过表达转移抑制基因KAI1对乳腺癌MDA-MB-231细胞上皮间质转化的影响

Effect of non-metastatic gene KAI1 overexpression on epithelial-mesenchymal transition in breast cancer cell line MDA-MB-231

目的探讨过表达转移抑制基因KAI1对人乳腺癌MDA-MB-231细胞上皮间质转化的影响。方法利用慢病毒感染乳腺癌MDA-MB-231细胞,获得稳定过表达KAI1的细胞系(过表达KAI1组),并设置空白慢病毒感染的阴性对照细胞系(阴性对照组)及未处理的人乳腺癌MDA-MB-231细胞为空白对照组。用Western blot法检测KAI1蛋白过表达的情况;采用RT-PCR法检测过表达KAI1对MDA-MB-231细胞间质标志物(波形蛋白、N-钙黏蛋白和纤粘蛋白)的mRNA表达水平的影响;并用Western blot检测过表达KAI1对MDA-MB-231细胞间质标志物(波形蛋白、N-钙黏蛋白)的蛋白表达水平的影响;采用明胶酶谱检测过表达KAI1对MDA-MB-231细胞基质金属蛋白酶2(MMP2)和MMP9活性的影响。多样本均数若满足方差齐性采用单因素方差分析,其两两比较采用LSD法进行,若不满足方差齐性则采用Dunnett's T3。结果慢病毒感染MDA-MB-231细胞后,各组间AKI1蛋白表达有差异有统计学意义(F=25.610,P=0.001),并且与阴性对照组(0.575±0.065)和空白对照组(0.458±0.0500)相比,过表达KAI1组(0.953±0.034)的KAI1蛋白表达水平明显提高(P均<0.050)。RT-PCR检测表明,细胞间质标志物波形蛋白(F=10.268,P=0.012)、N-钙黏蛋白(F=32.159,P=0.001)和纤粘蛋白的mRNA(F=38.364,P=0.000)在各组间表达有差异。与阴性对照组(0.937±0.102,0.998±0.064,1.093±0.083)和空白对照组(1.000±0.000,1.000±0.000,1.000±0.000)相比,过表达KAI1组(0.636±0.027,0.576±0.038,0.435±0.051)的波形蛋白、N-钙黏蛋白和纤粘蛋白mRNA表达水平明显降低(P均<0.050),且Western blot检测表明,各组间N-钙黏蛋白(F=9.172,P=0.015)和波形蛋白(F=14.441,P=0.005)表达有差异,与阴性对照组(1.068±0.032)和空白对照组(0.957±0.103)相比,过表达KAI1组(0.597±0.032)的波形蛋白表达水平明显降低(P均<0.050);与阴性对照组(1.452±0.036)和空白对照组(1.403±0.073)相比,过表达KAI1组(1.100±0.073)的N-钙黏蛋白表达水平相似(P=0.080,0.067)。酶活性分析发现,各组间MMP2(F=18.928,P=0.003)和MMP9(F=21.310,P=0.002)表达差异有统计学意义,与阴性对照组(1.090±0.160,1.091±0.107)和空白对照组(1.000±0.000,1.000±0.000)相比,过表达KAI1组(0.326±0.047,0.460±0.071)的MMP2和MMP9表达水平明显降低(P均<0.050)。结论过表达转移抑制基因KAI1能抑制人乳腺癌MDA-MB-231细胞的间质表型。

Objective To investigate the impact of overexpression of non-metastatic gene KAI1 on the epithelial-mesenchymal transition in human breast cancer cell line MDA-MB-231. Methods Lentivirus vector was transfected into MDA-MB-231 cells to obtain the KAI1-overexpressed cell line （ KAI1 overexpression group） . The cell line with blank lentivirus vector infection served as negative control group and untreated cell line as control group. Western blot analysis was used to verify KAI1 protein expression. RT-PCR was used to detect the mRNA expression levels of vimentin, N-cadherin and fibronectin in KAI1-overexpressed MDA-MB-231 cells. Western blot was used to detect the protein expression levels of vimentin and N-cadherin. The gelatin zymography was used to detect MMP2 and MMP9 activities. If the means of multi-sample met the homogeneity of variance, ANOVA was used, otherwise Dunnett’s T3 test was used. Pairwise comparison was performed using LSD method. Results After lentivirus infection of MDA-MB-231 cells, KAI1 protein expression showed a significant difference among groups （F=25. 610, P=0. 001）. Compared with the negative control group （0. 575 ± 0. 065） and control group （0. 458 ± 0. 0500）, KAI1 protein level （0. 953±0. 034）was significantly higher in KAI1 overexpression group （ all P values 〈0. 050 ） . RT-PCR showed that there were significant differences in the mRNA levels of stromal cells markers vimentin （F=10. 268, P=0. 012）, N-cadherin （F=32. 159, P=0. 001） and fibronectin （F=38. 364, P=0. 000） among groups. The mRNA levels of vimentin, N-cadherin and fibronectin in KAI1 overexpression group （ 0. 636 ± 0. 027, 0. 576 ± 0. 038, 0. 435 ± 0. 051 ） were significantly lower than those in negative control group （0. 937 ± 0. 102,0. 998 ± 0. 064,1. 093 ± 0. 083） and control group （1. 000 ± 0. 000,1. 000 ± 0. 000,1. 000 ± 0. 000） respectively （all P values〈0. 050）. Western blot analysis showed a significant difference in protein levels of N-cadherin （ F=9. 172, P=0. 015 ） and vimentin （F=14. 441, P=0. 005） among groups. Compared with the negative control group （1. 068 ± 0. 032） and control group （ 0. 957± 0. 103 ） , vimentin protein expression in KAI1 overexpression group （ 0. 597 ± 0. 032） was significantly lower （all P values 〈0. 050）, while N-cadherin expression showed no significant difference （1. 452 ± 0. 036 vs 1. 403 ± 0. 073 vs 1. 100 ± 0. 073, P=0. 080, 0. 067）. The gelatin zymography showed a significant difference in the expressions of MMP2 （F=18. 928, P=0. 003） and MMP9 （F=21. 310, P=0. 002） among the groups. Compared with the negative control group （1. 090 ± 0. 160, 1. 091 ± 0. 107） and control group （ 1. 000 ± 0. 000, 1. 000 ± 0. 000 ）, MMP2 and MMP9 expression levels in KAI1 overexpression group （0. 326 ± 0. 047,0. 460 ± 0. 071） were significantly lower （all P values 〈0. 050）. Conclusion The overexpression of KAI1 can inhibit the mesenchymal phenotype in human breast cancer cell line MDA-MB-231.