| [Objective and significance]Breast cancer is the most commonly seen malignant carcinoma of female breasts, and also one of the malignant carcinomas with the highest occurrence among females. Though our country is a region with lower occurrence rate of breast cancer, the incidence rate is increasing in recent years. It not only ranks at the first among female malignant carcinomas, and with an earlier peak of morbidity. Though the five year survival is improved because of comprehensive therapy targeting to breast cancer, distant metastasis is still the important prognostic factor for breast cancer. The genetic therapy against breast cancer is a new therapeutic measure following surgical approach, radiotherapy, chemotherapy and endocrine therapy, and it is a biological therapeutic method with good prospective. At present, some research achievements have been applied to clinical practice, with significant therapeutic effects obtained. For example:in Oct.,1998, FDA had formally approved to administer Trastuzumab (trade name, Herceptin, Genentech/Roche), the monoclonal antibody derived from high purity recombinant DNA and targeting Her-2 receptor, to treat metastatic breast cancer with over expression of Her-2/neu, which brought the first morning light to therapy against breast cancer. Though primary success has been obtained in genetic therapy against breast cancer, there are still many problems, and one of them is that, there are only few genes which can be applied into clinical therapy. Therefore, it is especially important to find a new target gene. S100A4 protein is a member of S100 protein family, and its coding genes are localized at chromosome 1q21 with other 14 members, and this region is the unstable region correlated with the occurrence of many malignant carcinomas. Therefore, coding gene of S100A4 protein has its special expression styles. It is indicated in the current study that, S100A4 protein participates in the signal transduction in and out of cells, cell cycle regulation, and it is involved in many life activities including cell proliferation and differentiation, cell adhesion, cell migration, etc., and it is deeply related with the pathological process of many malignant carcinomas including breast cancer, including genesis, infiltration and metastasis, etc. Moreover, S100A4 protein is nearly expressed in all carcinoma cells. In human body, S100A4 expression of varied degrees is only observed in monocyte, macrophage, polymorphonuclear granulocyte, keratinocyte, Langhans'cell and sweat gland cell till now. In normal cells of lung, kidney, breast cancer, thyroid, pancreas and colon, S100A4 is not expressed, so S100A4 protein is the ideal target for genetic therapy against carcinomas. Many studies in China and other countries have confirmed that, for many malignant carcinomas including breast cancer, S100A4 protein is a stable factor to predicting prognosis and lymphatic metastasis. In the aspect of genetic therapy, Shi Y et al applied RNA interference to silence the S100A4 expression in undifferentiated thyroid carcinoma cell line (ARO), and found that the proliferation of carcinoma cell was decreased by 46%+7.6%, migration of carcinoma cells was also inhibited obviously, and sensitivity of carcinoma cells to taxotere was improved. Saleem M et al performed RNA interference study targe-ting S100A4 in highly metastatic prostate carcinoma cell line (PC-3), and found that after S100A4 was silenced, proliferation, infiltration and metastasis of carcinoma cells were significantly inhibited. Besides of the abovementioned studies, similar conclusion has been obtained in related studies on other carcinomas, including pancr-eatic carcinoma, prostate carcinoma and esophageal cancer, etc., i.e., RNA interfere-nce is applied to silence expression of S100A4 gene, and proliferation and metastasis of carcinoma cells can be inhibited.Based on the study achievements in China and other countries, RNA interference was adopted in this study to synthesize S100A4-shRNA template strand with S100A4 as the target gene, and construct recombinant plasmid by linking two carriers of pGenesil-1.1 and S100A4shRNA. Liposome Lipofectamine 2000 was applied to me-diate cell transfect, and S100A4-shRNA was used to silence S100A4 gene expression, to observe the proliferation and apoptosis of MCF-7 cell, and changes of infiltrating and migrating capacity. Our study can provide a new approach for genetic therapy against breast cancer, and it has certain scientific significance and clinical practice.