| Objective:To observe the Effects of STAT3 gene silencing on Adriamycin resistance of human breast cancer cell line MCF-7/ADR, and explore its related mechanism.Methods:1. The Adriamycin IC50 of MCF-7/ADR and its parental cell line MCF-7 was detected by MTT assay. The expression of STAT3 and p-STAT3 protein in these two cell lines and MCF-7 cells that haved been treated with 3μg/mL Adriamycin for 12 hours and 36 hours were detected by Western blot technology.2. MCF-7/ADR cells were divided into 3 groups:the interference group, the negative control group and the blank group. For the interference group, MCF-7/ADR cells were infected with lentiviral particles targeting STAT3 gene (LV-shRNA-STAT3), while the negative control group with the negative control lentiviral particles targeting the no sense sequence (LV-shRNA-NC), the blank group with no treatment. After 72 hours, cells were screened for 5 days using 1 μg/mL puromycin, and the infection efficiency of lentiviral was observed with fluorescence microscope. The expression of STAT3 mRNA and protein and p-STAT3 protein in each group were detected by real-time PCR and Western blot.3. The chemosensitivity of every group cells to Adriamycin was detected by MTT assay. Apoptosis rate and cell cycle of every group cells that have been treated to Adriamycin for 48 hours were detected by flow cytometry assay, and cell apoptosis index was further detected by tunnel staining assay.4. The expressions level of apoptosis related proteins of bcl-2, survivin and c-myc were further detected by Western blotting technology.Results:1. The Adriamycin IC50 of MCF-7/ADR and MCF-7 was respectively (59.37+5.01) μg/mL and (5.50±0.82)μg/mL while cells was cultured in usual condition (P<0.05). The expression of STAT3 and p-STAT3 protein in MCF-7/ADR was significantly higher than that in MCF-7, and their expression was time dependently increased in MCF-7 cells after a certain concentration of adriamycin treatment for different time, they had significantly statistical difference(P<0.05).2. The infection efficiency of the interference group and negative control group cells was more than 90%. Compaired with the blank control group and negative group, the expression of STAT3 mRNA and protein and p-STAT3 protein in the interference group was significantly lower (P<0.05).3. MTT assay results showed that, the Adriamycin IC50 of the interference group, blank group and negative control group was respectively (7.64±0.20) , (56.12±3.01) μg/mL and (54.86±11.91) μg/mL (P<0.05). FCM results showed that the apoptosis rate of the interference group, blank group and negative control group was respectively (34.03±3.14)%, (10.53±0.67)% and (11.70±0.72)%. At the same time, the percentage of S phase in the interference group increased significantly while the G2/M phase and G1 phase percentage decreased (P<0.05). The Tunnel staining results further confirmed that the apoptosis index of the interference group was significantly increased (P<0.05).4. Western blot results showed that the expression of bcl-2, survivin and c-myc protein in the interference group were significantly lower than that in the blank control group and negative control group (P<0.05).Conclusions:1. The recombinant STAT3 interference lentiviral (LV-shRNA-STAT3) can specifically silence STAT3 gene in MCF-7/ADR, and effectively enhance the sensitivity of breast cancer cells to Adriamycin, promote apoptosis of resistant cells under the Adriamycin treatment, cause the cell cycle arresting in S phase, finally reverse drug resistance of breast cancer to Adriamycin effectively.2. The mechanism of STAT3 gene silencing increase the sensitivity of breast cancer cells to Adriamycin may be related to the down-regulation of bcl-2, survivin and c-myc proteins expressions that cause breast cancer resistant to chemotherapy. |
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