| ObjectiveTo investigate the effects of STAT5 gene silencing and overexpression on human breast cancer cells SKBR3 and MCF-7 paclitaxel resistance,we established paclitaxel-resistant strains SKBR3/PR and MCF-7/PR by the shock-induced method.Then we explored the role of STAT5 in breast cancer and the molecular mechanism of cell cycle related factors cyclin B1,CDK2 and c-myc in cell resistance,and provided new ideas for clinical treatment of breast cancer.MethodsThe paclitaxel-resistant strains of SKBR3/PR and MCF-7/PR were constructed,and then detected the expression of STAT5.SKBR3/PR and MCF-7/PR cells were transfected with STAT5 specific sh RNA,analyzed the sensitivity of SKBR3/PR-sh RNA and MCF-7/PR-sh RNA to paclitaxel.SKBR3 and MCF-7 cells were transfected with by STAT5 overexpression liposome vector and sh RNA,detected the effects of paclitaxel chemotherapy on survival and apoptosis rate of SKBR3-STAT,SKBR3-STAT-sh RNA,MCF-7-STAT and MCF-7-STAT-sh RNA cells by the method of MTS and flow cytometry,analyzed the level of cell cycle regulatory proteins cyclin B1,CDK2 and c-myc by q PCR and Western blot.Results1.With the increase of paclitaxel concentration(> 5n M)in SKBR3 and MCF-7 cells,the cell survival rate decreased in a dose-dependent manner(t=2.476 to 5.653,P<0.05;t=2.309 to 8.591,P<0.05).The cells in the SKBR3/PR group grew stably at a paclitaxel concentration of 80 n M,and the MCF-7/PR group grew stably in 640 n M paclitaxel.The optimal doses of paclitaxel for SKBR3 and MCF-7 were 80 n M and 640 n M respectively.2.The expression of STAT5 in SKBR3/PR and MCF-7/PR groups was higher than that of SKBR3 and MCF-7 groups(t=7.027,P=0.000;t=6.242,P=0.000).SKBR3/PR-sh RNA and MCF-7/PR-sh RNA cells were treated with80 n M and 640 n M paclitaxel for 24 h,48h and 72 h respectively.MTS results showed that the SKBR3/PR-sh RNA and MCF-7/PR-sh RNA groups were more sensitive to chemotherapy than the drug-resistant group at all time points(t=2.638～7.905,P <0.05;t=2.360～6.207,P<0.05)3.Paclitaxel(80n M/640 n M)acted on cells in SKBR3-STAT5 and MCF-7-STAT5 groups for 7 days,and the results showed that the sensitivities of paclitaxel were significantly lower in SKBR3-STAT5 and MCF-7-STAT5 groups than in SKBR3 and MCF-7 groups(t=2.532～4.109,P<0.05;t=2.297～3.254,P<0.05).When STAT5 was silenced,the chemotherapeutic sensitivities of SKBR3-STAT5-sh RNA and MCF-7-STAT5-sh RNA groups were higher than that of SKBR3 and MCF-7 groups(t=2.275～4.967,P <0.05;t=2.413～5.779,P<0.05).FCM results showed that the apoptosis rates of SKBR3-STAT5 and MCF-7-STAT5 groups were lower than that of SKBR3 and MCF-7 groups(t=3.248,P=0.004,t=4.517,P=0.001).The apoptosis rates of SKBR3-STAT5-sh RNA and MCF-7-STAT5-sh RNA groups were significantly higher than that of SKBR3 and MCF-7 groups(t=2.395,P=0.027,t=2.505,P=0.019).4.The levels of cyclin B1,CDK2 and c-myc protein and m RNA in SKBR3-STAT5 and MCF-7-STAT5 groups were up-regulated compared with SKBR3 and MCF-7-STAT5 groups(P<0.01).After silencing STAT5,compared with the SKBR3 and MCF-7 groups,the expressions of cyclin B1,CDK2,c-myc protein and m RNA were decreased in the SKBR3-STAT5-sh RNA and MCF-7-STAT5-sh RNA groups(P<0.01).Conclusions1.The expression of STAT5 was negatively correlnated with the sensitivity of paclitaxel chemotherapy in breast cancer.2.STAT5 gene regulated gene level and protein expression of cell cycle related factors of cyclin B1,CDK2 and c-myc,and then mediated paclitaxel resistance in breast cancer. |
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