[Statistical methods]Statistical analysis was performed by SPSS13.0 software.All data were presente-d as mean±standard deviation.one-way ANOVA was used to determine the statistical significance,first of all,doing the test of Homogeneity of Variances among the group-s. If the equal variances assumed, LSD was used to determine the statistical significa-nce. If the equal variances not assumed, Dunnett's T3 was used to determine the stati-stical significance.Repeated measures ANOVA was used to test the cross correlation between factor and groups in MTT.P<0.05 was considered statistically significant.[Methods and Results]Chapter 1 Construction of recombinant plasmid for target S100A4[Objective] To synthesize two recombinant plasmids as the expression vector of S100A4-shRNA to transfect into MCF-7 cells, and silence the expression of S100A4 gene.[Methods] Two shRNA sequences targeting S100A4 were designed and synthesized to obtain RNA interference sequence segment for S100A4, which was inserted into the stably screened and labeled plasmid carrier pGenesil-1.1 containing green fluorescent protein (EGFP), then recombinant plasmids pGenesil-1.1-S100A4-shRNA-1 and pGenesil-1.1-S100A4-shR.NA-2 were constructed, and amplified in great amount after enzyme digested and sequenced.[Results] After the recombinant plasmid extracted by using extraction kit was excised by Sac I enzyme, it was identified by using 1% agarose electrophoresis. A Sac I enzyme digestion site was designed in two target gene segments, and there was also a Sac I enzyme digestion site in plasmid pGenesil-1.1. Therefore, if the linkage was correct, a 916 bp DNA strap was excised from recombinant plasmid by Sac I enzyme. It was confirmed by enzyme excision that,2 recombinant plasmids conform-ed to design requirements, and the sequencing results obtained by Shanghai Invitroge-n Biological Technology Co., Ltd. confirmed that the sequence was correct.Chapter 2 In vivo experimental study on S100A4 gene expression in MCF-7 cell inhibited by RNA interference2.1 Expression of S100A4 protein in MCF-7 cell detected by immunohis-tochemical method[Objective] To detect the expression of S100A4 protein in MCF-7 cell.[Methods] The experiment had two groups including blank control group and test group. Inoculate MCF-7 cells with trypan blue staining activity>95% in sterile cover glass of 6-well plate at 1×106/well, Immunohistochemical SP method was applied to detect the expression of S100A4 protein in MCF-7 cell. [Results] Obvious expression of S100A4 protein was observed in human breast cancer MCF-7 cell.2.2 MCF-7 cell transfect test[Objective] To detect the transfect of liposome mediated recombinant plasmid into MCF-7 cell.[Methods] After transfect for 24h, the transfect of liposome mediated recombine-nt plasmid into MCF-7 cell was detected by using fluorescence microscope.[Results] Liposome mediated recombinant plasmid was successfully transfected into MCF-7 cells.2.3 Changes of S100A4 gene expression after transfect by QRT-PCR method[Objective] To detect S100A4 gene expression in MCF-7 cell 48h after transfect, and screen a recombinant plasmid with a higher S100A4 expression rate.[Methods] The experiment had four groups:blank control group, negative control group, S100A4-shRNA-1 group and S100A4-shRNA-2 group. The latter three groups were respectively transfected by negative control plasmid, recombinant plasmid pGenesil-1.1-S100A4-shRNA-1, recombinant plasmid pGenesil-1.1-S100A4-shRNA-2, and the expression of S100A4 gene in different groups were detected by QRT-PCR method 48h after transfect, with GAPDH as the internal reference gene.[Results] Results of QRT-PCR indicated that,48h after transfect, there are stastic-al significance among the three groups (F= 67.854, P=0.000).The expression amount of S100A4 mRNA in S100A4-shRNA-1 group and S100A4-shRNA-2 group was lower than those in negative control and blank control groups(P=0.000, P=0.000; P=0.000, P=0.000); expression amount of S100A4mRNA in S100A4-shRNA-1 group was lower than that in S100A4-shRNA-2 group (P=0.017). Thus, recombinant plasmid pGenesil-1.1-S100A4-shRNA-1 was used to perform the subsequent experi-ment.2.4 Changes of S100A4 protein expression detected by Westernblot method[Objective] To detect the expression of S100A4 protein in MCF-7 cells 48h after transfect.