Abstract:

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.

Key words: Breast neoplasms, Oncogenes, Genes,tumor suppressor, Cell line,transformed