Silencing BMI-1 Gene By RNAi Affect Biological Characteristics And Doxorubicin Sensitivity Of MCF-7 Cell

BACKGROUND: Breast cancer is one of the most common malignancies affecting women worldwide. Data reveal that there are 500,000 breast cancer-related death worldwide with approximately 1,200,000 new cases diagnosed annually. Breast cancer is the first most common cancer in developed countries such as Western Europe and North America. In China, the incidence rate increases a lot. More and more young women suffer from this cancer. Despite of combining various therapeutic strategies, including surgery, chemotherapy and radiotherapy or other therapy, the prognosis for patients with breast cancer remains poor. Thus, more efforts are needed to develop novel approaches.Bmi-1 (B-cell-specific moloney murine leukemia virus insertion site 1) is a component of the Polycomb repressive complex 1, which was involved in regulation of cell proliferation. It is extremely important to maitain stem cell self-renewing and differentiation potential. Study reveal that Bmi-1 is over-expressed in several malignancies such as breast cancer, non-small cell lung cancer, colorectal cancer and nasopharyngeal carcinoma and this overexpression is correlated with poor clinical outcome. Down-regulation of Bmi-1 was effective in suppressing cancer cells growth and tumorigenicity. Therefore, Bmi-1 may participate in tumorigenesis. Bmi-1 gene may be used as a potential therapeutic target for the treatment of cancer. Amplification of the Bmi-1 gene is detected in approximately 85% of human invasive ductal breast cancer and this genomic alteration is correlated with axillary lymph node metastases and positive estrogen receptor status. Overexpression of Bmi-1 induces telomerase activity and immortalizes human mammary epithelial cells, suggesting a very close relationship between Bmi-1 and human breast cancer. Chemotherapy is an important therapeutic strategy for breast cancer, especially for reoccurring and metastasis cases. Resistance to chemotherapeutic agent raised by cancer cells seriously influenced curative effect in clinical. Many studies indicate that the mechanism which chemotherapeutic agents cure cancer is inducing apoptosis. Doxorubicin is a common chemotherapeutic agent which has emerged as one of the most widely used chemotherapeutic agent for breast cancer. The effectiveness level is only 34% when doxorubicin is used for curing breast cancer alone. Heart toxicity is an important restricted cause for the use of doxorubicin. So, increase in chemosensitivity by increasing apoptosis is an important factor for breast cancer treatment. Thus, more efforts are needed to develope a strategy which increases doxorubicin sensitivity to lower doses without compromising its efficacy. In this study, RNAi was introduced to silence Bmi-1 gene aiming to study the effect on biological characteristics and doxorubicin sensitivity of MCF-7 cell. Results will provide theoretical basis for researching new target and strategy for treatment of breast cancer.OBJECTIVE: RNAi was introduced to silence Bmi-1 gene aiming to study the effect on biochemical characteristics of MCF-7 and it's role on tumorigenesis. On this basis, the effect of Bmi-1 on doxorubicin sensitivity and mechanism were studied. The study was divided into three parts.1. The siliencing of Bmi-1 gene by retroviral vector pSuper-retro/Bmi-1si affect MCF-7 cell. METHODS: Recombinant retroviral Bmi-1 gene short hairpin RNA (shRNA) expression vector pSuper-retro/Bmi-1si was transiently transfected into breast carcinoma MCF-7 cell, pSuper-retro/GFPsi vector expressing Green Fluorescent Protein (GFP) shRNA is used as negative control. The mRNA and protein expression of Bmi-1 was examined after transfection for 48 hours (h) with reverse transcriptional polymerase chain reaction (RT-PCR) and western blot assay, respectively. The proliferation of MCF-7 cell was examined by MTT. The cell cycle status and apoptosis of MCF-7 cell were detected by flow cytometry (FCM). The expression of p53, cyclinD1 and p21 was examined by western blot assay. RESULTS: The mRNA and protein