[Methods] The experiment had three groups, including test group, negative control group and blank control group. The first two groups were transfected by recombinant plasmids of pGenesil-1.1-S100A4-shR.NA-1 and negative control plasm-id. Western blot method was applied to detect the expression of S100A4 proteins in different groups 48h after transfect.[Results] Results of Western blot indicated that, compared to blank control group and negative control group, expression of S100A4 protein was obviously weakened, and Tanon systemic analysis indicated that, its protein strap gray scale was decreased by 57.21% and 54.82% compared to those of the first two groups.2.5 Changes of cell proliferation after transfect by using MTT method[Objective] To detect the changes of MCF-7 cell proliferation after transfect.[Methods] The experimental materials were grouped as abovementioned, and cells of test group, negative control group and blank control group were inoculated into a 96-well plate at 1×104 cells/well, MTT (5 mg/ml) 20μl was added into each well after the cells were transfected for 24,48 and 72 h. Then the plate was placed into a CO2 incubator for 4h, and the supernatant was discarded after the reaction was stopped. DMSO 200μl was added into each well, then the plate was shaken for 10min, and optical density (OD) value was read at 490 nm wavelength to draw the growth curves. The procedure was repeated for three times. [Results] Experimental results indicated that, there are stastical significance among the three groups at 24,48 and 72h(F=13.401, P=0.006; F=132.689, P= 0.000; F=3150.772, P=0.000).OD value of test group was lower than those of negative control and blank control group at 24,48 and 72h (P=0.006, P=0.003; P=0.015, P=0.015; P=0.000, P=0.000), while difference of OD value was insignificant betwe-en negative control and blank control groups at all time points (P=0.571; P=0.287; P= 0.211).2.6 Annexin V-FITC/PI double staining method was used to detect the changes of apoptosis rate after transfect.[Objective] To detect the changes of MCF-7 cell apoptosis 24 h after transfect.[Methods] The experimental materials were grouped as abovementioned, and cells were inoculated into a 6-well plate at 1×106 cells/plate. Triplicate wells were set for each group. Supernatant and cells were collected 24h after transfect. The cell density was adjusted to 3×105/mL, and then 2μl Annexin V-FITC and 5μl PI were added to determine the apoptosis of cells in different groups by using flow cytometry. The procedure was repeated for 3 times. Result judgment:normal cell was Annexin V-FITC (-)/PI (-), viable apoptotic cell was Annexin V-FITC (+)-/PI (-), non-viable apoptotic cell was Annexin V-FITC (+)/PI (+), and necrotic cell was Annexin V-FITC (-)/PI (+).[Results] Experimental results indicated that,24h after transfect, there are stastic-al significance among the three groups in the apoptosis rate of non-viable apoptotic cell (F= 5.895, P=0.038).the apoptosis rate of non-viable apoptotic cell in test group was higher than those of negative control and blank control groups (P=0.036, P=0.019), but no statistical difference was observed between the latter two groups (P=0.636).2.7 Construction and morphological observation of stable transfected cell strain[Objective] To construct stable transfected cell strain and observe their changes.[Methods] Firstly, use G418 to screen the concentration for stable transfect cell, and inoculate MCF-7 cells with cell activity>95% counted by trypan blue into a 6-well plate at 1×106 cells/well. After cells adhered to well wall, RPMI1640 solution containing 0,100,200,300,400,500,600,700,800,900 and 1000ug/ml G41810% FBS were used to culture the cells. The solution was replaced for every 3 days, growth of cells was observed between normal cells and G418 screened cells. G418 concentration with 70% death in one week and 100% death in the second week was selected to screen the concentration of stable transfect cell (in this experiment, G418 screening concentration was 500 ug/ml). The experiment had three groups including test control group, negative control group and blank control group. The first two groups were transfected with recombinant plasmid and negative control plasmid, and then MCF-7 cell was 1:10 passaged 24 h after transfect. Two control wells of normal cells were also set. After cells adhered to well wall,10% FBS RPMI1640 solution containing 500ug/ml G418 was adopted, and the solution was changed for every 3 days. After 2 