| Breast cancer is one of the most common female reproductive system malignant tumor, which has the highest incidence and the mortality of common female cancer. With the continuous development of our economy, and the transformation of the women’s life habits, except morbidity number rapid increase, morbidity crowd trend to younger gradually. There are many factors to cause breast cancer, BMI, family history of breast cancer, menarche age, menopausal age, and life stress, habits, etc,which are associated with a gland cancer risk. With the deepening development of molecular biology and genomics, people come to realize that the incidence of breast cancer is closely related to the genetic mutation, many kinds of genetic changes involve the occurrence and development of breast cancer.With the deepening research of the genetic role in the pathogenesis of breast cancer, it is eagerly to looking for target genes which can effectively reduce the incidence of breast cancer, to provide effective method for early breast cancer diagnosis and treatment. Gene therapy has become an important method for breast cancer treatment, such as oncogene and anti-oncogene therapy, suicide gene therapy and immune gene therapy method, which are all based on the role of genes in breast cancer molecular mechanisms. Combined utilization of the traditional surgery, traditional radiotherapy and chemotherapy, the success rate of breast cancer treatment increased effectively. Therefore, it is meaningful to study the pathogenic mechanism of genes in breast cancer.Under the impetus of the human genome research, genomics has been rapid developed, the subsequent gene chip technology has also been widely used in the field of genomics. Fast and efficient detection of gene expression characteristics in tissue or cell can be achieve by using the gene chip technology, is common to be used to find the particular expressed genes in cancer. That made the gene function research developed rapidly, and thus producing a large amount of gene chip information, needs to be further dig into its deep meaning.Pregnancy associated breast cancer is a special type of breast cancer, that diagnosed during pregnancy, lactation time or postpartum a year. In this study, we used the biological information database, searched for related gene expression microarray profile data set from existing pregnancy associated breast cancer gene chip, and screened differentially expressed genes between pregnancy associated breast cancer and normal tissues, then predicted the biological functions and signal transduction pathways these genes may involve by bioinformatics analysis. Combined with research papers retrieval, we finally selected a gene FBXW7 which may strongly relate to breast cancer for further in vitro cells experiment.The gene FBW7 belongs to the proteasome system and it is SCF ubiquitin ligase protein composition. FBXW7 participates in immune regulation in proteasome system, regulation of cell metabolism, and signals to other medicine regulatory processes. Research showed that FBXW7 has different expression in liver cancer, cervical cancer, gastric cancer and brain glioma and their pericarcinous tissue, and affects invasion in cells or tissue, metastasis regulation, cell cycle regulation, immune regulation and other important biological processes, which plays an important role in a variety of tumor diseases.In order to further explore the gene FBXW7 effects and the mechanism of action on breast cancer, breast cancer cell line MCF-7 were transfected with vector that up-regulated FBXW7, The proliferation ability, cell invasion and migration capacity of cells was observed by CCK8 assay and transwell assay, cell cycle and apoptosis were assayed by flow cytometry. Thus clarify FBXW7 biology effect on breast cancer cells on cellular level.Methods:1. The microarray data sets of pregnancy associated breast cancer were downloaded from the public gene expression database (Gene Expression Omnibus, GEO), Pregnancy Associated Breast Cancer used for search index words. The data set was imported to Qlucore Omics Explorer 3.0 software for statistical analysis. The increase or reduce expressed genes which compared with normal group were selected after effective filteraion.2. The increase or reduce expressed genes were imported biological information annotation databases DAVID, and analyzed the biological processes, molecular functions, the composition of cells and signal path of the differential genes by gene functional annotation in DAVID.3. Combined with the research papers, the gene which closely related to breast cancer was screened for the following research. The amino acid sequence was found and imported to PredictProtein online for predicting the molecular function and biological processes of the gene.4. Vector of up-regulated FBXW7 was built and transfected breast cancer cell line MCF-7 as the experimental group (FBXW7), At the same time set up empty plasmid transfection group as the negative control group (NC) and the cells without any infection as blank control group (Control). Transfection efficiency of FBXW7 gene and the expression of FBXW7 mRNA and protein was detected by qRT-PCR and WB.5. After up-regulated the expression of FBXW7 in MCF-7 cells, The proliferation ability of cells was observed by CCK8 assay, cell invasion and migration capacity were tested by transwell assay, cell cycle and apoptosis were assayed by flow cytometry. Preliminary