expression of Bmi-1 in Bmi-1si group which was transfected by pSuper-retro/Bmi-1si were significantly less. The inhibition rate at mRNA and protein level in Bmi-1si were 65.4% and 72.3% respectively, clearly indicating marked difference from that of blank control and GFPsi (p<0.05). MTT assay indicated strong inhibition in the growth rate of MCF-7 cells of Bmi-1si as compared to that of blank control and GFPsi. There were significant differences between Bmi-1si and controls (p<0.05). The effect of Bmi-1 silencing on cell cycle progression was analysed using FCM. 77.10% cells in Bmi-1si were noticed at G0/G1 phase, compared to 63.75% and 64.84% cells in blank control and GFPsi respectively. There were 32.95% and 29.90% cells noticed at S phase in blank control and GFPsi respectively, while 17.17% cells of Bmi-1si in S phase. There were significant differences between Bmi-1si and controls (p<0.05). Compaired with controls, Bmi-1 silencing induced an obvious increase in the number of cells at G0/G1 phase and reduction in S phase. Western blot results clearly showed a reduction in the expression of cyclin D1 and an increase of p53 and p21 in Bmi-1si compared with blank control and GFPsi. There were significant differences between Bmi-1si and controls (p<0.05). There were not significant differences between GFPsi and blank control (P>0.05). 48h after transfection, the basal levels of apoptosis in Bmi-1si and GFPsi were 4.94% and 2.84% respectively. Compared with untransfected control (3.18%) and negative control, no significant increase was found in the basal level of apoptosis in Bmi-1si. CONCLUTION: Retroviral vector pSuper-retro/Bmi-1si can significantly inhibit the expression of Bmi-1 gene. Further, silencing of Bmi-1 resulted in a drastic inhibition in the growth of MCF-7 cells as well as G1/S phase transition which mediated by up-regulation of p53, p21 and down-regulation of cyclinD1.2. A stable cell line with a persistent silencing of Bmi-1 gene was established. Biochemical characteristics and doxorubicin sensitivity of this cell line were studied. METHODS: Retroviruses were produced using the retroviral vector and PT67 packaging cell line. Retroviruses were used to infect MCF-7 cell, then puromycin-resistant cells were pooled. A stable cell line with a persistent silencing of Bmi-1 was established,which was named as MCF-7/Bmi-1si. The expression of Bmi-1 was appraised with RT-PCR and western blot assays, respectively. The colony-forming unit assay was used to measure the ability of cell proliferation, and Transwell chamber model was employed to test the ability of cell invasion and metastasis in vitro. MTT assay was performed to evaluate the 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by DAPI staining and the expression of related genes (p53, phospho-Akt (ser473) (pAkt), totle-Akt (tAkt), Bcl-2 and Bax) were studied by western blot. RESULTS: Compared with MCF-7 and MCF-7/GFPsi, the mRNA and protein expression of Bmi-1 in MCF-7/Bmi-1s were significantly less (P<0.05). The inhibition rate is 72.1% and 76.7% respectively, clearly indicating marked difference from that of MCF-7 and MCF-7/GFPsi (p<0.05). Compared with MCF-7 and MCF-7/GFPsi, the number and rate of colony formation in MCF-7/Bmi-1si were largely decreased (p<0.05). The results of invasion and metastasis assays indicated: the cells in MCF-7/Bmi-1s moved from the upper chamber into the lower one for 24h were less than those in the control groups MCF-7 and MCF-7/GFPsi (p<0.05). After treatment with doxorubicin for 72h, we found that cells in MCF-7/Bmi-1s showed higher IR than that in MCF-7 and MCF-7/GFPsi. The IC50 value of doxorubicin in MCF-7 and MCF-7/GFPsi were 0.86±0.02μg/ml and 0.84±0.02μg/ml. They were significantly higher, while the IC50 was 0.12±0.07μg/ml in MCF-7/Bmi-1si. There were significant differences between the IC50 of MCF-7/Bmi-1si and that of controls. Apoptosis was examined after treating each group cells with 1μg/ml doxorubicin for 48h. The Apoptosis Index (AI) in MCF-7/Bmi-1si cells treated with doxorubicin was great, and apoptosis cells were frequently observed which appeared typical