| Breast cancer is the most common malignancy in women. Worldwide, it is estimated that more than one million women are diagnosed with breast cancer per year, and more than 410,000 will die from the disease. In recent years, the incidence of breast cancer is increasing in China. And the occurrence of invasion and metastasis accounts for poor prognosis and high mortality. Although the treatment of breast cancer has been a great progress, the survival rate of breast cancer was not significantly increased increase. Therefore, it is current research focus to understand Breast Cancer pathogenesis in the molecular biology search of the targets of malignant transformation of breast cancer and target therapy of breast cancer.BCSG1, breast cancer-specific gene 1, is a specific selective expression in breast cancer gene, localized to human chromosome 10q23, and the DNA length is about 5 kb. BCSG1 can also be called spersyn, the same gene family members SNCG with SNCs. BCSG1 gene can encode and syntheticen a protein sequence with 127 amino acids, which is closely related to breast and ovarian cancer associated with sex hormones. BCSG1 is a tumor marker related to progression of breast cancer and the expression of CBCSG1 has a especial period and it was over expression may indicate breast cancer malignant process.S100A4, Ets-1, VEGT and MVD were associated with tumor angiogenesis, invasion and metastasis.S-100A4, one of the S-100 calcium binding protein super families, is a potential molecular marker, which has a great significance on prognosis of tumor. S-100A4 mainly distributes in fibroblasts, myoepithelial cells, cytoplasm of tumor cells and extracellular fluid. It can promote extracellular matrix degradation and remodeling, increasing athletic ability of tumor cells, inhibit apoptosis, and promoting abnormal cell proliferation and angiogenesis. Therefore, it is mainly used in the research of metastatic cancer.Ets1, E26 transcription factor, involves in a variety of tumor invasion and metastasis through the regulation of cell adhesion, hydrolysis and matrix proteins to promote angiogenesis and increase tumor cell hypoxia and other mechanisms. VEGF, a general kind of vascular endothelial growth factor, takes part in all aspects of lymphatic and vessel formation. As a vascular growth factor, it is the strongest. It can specifically bind vascular endothelial cells, promote endothelial cell growth, and help tumor cells enter the vasculature. It is closely related to tumor metastasis and invasion and also affects the efficacy and prognosis.Tumor growth and metastasis depends on tumor angiogenesis. Tumor minute vessel density (MVD) assessed by CD 105 staining is the current gold standard for evaluating tumor angiogenesis in the clinic.It is a membrane-bound glycoprotein, a disulfide linked homodimer,180 KD, composed by 633 amino acids. CD 105 is one of TGF-?receptor complex. CD105 is the specific marker of endothelial cells of no-newly-born vessels in tumor tissue, which can differentiate between newly- born vessels and no-newly-born vessels, to avoid false positives, resulting in correct evaluation tumor angiogenesis.RNAi, RNA interference, is a kind of double-stranded RNA molecules, which can silence the expression of corresponding gene sequence of at the mRNA level. RNAi can result in the degradation of the target gene mRNA through the bind between 21-23nt of dsRNA or hairpin RNA with the specific mRNA, and then lead to the reduced expression of the purposed products. RNAi is widely used in the study of gene function and disease gene therapy because of its fast, reliable and easy-to-features.To investigate the influence of BCSG-1 siRNA on gene expression of BCSG-1 in human breast cancer and cell invasion, BCSG1 siRNA vector was constructed and transfected MCF-7 breast cancer cells. The use of Immunohistochemistry(SP) and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in the cells; plate cloning experiments and Boyden chamber were used to detect cell proliferation and migration in each group cells. In addition, breast cancer xenograft model in nude mice were built with MCF-7 cells transfected BCSG1 siRNA immunohistochemistry and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in the tissue of nude mice transplanted tumor. Finally, immunohistochemical (SP way) was used to detect the expressions of BCSG1, S100A4, Ets-1, VEGT and CD 105 protein in the tissues of breast cancer and breast benign lesions. In this study, we have investigated the mechanism of invasion and metastasis of breast cancer mechanism in molecular level, which provides a theoretical basis and experimental evidence for targeted therapy of breast cancer.Materials and methods1. Effect of BCSG-1 siRNA on the expression of BCSG-1 gene and invasionof breast cancer MCF-7 cellsHuman breast cancer cells MCF-7 were purchased from Kono Bio Co. Ltd. of Nanjing.MCF-7 cells were cultured in the RPMI-1640 medium containing 100g/L fetal bovine serum, at 37?in a 5% CO2 humidified atmosphere.BCSG1 siRNA