discussion of the role of FBXW7 in breast cancer development.Results:1. Pregnancy associated breast cancer gene screeningThe data set GSE31192 was screened in GEO Datasets by using the key words Pregnancy Associated Breast Cancer.148 differentially expressed genes were screened by Qlucore Omics Explore 3.0, among which 24 were up-regulated and the other 124 were down-regulated.2. Bioinformatics analysis of differential genes in pregnancy associated Breast Cancer148 differentially expressed genes were uploaded to DAVID, The results of these 148 differential genes bioinformatics analysis shown that the genes function mainly related to phosphorus metabolic process, cytoskeleton protein binding,cell differentiation, mitotic cycle, (blood vessel development, regulation of protein kinase activity, positive regulation of transcription from RNA polymerase II promoter and membrane-bounded vesicle. Mapk signaling pathway, focal adhesion pathway, vascular smooth muscle contraction pathway may play important roles in the development of pregnancy associated breast cancer.3. The certain gene screening and function predictionCombined with the results of bioinformatics analysis and research papers founding, we chose the gene FBXW7 which may play a role in breast cancer as the research objective. The molecular function and biological process of FBXW7 were predicted online by PredictProtein. The outcomes showed that FBXW7 mainly involved in nucleic acid binding, ribonucleo protein complex binding, cyclin binding and dynein binding. The biological process mainly related to primary metabolic process, cellular process and regulation of cellular process.4. Up-regulated FBXW7 vector building and its transfection effect detectionThe over-expressed FBXW7 vector pEZ-M02-FBXW7 was builded and screening positive clone. The results of PCR and sequencing showed that recombinant clone sequences are consistent with theoretical predictions, which indicated that the up-regulated FBXW7 vector pEZ-M02-FBXW7 was available. Breast cancer cell line MCF-7 transfected with pEZ-M02-FBXW7, the results of qRT-PCR and WB showed that the expression levels of FBXW7 mRNA and protein in MCF-7 cells experimental group (FBXW7) were significantly improved compared with the negative control group (NC) and the blank control group (Control)5. The effects of over-expressed FBXW7 on MCF-7 cell line(1) The proliferation ability of of MCF-7 cells transfected with pEZ-M02-FBXW7 was observed by CCK8 assay. The results showed that the OD value on 36h was significantly lower in MCF-7 cells experimental group (FBXW7) were significantly improved compared with the negative control group (NC) and the blank control group (Control) (P<0.05); There was no significant difference between the negative control group (NC) and the blank control group (Control) (P> 0.05).(2) The migration and invasion capacity of MCF-7 cells transfected with pEZ-M02-FBXW7 were detected by transwell assay(Giemsa staining,×200). The result of migration transwell assay displayed that the amount of migrated cells in experimental group (FBXW7) was significantly less than that in the negative control group (NC) and the blank control group (Control) (P< 0.05); There was no significant difference between the negative control group (NC) and the blank control group (Control) (P> 0.05). The result of invasion transwell assay displayed that the amount of invaded cells in experimental group (FBXW7) was significantly less than that in the negative control group (NC) and the blank control group (Control) (P<0.05); There was no significant difference between the negative control group(NC)and the blank control group (Control) (P>0.05).(3) The cell cycle distribution of MCF-7 cells transfected with pEZ-M02-FBXW7 was assayed by flow cytometry (FCM). The results showed that the percentage of cells in G0/G1 phase were significantly higher than that in the negative control group (NC) and the blank control group (Control) (P<0.05); There was no significant difference between the negative control group (NC) and the blank control group (Control)(P> 0.05). There was no statistical difference on percentage of cells in G2/M phase in three groups.(4) The cell apoptosis of MCF-7 cells transfected with pEZ-M02-FBXW7 was tested by flow cytometry (FCM). The results showed that the early apoptosis rate, late apoptosis rate and total apoptosis rate were significantly higher than that in the negative control group(NC)and the blank control group(Control)(P<0.05); There was no significant difference between the negative control group(NC)and the blank control group(Control)(P>0.05).Results:148 differentially expressed genes of pregnancy associated breast cancer patients and normal people were screened, mong which 24 were up-regulated and the other 124 were down-regulated. These genes participated in numerous biological processes and signaling pathways which associated with breast cancer. Combined deeper literature mining, the gene FBXW7 was selected which may related to the occurrence and development of breast cancer. On the basis of bioinformatics analysis of FBX W7 gene function, we chose breast cancer cell line MCF-7 for the experiment in vitro. The experimental results show that over-expressed FBXW7 could inhibit the proliferation, invasion and migration ability of MCF-7 cells, and can arrest the cells in G0/G1 phase, and promote its apoptosis. |
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