高表达twist基因促进三阴性乳腺癌SUM159细胞干性特征的初步研究

Preliminary study on high expression of twist gene promoting the stemness related characteristics of triple negative breast cancer SUM159 cells

目的探讨高表达twist基因对乳腺癌SUM159细胞干性特征的影响。方法用慢病毒构建过表达twist基因的乳腺癌SUM159细胞作为实验组(SUM159/twist),空载体构建对照组细胞(SUM159/vector)。利用RT-PCR检测twist转染前后mRNA水平变化;通过平板克隆形成、成球实验、流式细胞仪检测肿瘤干细胞比例,并用RT-PCR检测干性相关基因SOX2、OCT4、BMI1的mRNA表达水平。mRNA及细胞克隆数等计量资料以x珋±s表示,组间比较采用t检验。结果成功构建过表达twist基因的乳腺癌SUM159细胞,SUM159/twist细胞中的twist mRNA表达为2.04±0.15,高于SUM159/vector细胞的0.49±0.45(t=-16.33,P<0.001);SUM159/twist细胞的克隆菌落数目(92.75±4.85)明显高于SUM159/vector细胞(44.50±5.19)(t=13.56,P<0.001);成球实验显示:SUM159/twist细胞形成的细胞球数目(46.75±4.11)显著高于SUM159/vector细胞(22.50±3.41)(t=9.07,P<0.001);利用流式细胞仪检测SUM159/twist和SUM159/vector细胞中的ALDH1+细胞比例发现:SUM159/twist细胞中ALDH1+细胞比例为(9.63±0.89)%,显著高于SUM159/vector细胞中的(2.31±0.21)%(t=13.85,P<0.001);SUM159/twist细胞中SOX2、OCT4、BMI1的表达(12.39±0.63、13.35±1.56、6.48±0.96)均高于SUM159/vector细胞(1.61±0.33、2.67±0.28、2.70±0.35)(t=-29.68,-11.61,6.43;P均<0.001)。结论 twist基因能够增强乳腺癌SUM159细胞干性能力。

Objective To investigate the impact of high expression of twist gene on the stemness related characteristics of breast cancer SUM159 cells. Methods We established a twist-overexpression breast cancer SUM159 cell line as experimental group（ SUM159 / twist）,and the cells with blank vector construction served as control group（ SUM159 / vector）. The twist mRNA level were detected before and after twist gene transfection using RT-PCR. The proportion of cancer stem cells was investigated by flat clone formation,mammosphere assay and flow cytometry. The mRNA expressions of stemness-related genes SOX2,OCT4 and BMI1 were detected by RT-PCR. All data were expressed as x珋± s and processed by t test. Results SUM159 cells with twist gene overexpression were successfully established. The relative expression of twist mRNA in SUM159 / twist cells was 2. 04 ± 0. 15,significantly higher than 0. 49 ± 0. 45 in SUM159 / vector cells（ t =- 16. 33,P〈0. 001）. The colony number of clone formation was 92. 75 ± 4. 85 in SUM159 / twist cells,significantly higher than 44. 50 ± 5. 19 in SUM159 / vector cells（ t = 13. 56,P〈0. 001）. The mammosphere number was 46. 75 ± 4. 11 in SUM159 / twist cells and 22. 50 ± 3. 41 in SUM159 / vector cells,and the difference was statistically significant（ t = 9. 07,P〈0. 001）. Flow cytometry results showed that the proportion of ALDH1＋was（ 9. 63 ± 0. 89） % in SUM159 / twist cells and（ 2. 31 ± 0. 21） % in SUM159 / vector cells,indicating a significant difference（ t = 13. 85,P〈0. 001）. The relative mRNA expressions of SOX2,OCT4 and BMI1 in SUM159 / twist cells were 12. 39 ± 0. 63,13. 35 ± 1. 56 and 6. 48 ± 0. 96,significantly higher than 1. 61 ±0. 33,2. 67 ± 0. 28 and 2. 70 ± 0. 35 in SUM159 / vector cells respectively（ t =- 29. 68,- 11. 61,6. 43,all P〈0. 001）.Conclusion The twist gene promotes the stemness related capability of breast cancer SUM159 cells.