| Background/Objective Breast cancer is one main threat to women ’s health. At present,the main treatment measure is surgical resection combined with postoperative chemotherapy and radiotherapy. However, considering the severity of side effects,traditional Chinese medicine and gene targeted therapy gradually become hot points in the treatment of breast cancer. The aim of this study was to investigate the effect of Zinc-finger protein 1(ZIC1)gene associated with matrine on proliferation, adhesion, migration and apoptosis in highly metastatic MDA-MB-231 human breast cancer cell, and then to explore a new idea for the treatment of breast cancer.Methods 1. ZIC1 gene was firstly transfected into human breast cancer MDA-MB-231 cell line stably by lentiviral vector p LV-Zic1-PGK-puro. After 48 hours of transfection, the expression of green fluorescent protein(GFP) was observed under the fluorescence microscope, and the cells were cultured as well as screened, so we get a stably transfected cell strain.2. Taking GAPDH as a loading control, the expression of ZIC1 in MDA-MB-231 cells was detected by Western blot.3. Empty vector transfected human breast cancer MDA-MB-231 cells and stable ZIC1 gene transfected cells were taken as the object of cytotoxicity test.Selected concentration gradient of 50,100,200,400,800mg/L of matrine applicated respectively empty vector group and transfection group for 48 h, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) was used to detect cell proliferation inhibition rate of different concentrations of matrine on cells and I got out half maximal inhibitory concentration(IC50).4. The IC50 of matrine(48h)was taken as experimental standard concentration in follow tests.The cells were artificially divided into four groups, namely(A)no-load/no-dosing group(negative control group),(B) no-load/dosing groups,(C)no-dosing/transfection group and(D) dosing / transfection group. Then we treat with cells as follows:(1) cell viability was detected by cell adhesion test, and the adhesion rate of cell was calculated with MTT method;(2) cell migration was analyzed by wound-healing assay, and the migration ability of cells in different groups was observed at 24 h,48h,72h;(3)the apoptosis rates were measured by flow cytometry(FCM), the apoptosis rate and distribution of the cells in each group were analyzed.Results 1. The expression of ZIC1 in stable-transfection human breast cancer MDA-MB-231 cells: a large number of green fluorescent protein was detected by 48 h fluorescence microscope, and the transfection rate was 90%.2. Western blot showed that ZIC1 protein was expressed in stably transfected cells, and no expression was found in non transfected cells.3. With the effect of matrine, drug cytotoxicity test indicated that the proliferation inhibition rate gradually increased with drug concentrations increasing,and there was a significant difference(P<0.05). Among them, the inhibitory rate of cell proliferation was respectively 49.21% and 52.42% after the no-load and transfected cells were treated with matrine at concentration of 200mg/L for 48 h, close to 50%(IC50). So we select 200mg/L as concentration for subsequent experiment.4. In the cell adhesion assay, after cells were treated with 200mg/L matrine for48 h, adhesion rate of no-dosing/transfection group is about 64.13% and no-load/dosing49.17%, while adhesion rate of dosing/transfection group is about 31.23%, significantly lower than the other groups(P<0.05). In cell migration experiment, migration distance ofeach group cells increased gradually with the extension of time,and in the same period,migration distance of dosing/transfection group cells is the shortest and the relative migration rate was the lowest(P < 0.05). In cell apoptosis of 48 h, the highest apoptosis rate was 34.78% + 1.82, for dosing/transfection cells, secondly for no-load/dosing group(26.3% + 1.28) and no-dosing/transfection group(24.24% + 1.14), the lowest for the negative control group(7.99% + 1.07).Conclusion1. When transfected stably with the slow virus vector p Lv-zic1-pgk-puro,the expression of zic1 gene in human mammary malignant tumor MDA-MB-231 cells was significantly enhanced.2. After treating with matrine of 200mg/L for 48 h, the suppression rate of human mammary malignant tumor MDA-MB-231 in the ZIC1 transfected vector and empty vector test groups was about 50%.3. Up regulation of ZIC1 gene expression or matrine alone can significantly inhibit the proliferation, adhesion and migration of human breast cancer MDA-MB-231 cells, and induce the apoptosis of human breast cancer cells.4. The anti-tumor effect of ZIC1 gene combined with matrine is more obvious, that is they can play an apparent role in supressing cell growth, migration and promoting apoptosis of MDA-MB-231.Therefore, it can be considered that the traditional Chinese medicine matrine combined with ZIC1 gene targeted therapy may play a synergistic inhibitory effect on breast cancer,and it is speculated that they will produce a more ideal anti-tumor therapeutic effect. |
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