| THESIS:About ninety percent of breast cancer which is the most common malignant in women, express estrogen receptor a (ERa), progesterone receptor (PR) or human epidermal growth factor receptor 2 (HER2), can largely benefit from the endocrine therapy or the HER2 molecular targeted therapy. However, the primary or acquired resistances contribute to the tumor recurrence and progress, and greatly challenge the treatment of the breast cancer patients. Moreover, approximately ten percent breast cancer which is known as triple-negative breast cancer (TNBC), defined being negative for ER, PR and HER2, was poor in prognosis owing to the deficiency of specific therapeutic targets. Therefore it is crucial important to identify new therapeutic targets to improve the prognosis of breast cancer patients. Currently, there have been quite a number of researches focusing on the mechanism of drug resistance during the treatment of breast cancer patients, and has established some new molecular targets which are probably useful in breast cancer, such as cyclin-dependent kinase 4 and 6 (CDK4/6), Src and androgen receptor (AR). In recent years, quite a lot of studies about genes and miRNAs focused on the pathogenesis of breast cancer, and some of them have proven that miRNAs played crucial role in different breast cancer subtypes, provided the experiment basis for the treatment of breast cancer. It has been widely considered that miRNAs were involved in the occurance, development and drug resistance, and we could take this kind of small non-coding RNA as biomarkers in breast cancer. Importantly, miRNAs may become a novel treatment way for the cancer lack of effective and specific therapeutic target, such as TNBC. It also seems promising to apply miRNA in the treatment of cancer. For the miRNA with low expression level, increase its content by designed drug; for the miRNA with high expression level, reduce its content by the corresponding drug, so as to achieve the purpose of inhibiting tumor.Our present study explored the role of novel targeted drug CDK4/6 inhibitor Palbociclib in breast cancer, and screening the related miRNAs to lay a foundation for the further study on the mechanism. Moreover, we also screened the AR-related miRNAs, explored the signaling pathways by the use of bioinformatics tools and figured out the miRNA-target genes network, provided a basis choosing AR as the therapeutic target in breast cancer. In addition, we tried to explore the role of miRNAs in TNBC occurance and progression, which may contribute to the treatment of this subtype of breast cancer with the poorest prognosis. Last but not least, we established a breast cancer cell line resistant to Src inhibitor, Saracatinib, then screened the differentially expressed miRNAs between the resistant and sensitive cell lines, to provide a foundation for the further exploration of the mechanism of drug resistance.MATERIALS AND METHODS:Four different subtypes of breast cancer cell lines were used in our study:MCF-7 (ER+, HER2-, AR+), SK-BR-3 (ER-, HER2+, AR+), MDA-MB-231 (ER-, HER2-, AR+), Hs578t (ER-, HER2-, AR+), and did the experiments as following:1. Palbociclib (24h) to treat the four cell lines, then harvested them for the miRNA expression profiling analysis;2. Analysed the commonly up-regulated or down-regulated miRNAs in the four groups, and verified by PCR;3. Analysed the differentially expressed miRNAs between AR-positive and AR-negative breast cancer cell lines, and explored the related signaling pathways and miRNA-target genes network;4. Analysed the differentially expressed miRNAs between TNBC and non-TNBC cell lines, then combine the references to review the role of miRNAs in TNBC;5. Established and identified the resistant breast cell line by the induction with low concentration of Saracatinib, and analysed the differentially expressed miRNAs between resistant and sensitive cell lines.RESULTS:1. There was no up-regulated or down-regulated miRNAs in the four group at the same time, indicated that this drug had different mechanisms of effect on the different types of breast cancer cell lines; for the treated cells in the groups of MCF-7 and SK-BR-3, miR-29b-3p and miR-1321 was down-regulated and up-regulated respectively;2. Compared the AR-positive cell lines (MCF-7, SK-BR-3, MDA-MB-231) to the AR-negative one (Hs578t), there were 52 miRNAs up-regulated and 101 ones down-regulated; these miRNAs involved in varieties of cancer signaling pathways, such as EGFR and mTOR, and we built up a VEGF-miRNA-target genes network;3. Compared the TNBC cell lines (MDA-MB-231, Hs578t) to the non-TNBC ones (MCF-7, SK-BR-3), there were four miRNAs up-regulated (miR-138-5p, miR-4324, miR-4800-3p, miR-6836-3p) and eight down-regulated (miR-363-5p, miR-182-5p, miR-141-3p, miR-339-5p, miR-4655-3p, miR-4784, miR-664b-5p, miR-6787-5p); some miRNAs were involved in the EMT (miR-155, miR-221) and metastasis (miR-17,miR-182);4. The IC50 were 9.52μmol/L and 4.85μmol/L in Saracatinib resistant cell line (SK-BR-3/SI) and sensitive cell line (SK-BR-3) respectively; there were 32 miRNAs up-regulated, and 36 down-regulated in SK-BR-3/SI compared to the SK-BR-3.CONCLUSIONS:As the increasing incidence of recurrence and progression during the cancer treatment, and lack of specific therapeutic targets for TNBC, it has become a key problem to identify new molecular targets in breast cancer. Therefore, CDK4/6, AR and Src have become the potential molecular targets. Taking use of the high throughput technology means, we screened differentially expressed miRNAs in breast cancer cells before and after treatment with CDK4/6 inhibitor, provided a basis for further exploration of the mechanism; anslysed the differentially expressed miRNAs in AR-positive/-negative and TNBC/non-TNBC cell lines, then predicted the target genes and the involved signaling pathways; meanwhile, we established the breast cell line resistant to Sre inhibitor, and analysed the differentially expressed miRNAs between resistant and sensitive cell lines, which may provide a foundation for studying the mechanism of drug resistance. |
PDF Full Text Request