sequence were designed, synthesized and the synthesis of plasmid vector.Groups:The normal control group, Empty liposome, Negative control group, BCSG1 siRNA 1 group, BCSG1 siRNA2 group, BCSG1 siRNA3 group, BCSG1 siRNA4 group.The immunocytochemistry and RT-PCR were used to detect the expression of BCSG1 protein and mRNA.Plate cloning experiments and Boyden chamber were used to detect proliferation and invasion of MCF-7 cells transfected with the BCSG1 siRNA which inhibits MCF-7 cells according to the results of immunohistochemistry and RT-PCT.2. Effect of BCSG-1 siRNA on the expression of BCSG-1 gene in tumor tissue of nude mice transplanted with human breast cancer cells2.1 BALB/C nude mice, female,2 weeks old, were purchased from Hayes Lake Animal Company of limited liability. They were Reared in the conditions of SPF (specific pathogen Free), kept in constant humidity (relative humidity40-60%), constant temperature (26-28?).2.2 Groups:The mice were randomly divided into 6 groups,5 each group: group of the mice were inoculated with MCF-7 cells untransfected, group of the mice were inoculated with MCF-7 cells transfected nonsense siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG1 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG2 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG3 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG4 siRNA sequences.2.3 Construction of human breast cancer xenograft model in nude mice MCF-7 cells were inoculated subcutaneously into nude mice according to request.Sclerosis, grain of rice, was shown at injection site gradually after inoculating 2 weeks.2.4 Tumor volume and weight each group were measured.2.5 Immunohistochemical and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in nude mice transplanted tumor tissue.3. The expressions of BCSG1,S100A4,ETS1,VEGF and CD105 protein in human breast cancer tissue.3.1 Collecting 40 cases of breast fibroadenoma,62 cases of invasive ductal carcinoma, divided into 2 groups according to whether there are lymph node metastasis (respectively 13 and 49 cases), and 48 cases of adjacent, In which 1 group of 13 cases,49 cases of group 2.3.2 Immunohistochemical was used to detect the expressions of BCSG1, S100A4, Ets1, VEGF and CD 105 protein.4. Statistical analysis:Statistical analysis:measurement data were presented as mean value±tandard error of the mean. The differences between two groups were determined with the Student's t test and LSD test. The differences among three or more groups were determined with a one-way ANOVA. Computations were performed using the SPSS 10.0 software. The cutoff for statistical significance was set as P?0.05.Results:1 The changes of the cell morphologyThe control group had normal cells morphology. Cells are irregular polygon, and have a clear outline. After BCSG1 siRNA transfection, compared with the control groups, some cells are shrunken, the size become smaller, the cell boundaries become blur, the gap become widened, a small amount of cells are suspended in the culture medium.2 The immunocytochemistry resultsPositive signal of BCGS-1 protein showed brown and located in the cytoplasm. Under the Microscope,10 high power fields were randomly selected, according to the percentage of positive cells and the color depth the results were determined. Rating criteria:(1) the scores according to the percentage of positive:"1%for 0; 1% to 25% for 1 point; 26% to 50%for 2 points; 51% to 75% for 3 points;> 76% to 4 points. (2) the scores according to the color depth of positive cells:no coloring is 0; yellow is 1; brown for 2; tan color is 3. Finally, multiply the final integrated score of two parts. The expressions of BCSG protein in cells of BCSG-1-siRNA-1 group was 4.27±0.12, that of BCSG-1-siRNA-2 group was 4.19±0.22, that of BCSG-1-siRNA-3 group was 4.15±0.14 and that of BCSG-1-siRNA-4 group was 4.17±0.13, independent sequence group was 7.92±0.22, control group was 8.02±0.13, MCF7 control group was 8.16±0.32. Compared with the control groups, the expression of BCSG-1 protein in four groups of specific siRNA transfected cells were decreased, and the differences were statistically significant (P all<0.05), but there was no significant difference four groups of specific siRNA transfection group each other (P>0.05), and there also no significant difference among the control groups, (P> 0.05).3 Cell RT-PCR results5?l PCR amplification products and 2?l 6 x Loading Buffer were mixed, spotting in 1%agarose containing ethidium bromide gel, electrophoresis for 20?30min (in 1 x TAE electrophoresis buffer, in 80v voltage; shooting with gel Imaging and recording the scan results; using Quantity One software (BIO-RAD company) to take measurements of the gray value of each band, compared with the calculation of internal reference?