weeks, normal cells had a death rate of 100%, and 30% transfected cells were survived. Then 10% FBS RPMI1640 solution containing 350ug/ml G418 was changed, and single cells appeared 1 week later. Under the fluorescent microscope, living MCF-7 cells with fluorescence were labeled. The cells were digested and collected to adjust the solution concentration<=1 cell/100ul, and cells were inoculated into a 96-well plate at 100ul/well. Then expand the culture, the cells formed cell cluster 2 weeks later. After 5 weeks,50% wells were covered. When cells covered 24 wells, transfer the cells gradually to a 6-well plate and a 25cm2 culture flask. Respectively name the stable transfect cell as MCF-7-S100A4 cell in test group, MCF-7-HK cell in negative control group. Immunohistochemical SP method was used to observe the differences of morphologies and size, etc. between MCF-7-S100-A4 cells, MCF-7-HK cells and MCF-7 cells.[Results]After stable transfect cells were screened, it was found by immunohisto-chemical method that, MCF-7 cell and MCF-7-HK cell also had irregular morpholo-gies, varied sizes, big nuclei deeply staining, in round of oval shapes. The cell had no cell polarity, with confused arrangement. There was no significant difference between MCF-7-S100A4 cells and the above cells in cell size, morphology and living styles.2.8 Migrating capacity of stable transfect cells by using scratch adhesion test[Objective] To detect the changes of migrating capacity of stable transfect cells.[Methods] Scratch adhesion test was used to detect the changes of migrating capacity of stable transfect cells. The experiment had three groups including test group (MCF-7-S100A4 cell group), negative control group (MCF-7-HK cell group) and blank control group. Cells at logarithmic phase were collected and inoculated into a 6-well plate at 1×106 cells/well. On single cell layer surface, scratch with 100μl transferpettor probe vertically, and flush with PBS for 2 times, then observe the migrating cells under a microscope for scratches made at 0,6,24 and 48 h, then take the images.[Results] Results of scratch adhesion test indicated that, the migrating rate of MCF-7-S100A4 in test group was obviously smaller than those of MCF-7 cell in blank control group and MCF-7-HK cell in negative control group. After the cells were inoculated for 48 h, MCF-7 cells and MCF-7-HK cells covered the scratches, and MCF-7-S100A4 cells had not spread over the scratch.2.9 Transwell chamber method was applied to determine the infiltrating capacity of stable transfect cells. [Objective] To determine the changes of infiltrating capacity of stable transfect cells.[Methods] The materials of the experiment were grouped as abovementioned, and Transwell chamber method was applied to determine the changes of infiltrating capacity of stable transfect cells.[Results] Results of in vitro infiltrating experiment indicated that, there are stasti-cal significance among the three groups (F= 826.583, P=0.000), and the stastical dif-ference was observed between test group and the other two groups (P=0.000, P= 0.000), while no statistical difference was observed between negative and blank cont-rol groups (P=0.128). The results of the above experiment indicated that, after S100-A4 was silenced, the infiltrating and migrating capacities of MCF-7 cells were signif-icantly decreased.Chapter 3 In vivo study on expression of S100A4 gene in MCF-7 cell inhibited by RNA interference3.1 Tumorigenicity experiment on stable transfect cell in naked mice[Objective] To determine the tumorigenicity of transplanted tumor by inoculating stable transfect cell into the back of naked mice.[Methods] The experiment had three groups including negative control group (MCF-7-HK cell group),test group (MCF-7-S100A4 cell group) and blank control group, with 10 mice in each group. Take cells from the above groups and prepare single cell suspension, adjust the cell density to 1×107/mL, and 0.4 ml solution was injected subcutaneously into the back of naked mice. After the carcinoma cells were implanted, the diet, mental activities and movements were observed, tumor size was determined for every 2 days, and the maximal diameter (a) and maximal vertical transverse diameter of the tumor were determined. The tumor volume was calculated following V (mm3)=axb2/2, and growth curve was drawn to calculate the tumor inhibition rate. After observed for 35 d, the naked mice were decapitated, then the transplanted tumor was weighed, and the results were analyzed.