| Breast cancer is the most prevalent malignant tumor in women worldwide, with the highest morbidity and mortality, which has enormously affected their lives and qualities. In the past two decades, morbidity and mortality of breast cancer in Chinese women were lower, but it increased rapidly recent years. Chemotherapy is the main strategy during breast cancer therapy procedure, and the reasonable and optimal chemotherapy regimens can significantly improve the efficiency and complete remission of metastatic breast cancer (MBC). There are variety of drugs in MBC chemotherapy, among which docetaxel is the most attractive due to its lower toxicity and has become the first-line treatment. However, the drug resistance phenomenon has seriously affected the clinical effects of docetaxel and the outcome. Hence, it is pivotal to find the new target that mediates drug resistance.Alternative splicing variant is a serial procedure of slicing one pre-mRNA into various mature mRNA splicing isomers to fabricate proteins with different structures. The protein products might exhibit different phenotype in the same cell due to the variant expression and even function antagonistically. Alternative slicing variant widely exists in eukaryote biology and is considered to be the main reason of the complexity and diversity of proteins. It can generate many primary transcripts from one gene thus leads to a number of proteins that far more than genes number to accomplish the complicated function and fine regulation of body. Approximately75%happens in the translation area and have influence on the protein sequence, which results in the diversity proteins in eukaryote biology. It affects various aspects of physiology, development, disease etc. so that it is critical to understand the mechanism and guide tumor diagnosis and therapy.FoxMl is a novel member of Forkhead transcription factors family and generally exists in mitotic cells. The common character of the family is the evolutionary conserved DNA binding domain, named "fork head" or "winged helical structure". FoxMl plays an important role in cell proliferation, differentiation, senium, apoptosis etc. and has close relationship with mitosis and cell cycle. In addition, FoxMl is an oncogene that highly expressed in the majority of malignant tumors and has close relationship with the prognosis of many tumors. There are three kind of splicing in FoxMl:FoxM1a, b and c. FoxMl a has lost the transactivation activity because of the exon A2in C terminal, while FoxMlb and c not. Consequently, study the expression, function and mechanism of FoxMlb splicing isomer is essential.Our present study aims at revealing the expression of FoxMlb in breast cancer and understanding the relationship between FoxMlb and docetaxel resistance and its mechanism. The study can be divided into three parts:Part1:Expression and identification of FoxMl isomers in breast cancer. We firstly use a certain concentration of docetaxel to long term intermittently stimulate breast cancer MDA-MB-231cell line and validate the drug resistant MDA-MB-231/Doc cell line. We found that the cell morphology and growth status of resistant cell line were significantly better than normal MDA-MB-231under all the concentrations of docetaxel. The MTT assay got the similar data that the docetaxel resistance of MDA-MB-231/Doc was much enhanced than MDA-MB-231. The immunohistochemical staining revealed that FoxMl was over expressed in most human breast cancer tissues, mainly concentrated in cytoplasm and nucleus. The FoxMl expression in several human breast cancer cells including six cell lines:MDA-MB-468, MDA-MB-453, BT-549, MCF-7, MDA-MB-231, MDA-MB-231/Doc was detected by RT-PCR, Real-time PCR and Western blot. The results demonstrated that all FoxM1a, b and c were expressed in the six cell lines and expression of FoxMlb was much more than FoxMla or c. in the meantime, expression of FoxMlb in MDA-MB-231/Doc was much higher than MDA-MB-231.Part2:The function of FoxMlb in breast cancer. We firstly tried to establish the FoxMlb over expressed MDA-MB-231cell line and silenced MDA-MB-231/Doc cell line by transfection of pcDNA3.1-FoxMlb and FoxMl shRNA separately and the stability of transfected cell line was validated by G418. Real-time PCR and Western blot were used to check the expression of FoxMl in mRNA and protein level. The MTT assay and plane clone formation were used to test the resistance. Flow cytometer was performed to monitor the alteration of cell cycle and apoptosis. Our results showed that the proliferation and resistance in FoxMlb over expressed MDA-MB-231(FoxMlb) cell line were significantly improved compared to MDA-MB-231(Mock). However, when the FoxMl was down regulated in MDA-MB-231/Doc(shRNA), they were dramatically decreased. In the meanwhile, once FoxMlb was elevated, G2ratio in mitosis and apoptosis ratio were down regulated.Part3:The mechanism of FoxMlb involution in docetaxel resistance breast cancer. To explicit the regulation mechanism of FoxMlb in docetaxel resistance breast cancer, the probable downstream molecules of FoxMlb including MDR1、XRCC1、ERCC1、BRCA1and BRCA2were determined by Real-time PCR and Western blot. The data implied that FoxMlb expression was positively correlated with BRCA1and Bcl-2. The immunohistochemistry further confirmed that FoxMl, BRCA1and Bcl-2proteins were highly expressed in cytoplasm and nucleus in the same human breast cancer tissue. The transfection assessments revealed that when FoxMlb was upregulated, BRCA1and Bcl-2were increased consequently and vice versa. Therefore, it was very likely that FoxMlb acts as the upstream molecular of BRCA1and Bcl-2and regulates their expression.Above all, FoxMl protein is seldom expressed in normal human tissues but highly expressed in breast cancer tissue. All the three splicing isomers of FoxMl are upregulated and FoxMlb is the most. The over expression of FoxMlb is closely correlated with docetaxel resistance breast cancer and can control the expression of BRCA1and Bcl-2. Hence, FoxMlb mRNA expression might be used to judge the prognosis of docetaxel resistance breast cancer patients. In addition, FoxMlb mediates drug resistance through regulation of BRCA1and Bcl-2, which might become a promising therapeutic target in clinic as well as a new target for shifting docetaxel resistance. |
PDF Full Text Request