-actin and got the relative expression level of each gene. BCSG-1 mRNA was all expressed in the MCF-7 cells each group. The relative expression level of BCSG-1 mRNA in BCSG-1-siRNA-1 group was 0.624±0.010, that BCSG-1-siRNA-2 group was 0.626±0.013, BCSG-1-siRNA-3 group was 0.634±0.008, BCSG-1-siRNA-4 group was 0.631±0.010, independent of sequence group was 0.97±0.076, control group was 0.983±0.052, MCF7 control group was 1.014±0.034. Compared with the control groups, the expressions of BCSG-1 mRNA in the groups transfected the four specific siRNA were significantly lower (P all<0.05), and the differences were statistically significant (P all<0.05). There were no statistically significant between the groups transfected four group-specific sequence each other (P all> 0.05) and there no significant difference between the control groups (P> 0.05).4 Plate cloning experiments resultsSingle cell suspension of MCF-7 cells were seeded in 60m2 Petri dish, each dish separately with 5×103,10×103 and 20×103 cells. Adding culture medium, gently shaking, the cells dispersed. After 2-3 weeks, terminating cultivation when there is visible cloning cluster. Washed 3 times with PBS solution; Fixed with methanol for 15min, and stained with hematoxylin for 2-3min, Rinsed with running water, dry in air. Colonies including at least 50 cells were counted under microscope. The number of clones 3 dish will be converted into cloning efficiency, and taking the mean. Cloning efficiency = clone number/inoculation cell number×100%. The results showed that:MCF7 control group was 32.33±.52%, empty liposome group was 28.33±3.51%, independent of the control group was 32.00±4.36%, BCSG1 siRNA-3 group was 4.17±0.13%. Compared with the control group, the formation of transfected clones was significantly higher than the control group, and differences were statistically significant (P all<0.05).5 Boyden chamber experimentsMatrigel glue, diluted with 50?l serum free RPMI-1640 medium, was to uniform evenly blanketed in polycarbonate membrane, irradiated by Uv for 30min when it to become gelatinous after standing for 30min in air 37?. The cell suspension was diluted to single-cell suspension which concentration was 5×105/ml. According to the requirement, each small room plus 400?l diluted single-cell suspension and each cell plus the three small rooms. They were cultured for 24h under normal conditions; then removing the small room, carefully wiping the film with a cotton swab, fixed with 4% paraformaldehyde,0.1% crystal violet staining, the cells attached to the slide on the side; 5 high power field of view (400×) were randomly selected under microscope, the counting their penetrating cells, Averaging the results. The results show that:penetrating cells in each group, MCF7 control group was 103.53±1.33, empty liposome group was 100.93±1.81, independent of the control group was 101.20±1.71, BCSG1siRNA-3 group was 68.80±10.43. The interference group and control group, the number of penetrating cells significantly decreased, and the difference was statistically significant (P<0.05).6 The growth of nude mice implanted tumor in each groupThe six groups of cells were inoculated into the subcutaneous of the right anterior axillary of nude mice in each group. Scleroma, the size of a grain of rice, gradually appeared at the inoculation site after 3 weeks in the control group, and that appeared in all the groups one week later. And then after 4 weeks, the tumor formation rate in each group is 100%. After the mice were killed, taking out the tumor in each group, weighing, and calculating the rate of tumor growth inhibition (tumor inhibition rate=(control group, tumor weight of a tumor weight of experimental group)/tumor weight of control group x 100%). The results showed:two control groups (A, B) of the tumor mass were 122.34±6.79 and 116.04±5.20, four specific siRNA transfection group (C, D, E, F) the quality of tumor tissue were 20.12±0.85, 21.44±1.43,20.28±0.94 and 20.96±1.42, after the tumor was less than four groups of two control groups, application of statistical methods to compare LSD; four specific siRNA transfection group was no statistical difference between Significance (P> 0.05), the first two control groups no significant difference between (P> 0.05). Compared with the control group, four groups of specific siRNA transfection group nude mice is small and the differences were statistically significant (P?0.05). The rate of tumor growth inhibition compared to the four specific siRNA transfection group was significantly smaller than the tumor inhibition rate independent of the control group, and the difference was statistically significant (P?0.05), but four specific siRNA transfection group Tumor inhibition was no significant difference between the rate (P> 0.05).7 The expression of BCSG1 mRNA in each tumor tissue of nude mice transplanted with MCF-7 cellsThe expressions of BCSG1 mRNA in tumor tissue of nude mice transplanted with MCF-7 cells were respectively:BCSG-1-siRNA-1 group was 1.28±0.36, BCSG-1-siRNA-2 group was 1.21±0.38, BCSG-1-siRNA-3 group 1.21±0.47, BCSG-1-siRNA-4 group was 0.86±.17, independent of sequence group 2.19±0.37, control group 2.62±0.68. The implanted tumor in nude mice in unrelated sequence group and