、E-cadherin和Vimentin的表达;同时应用酶联免疫吸附实验检测间皮素和CA125的表达变化。结果:miR200c表达与空白对照组比较,SKOV3-mimic组为297.40±5.26倍,F=77.571,P<0.001;HO-8910-mimic组为348.57±6.50倍,F=63.563,P<0.001。侵袭到滤膜下的细胞数SKOV3-mimic组为74.20±7.44,低于空白对照组和阴性对照组,F=48.413,P<0.001;HO-8910-mimic组为98.67±7.62,低于空白对照组和阴性对照组,F=55.133,P<0.001。迁移到滤膜下的细胞数SKOV3-mimic组为56.27±6.36,低于空白对照组和阴性对照组,F=55.354,P<0.001;HO-8910-mimic组为91.33±5.75,亦低于空白对照组和阴性对照组,F=60.374,P<0.001。SKOV3-mimic组E-cadherin蛋白相对表达量为155.36±18.52,较对照组高,F=62.451,P=0.002,ZEB1和Vimentin蛋白表达降低,P值分别为0.003和0.005;HO-8910-mimic组E-cadherin蛋白相对表达量为149.90±38.53,较对照组高,F=54.536,P=0.002,ZEB1和Vimentin蛋白表达降低,P值分别为0.006和0.005。转染miR200cmimic后,SKOV3中CA125表达降低了33.2%,F=35.646,P=0.005,间皮素表达降低了39.6%,F=25.625,P=0.006;HO-8910中CA125表达降低了37.2%,F=27.426,P=0.007,间皮素表达降低了41.2%,F=33.735,P=0.005。结论:miR200c可通过miR200c/ZEB1/E-cadherin和间皮素/CA125通路抑制卵巢癌细胞侵袭和迁移能力。
Objective: To investigate the effect of miR200c on the metastatic potential of ovarian cancer cells SKOV3 and HO-8910 and its regulatory mechanism. METHODS: miR200cmimic (experimental group) and control unrelated sequence (negative control group) were synthesized and transfected into SKOV3 and HO-8910 cells by lipofectamine. SKOV3 and HO-8910 were used as blank control. After 48h incubation, RT- The expression of miR200c in epithelial cells was detected by RT-PCR and Transwell chamber assay. The expression of ZEB1, E-cadherin and Vimentin in epithelial cells was detected by Western blotting. At the same time, The changes of the expression of mesothelin and CA125 were detected by immunoadsorption assay. Results: Compared with the blank control group, the expression of miR200c in the SKOV3-mimic group was 297.40 ± 5.26 folds, F = 77.571, P <0.001; the HO-8910-mimic group was 348.57 ± 6.50 folds, F = 63.563, P <0.001. The cell number in the SKOV3-mimic group was 74.20 ± 7.44, which was lower than that in the blank control group and the negative control group (F = 48.413, P <0.001). The cell number in the HO-8910-mimic group was 98.67 ± 7.62, Group and negative control group, F = 55.133, P <0.001. The number of cells migrated to the membrane was 56.27 ± 6.36 in the SKOV3-mimic group, which was lower than that in the blank control group and the negative control group (F = 55.354, P <0.001) and 91.33 ± 5.75 in the HO-8910-mimic group Control group and negative control group, F = 60.374, P <0.001. The relative expression of E-cadherin protein in SKOV3-mimic group was 155.36 ± 18.52, higher than that in control group (F = 62.451, P = 0.002). The expression of ZEB1 and Vimentin protein were decreased The relative expression of E-cadherin protein was 149.90 ± 38.53, higher than that of the control group (F = 54.536, P = 0.002). The expression of ZEB1 and Vimentin protein were decreased. The P values were 0.006 and 0.005 respectively. The expression of CA125 decreased by 33.2%, F = 35.646, P = 0.005, decreased by 39.6%, F = 25.625, P = 0.006 in SKOV3 cells transfected with miR200cmimic; decreased by 37.2% in HO-8910 cells, F = 27.426, P = 0.007, decreased mesothelin expression by 41.2%, F = 33.735, P = 0.005. Conclusion: miR200c can inhibit the invasion and migration of ovarian cancer cells through the miR200c / ZEB1 / E-cadherin and mesothelin / CA125 pathways.