[Results]In experiment in vivo, after the naked mice were inoculated for 35 d, the average tumor volume was more than 900 mm3 in mice of blank and negative control groups, with a tumor formation rate of 100%. After observed for 2-3 weeks on mice of test group, tiny tumor (about 12mm3) could only observed touched subcutaneously, and the tumors would not growth as times went by, or they even shrunk (<12 mm3), and the tumor formation rate was 90%. After tumor cell was inoculated for 35d, the animals were executed, and subcutaneous tumors were isolated and weighed. Experimental results indicated that, there are stastical significance among the three groups in tumor mass and volume of naked mice (F= 102201.907, P=0.000; F= 8859.118, P=0.000).tumor mass and volume of naked mice in test group were significantly lower than those of negative and blank control groups (P=0.000, P=0.000; P= 0.000, P=0.000). while no significant difference was observed between two control groups(P=0.991, P=0.062). The following formula was used to calculate tumor inhibition rate (%)= [(average tumor weight in blank control group-average tumor weight in test group)/average tumor weight in blank control group]×100%, and the inhibition rate was 98.64%.3.2 Expression of S100A4 gene in transplanted carcinoma determined by QRT-PCR method[Objective] To determine the expression of S100A4 gene in transplanted carcinoma.[Methods] Animals in this experiment were groups as abovementioned, with 4 naked mice with carcinomas randomized in each group. The transplanted carcinoma tissues were collected to perform the experiment, and normal tissues were also taken as controls. By using QRT-PCR and Sybrgreen staining method, GAPDH was adopted as the internal reference gene, and expression amount of S100A4 gene was determined in tissues of transplanted carcinoma on back of naked mice.[Results] Results of QRT-PCR indicated that, there are stastical significance among the three groups (F=83.598, P=0.000) expression amount of S100A4 mRNA in test group was far lower than those of negative and blank control groups (P=0.000, P=0.000), while no significant difference was observed between the two control groups (P=0.915)。3.3 Expression of S100A4 protein in transplanted carcinoma on back of naked mice determined by Westernblot[Objective] To determine the expression of S100A4 protein in transplanted carcinoma on back of naked mice.[Methods] Animals in this experiment were groups as abovementioned, with 4 naked mice with carcinomas randomized in each group. The transplanted carcinoma tissues were collected to perform the experiment, and normal tissues were also taken as controls. And Western blotting method was adopted to determine the expression of S100A4 protein in transplanted carcinoma on back of naked mice.[Results] Results of Western blot indicated that, there are stastical significance among the three groups (F= 90.297, P=0.000).expression of S100A4 protein was significantly higher in blank and negative control groups compared to test group (P=0.000, P= 0.000), while no significant difference was observed between control groups(P=0.643).[Conclusion]In this study, breast cancer MCF-7 cell was adopted as the study model to construct S100A4-shRNA with specific interfering sequence S100A4 as the tool to perform RNA interference, and the expression of S100A4 gene was silenced. Accor-ding to the results, the study explained the following points:1) Silencing S100A by using S100A4-shRNA can effectively inhibit expression S100A4 in MCF-7 cells.2) Silencing S100A by using S100A4-shRNA can effectively inhibit MCF-7 proliferation and promote apoptosis.3) Silencing S100A by using S100A4-shRNA can effectively inhibit cell migration and infiltration of MCF-7 cells.4) Silencing S100A by using S100A4-shRNA can effectively inhibit the Tumorigenicity of MCF-7 cells in naked mice.The following results are obtained according to the above results:the construc-ted S100A4-shRNA carrying S100A4 special interference sequence can effectively silence S100A4 gene, inhibit proliferation, infiltration and migration of MCF-7 cells, and promote their apoptosis. These results indicate that S100A4 protein participates in cell proliferation, differentiation and cell migration, and it is closely correlated with proliferation, infiltration and migration of breast cancer MCF-7 cells.
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