blank control group showed obvious BCSG1 mRNA positive band, and that in each BCSG1-siRNA transfected group, only a small amount of mRNA BCSG1 weak positive bands. Compared with each controlled group, the expressions of BCSG1 mRNA were significantly lower (P<0.05). Compared by LSD method, there were no significant difference between groups transfected four specific sequence (P>0.05), the group transfected independent of sequence and the non-transfected control group (P> 0.05).8 The expression of BCSG1 protein in each tumor tissue of nude mice transplanted wih MCF-7 cellsHigh-definition image processing system was used to analysis immunohisto chemical image. The results showed:BCSG-1-siRNA-1 group was 8.06±0.21, BCSG-1-siRNA-2 group was 6.98±0.43, BCSG-1-siRNA-3 group was 6.98±0.72, BCSG-1-siRNA-4 group was 7.70?0.47, independent of sequence group 16.56±0.86, control group 17.90±1.38. Compared with each control group, the expressions of BCSG1 protein were significantly lower (P<0.05),and there were no significant difference between groups transfected four specific sequence (P> 0.05), the group transfected independent of sequence and the non-transfected control group (P> 0.05).9 The expression of BCSG1 protein in human breast tumor tissue.The positive rate of BCSG1 protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma with axillary lymph node metastasis were 2.50%,12.50%, 69.23%, and 77.55%. BCSG1 protein in human breast cancer tissue and normal expression was significantly higher than that of benign (noncancerous) breast tissue, and the differences were statistically significant (P<0.05); the differences in positive rates between benign and malignant tumor were significant (P<0.05); the expression of BCSG1 protein was positively correlated with tumor grade, and increased with the increasing of the degree of tumor invasion and metastasis.10 The expression of S100A4 protein in human breast tumor tissue.The positive rate of S100A4 protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma axillary lymph node metastasis were 5.00%,6.25%, 53.85%,59.18%. Compared with with the benign (noncancerous) breast tissue, the expression of S100A4 protein in human breast cancer tissue and normal expression were significantly higher, and the differences were statistically significant (P<0.05); the differences in positive rates between benign and malignant tumor were significant (p?0.05).11 The expression of Etsl protein in human breast tumor tissue.The positive rate of Etslprotein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma with axillary lymph node metastasis were 12.50%,25.00% and 53.85%,61.22%. The difference in positive rate between the four groups was statistically significant (P<0.05). Compared with the invasive ductal carcinoma with axillary lymph node metastasis, the expression of S100A4 protein in ductal carcinoma without lymph node metastasis no significant changes (P>0.05)12 The expression of VEGF protein in human breast tumor tissue.The positive rate of VEGF protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma axillary lymph node metastasis were 10.00%,8.33%, 53.85%,73.47%.Compared with the benign (noncancerous) breast tissue, the expression of VEGF protein in human breast cancer tissue and normal expression were significantly higher, and the differences were statistically significant (P <0.05);the differences in positive rates between benign and malignant tumor were significant (P<0.05).13 The expression of CD105 protein in human breast tumor tissue.MVD values (X±S) In breast fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma were 9.33±2.08,18.69±3.37,42.67±14.02. The differences between Benign and malignant was statistically significant (P<0.05), MVD in invasive ductal carcinoma increased significantly.Conclusion1. The expression of BCSG1 protein and mRNA in human breast cancer MCF-7 cells and in tumor transplants of human breast cancer cell line in nude mice were high.2. BCSG1 siRNA can specifically inhibit the expressions of BCSG1 protein and mRNA in the human breast cancer MCF-7 cell line and in the tumor transplants ofhuman breast cancer cell line in nude mice.3. BCSG1 siRNA can inhibit breast cancer MCF-7 cells invasive. It guess BCSG1 siRNA treating breast cancer is feasible and provides a new idea for the treatment of breast cancer. 4. BCSG1 siRNA can inhabit the growth of the tumor transplants of human breast cancer cell line in nude mice.5. The expression of BCSG1 gene is high in human breast cancer.6. The expression of BCSG1 gene was positively correlated to tumor pathological grade, and with the degree of cancer invasion and metastasis increase, the positive rate of BCSG1 increased.7. The expression of S100A4?Ets1?VEGF and CD105 protein are all high. BCSG1, S100A4, Ets1, VEGF, CD 105 are closely related to the generate, development and prognosis of breast cancer, and there is great significance on breast cancer prognosis if these five factors are joint detected. |
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