apoptotic morphological changes, such as condensed chromatin, shrunken nuclei and loss of cell volume. In contrast, only less apoptotic cells with the same apoptotic morphological changes were observed in MCF-7 and MCF-7/GFPsi cells. The AI of these two groups were low. There were significant differences between the apoptosis of MCF-7/Bmi-1si and that of controls. The expression levels of p53, pAkt, tAkt, Bcl-2 and Bax were examined after cells were treated with doxorubicin for 48h. Compared with MCF-7 and MCF-7/GFPsi, there were increased expression of p53 and decreased Akt phosphorylation without affecting tAkt expression. There were decreased expression of Bcl-2 and increasion of Bax, and Bcl-2/Bax ratio was down-regulated (p<0.05). CONCLUTION: The stable cell line with a persistent silencing of Bmi-1 gene was successfully established. Silencing of Bmi-1 gene can inhibit the ability of proliferation, invasion and metastasis in MCF-7 cells in vitro. The silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin by inducing a significantly higher percentage of apoptosis cells.3. Silencing of Bmi-1 gene affect the oncogenicity of MCF-7 cell and sensitivity of tumor tissues against doxorubicin. METHODS: stable cell lines which were established at the second part were injected subcutaneously into the right flank of the male BALB/c nude mice. It was judged as tumorigenesis, when the diameter of tumor was 0.5cm approximately. Tumor growth was monitored in all the groups. Two orthogonal diameters of the tumors were measured with vernier calipers at a certain time (7d, 14d, 21d, 28d). On the 28d after the injection, all mice were sacrificed and the tumor tissues were removed, weighed. The inhibition ratio of tumor growth was calculated. The pathological change and expression of Bmi-1 in tumor tissue were observed. In order to study the effect of Bmi-1 silencing on doxorubicin sensitivity at animal level, cells (MCF-7, MCF-7/Bmi-1si and MCF-7/GFPsi) were injected subcutaneously into the right flank of the male nude mice respectively, with 12 animals in each group. On the 15d after the postinoculation, the mice of each group were divided into two groups randomly. The mice in one group were used as control which received normal saline intraperitoneally, where as mice in the other group received doxorubicin. Tumor growth and volume were monitored. On the 21d after the injection, all mice were sacrificed and the tumor tissues were removed, weighed. The inhibition ratio of tumor growth was calculated. The apoptosis of tissue cells was detected by TUNEL assay. RESULTS: Compared with MCF-7 and MCF-7/GFPsi, the time of tumorigenesis in MCF-7/Bmi-1si was longer, the growth rate and volume of tumor was significantly lower (p<0.05). The cells in MCF-7 and MCF-7/GFPsi xenograft tumor tissue appeared large cell volume, abundant cytoplasm and nucleus deeply stained. In MCF-7/Bmi-1si group, structure was loosely and disorderly arranged. The expression of Bmi-1 in parent MCF-7 and MCF-7/GFPsi negative control solid tumors was strong, whereas it was weak in MCF-7/Bmi-1si solid tumors. The growth speed of MCF-7/Bmi-1si tumor after doxorubicin treatment was notably inhibited compared to other two control groups (p<0.05). Compared with MCF-7 and MCF-7/GFPsi, the T/C ratio of MCF-7/Bmi-1si was significantly low. Compared the weigh of MCF-7/Bmi-1si+doxorubicin tumor with that of MCF-7/Bmi-1si+normal saline, the inhibition rate is 63.3%. The inhibition rate of MCF-7+doxorubicin and MCF-7/GFPsi+doxorubicin were 35.2% and 37.8% respectively. There were significant differences between MCF-7/Bmi-1si+doxorubicin and controls (p<0.05). AI of MCF-7/Bmi-1si solid tumor treated with doxorubicin was significantly higher than control groups which indicated that MCF-7/Bmi-1si tumor treated with doxorubicin exhibited significantly increased apoptosis than MCF-7 tumor treated with doxorubicin and MCF-7/GFPsi tumor treated with doxorubicin (p<0.05). CONCLUTION: Silencing of Bmi-1 gene could degrade the oncogenicity of MCF-7 cell and render MCF-7/Bmi-1si tumor more sensitive to doxorubicin by increasion of apoptosis.