S100A6基因作用对宫颈癌细胞增殖迁移能力的影响及其机制

Effects of S100A6 transfection and inhibition on proliferation and migration of human cervical cancer cells

目的观察S100A6基因转染、抑制对宫颈癌细胞增殖迁移能力的影响,并探讨其机制。方法将宫颈癌Hela细胞分为观察1组、对照1组和空白1组。观察1组转染pc DNA3.0-S100A6真核表达载体,对照1组转染pc DNA3.0空载体质粒,空白1组不转染。将宫颈癌Ca Ski细胞分为观察2组、对照2组及空白2组。观察2组转染S100A6 siRNA,对照2组转染无关序列,空白2组不转染。转染24 h后收集各组细胞。(1)采用实时荧光定量PCR法检测各组细胞S100A6 mRNA相对表达量;(2)采用细胞迁移实验观察各组细胞迁移能力(以穿膜细胞数表示);(3)采用Western blotting法检测各组细胞中上皮间质转化(EMT)相关蛋白(E-cadherin、N-cadherin)和PI3K-Akt信号通路相关蛋白(p-AKT、t-AKT、Snail、Twist)表达量;(4)继续培养24、48、72 h,采用CCK-8法观察各组细胞增殖能力(以OD450表示)。结果 (1)转染24 h观察1组、对照1组、空白1组Hela细胞S100A6 mRNA相对表达量分别为3.87±0.86、1.04±0.08、0.97±0.11,观察1组Hela细胞S100A6 mRNA相对表达量与对照1组和空白1组相比,P均<0.05;观察2组、对照2组、空白2组Ca Ski细胞S100A6 mRNA相对表达量分别为0.43±0.07、1.12±0.09、0.98±0.12,观察2组Ca Ski细胞S100A6 mRNA相对表达量与对照2组和空白2组相比,P均<0.05。(2)转染24 h行细胞迁移实验,观察1组、对照1组、空白1组穿膜细胞数分别为(48.26±4.89)、(21.31±4.27)、(20.12±6.23)个,观察1组穿膜细胞数与对照1组和空白1组相比,P均<0.05;观察2组、对照2组、空白2组穿膜细胞数分别为(81.36±8.33)、(147.49±6.98)、(142.23±9.16)个,观察2组穿膜细胞数与对照2组和空白2组相比,P均<0.05。(3)转染24 h,与对照1组和空白1组相比,观察1组Hela细胞E-cadherin蛋白表达量较低(P均<0.05),N-cadherin、p-AKT、Snail、Twist蛋白表达量较高(P均<0.05);与对照2组和空白2组相比,观察2组Ca Ski细胞E-cadherin蛋白表达量较高(P均<0.05),N-cadherin、p-AKT、Snail、Twist蛋白表达量较低(P均<0.05)。(4)转染24 h后继续培养24、48 h,三组细胞OD450相比,P均>0.05。继续培养72 h,观察1组Hela细胞OD450高于对照组和空白组(P均<0.05),观察2组Ca Ski细胞OD450低于对照2组和空白2组(P均<0.05)。结论 S100A6促进宫颈癌细胞增殖和迁移,这可能是通过激活PI3K-Akt信号通路进而促宫颈癌细胞EMT实现的。

Objective To observe the effects of S100 A6 transfection and inhibition on the proliferation and migration of human cervical cancer cells,and to explore the mechanism. Methods The cervical cancer Hela cells were divided into the observation group 1,the control group 1,and the blank group 1. The observation group 1 was transfected by the pc DNA3. 0-S100 A6 eukaryotic expressive vector,the control group 1 was transfected by the empty vector,and the blank group1 was not transfected. The cervical cancer Ca Ski cells were divided into the observation group 2,the control group 2,and the blank group 2. The observation group 2 was transfected by the S100 A6 siRNA,the control group 2 was transfected by the siRNA-negative controls and the blank group 2 was not transfected. At 24 h after transfection,the cells were collected.（1） The real-time PCR assay was used to detect the relative expression levels of S100 A6 mRNA in each group.（2） The cell migration assay was employed to detect the cell migration（ the number of migration cells）.（3） Western blotting was utilized to determine the levels of the epithelial-mesenchymal transition（ EMT）-related proteins（ E-cadherin and N-cadherin）,and the levels of the PI3 K-Akt signing pathway-related proteins（ p-Akt,t-Akt,Snail,and Twist）.（4） At 24,48,and 72 h after 24-hour transfection,CCK-8 was applied to analyze the cell proliferation ability（ OD450）. Results（1） At 24 h after the transfection,the relative expression levels of S100 A6 mRNA in Hela cells of the observation group 1,the control group1,and the blank group 1 were 3. 87 ± 0. 86,1. 04 ± 0. 08,and 0. 97 ± 0. 11,respectively. Compared with the control group 1 and the blank group 1,the relative expression level of S100 A6 mRNA in the observation group 1 was higher（ P〈0. 05）. The relative expression levels of S100 A6 mRNA in the Ca Ski cells of the observation group 2,the control group 2,and the blank group 2 were 0. 43 ± 0. 07,1. 12 ± 0. 09,and 0. 98 ± 0. 12,respectively. Compared with the control group 2 and the blank group 2,the relative expression level of S100 A6 mRNA in the observation group 2 was lower（ P〈0. 05）.（2）At 24 h after the transfection,the migration cells in the observation group 1,the control group 1,and the blank group 1 were 48. 26 ± 4. 89,21. 31 ± 4. 27,and 20. 12 ± 6. 23,respectively. Compared with the control group 1 and the blank group 1,the number of migration cells in the observation group 1 was higher（ P〈0. 05）. The migration cells in the observation group 1,the control group 1,and the blank group 1 were 81. 36 ± 8. 33,147. 49 ± 6. 98,and 142. 23 ± 9. 16,respectively. Compared with the control group 2 and the blank group 2,the number of migration cells in the observation group2 was lower（ P〈0. 05）.（3） At 24 h,compared with the control group 1 and the blank group 1,the protein expression of E-cadherin in the observation group 1 was lower（ P〈0. 05）,while the protein expression of N-cadherin,p-AKT,Snail,and Twist was higher（ all P〈0. 05）. Compared with the control group 2 and the blank group 2,the protein expression of E-cadherin in the observation group 2 was higher（ P〈0. 05）,while the protein expression of N-cadherin,p-AKT,Snail,and Twist was lower（ all P〈0. 05）.（4） At 24 and 48 h after 24-hour transfection,the OD450 in each group was not different（ all P〈0. 05）. At 72 h,the OD450 of Hela cells in the observation group 1 was higher than that in the control group 1 and the blank group 1（ P〈0. 05）; the OD450 of the Ca Ski cells in the observation group 2 was lower than that in the control group 2 and the blank group 2（ P〈0. 05）. Conclusion S100 A6 could promote the proliferation and migration of human cervical cancer cells,which may be achieved by activating the PI3 K-Akt pathway and enhancing the EMT of cervical cancer cells.