Research Of Histone Deacetylation Regulation On Cell Proliferation Of Triple Negative Breast Cancer

[Background and Objective]Breast cancer is one of the higher mortality rates of malignant tumors for woman. There are about1,300,000new cases of breast cancer all over the world. Among these new patients, there are over15%are triple negative breast cancer. Breast cancer, which show estrogen receptor(ER), progesterone receptor (PR) and human epidermal growth factor receptor2(Her-2) all negative, is called tripe negative breast cancer (TNBC). This kind of breast tumor always has higher histologic grade, stronger invasion and poor prognosis. It is invalid to endocrine drugs and targeted therapy drugs for Her-2. Compared to other kinds of breast cancer, patience of TNBC can get higher pathologic complete remission and clinical response rates to the neoadjuvant chemotherapy which contains paclitaxel and anthracycline, but also would get lower recurrence-free and overall survival rates. It has an easier shorten recurrence, a higher rate of brain metastases and internal metastases than bone metastases, and also a higher death rate. So it becomes an urgent problem to find an effective therapeutic drug for TNBC patients.In recent years, people gradually pay attention to the research on Epigenetics. Epigenetics, which is a branch of genetics and can transfer stably during development and cell proliferation, is not related to DNA sequence alterations of gene or protein expression changes. It is found that epigenetics related to the occurrence and development of many diseases and cancers. Histone acetylation is an important part of epigenetics. The dynamic equilibrium between Histone acetyltransferase (HAT) and histone deacetylase (HDAC) can control the chromatin structure and gene expression. The disorders of their dynamic balance can lead to tumor. HDAC1, type I of HDAC, play an important role on the proliferation of tumor cell. HDAC inhibitors can inhibit the growth and survival of tumor cell through regulating the abnormal proliferation and (or) the expression of apoptosis-related gene. Research shows that HDAC inhibitors have shown a good antitumor effect on a variety of tumor cells including bladder, ovarian, lung, bone, esophageal. Studies have confirmed that HDAC inhibitors can inhibit estrogen receptor positive or negative breast cancer cells. The animal experiment shows that the combination between HDAC inhibitor and chemotherapeutic drugs may enhance the curative effect. Our experiment is to make the construction of HDAC1plasmid and transfer it to the TNBC cells (positive intervention). Meanwhile we put HDAC inhibitor on the other TNBC cells and look for the mechanism by observing the changes of expression on cell cyclin cyclinA2, cyclin D2and tumor suppressor gene P21,14-3-3sigma protein. It can provide a basic study reference for developing HDAC inhibitor as a new molecular targeted drug for TNBC patients.【Methods】 The study includes two parts1. Effect of histone deacetylase1(HDAC1) on triple negative breast cancer cell proliferation (Positive intervention)1.1To construct histone deacetylase1(HDAC1) plasimid (pcDNA3.1-hdac1) by subcloning technology;(our previous work has been completed)1.2To make competence bacterium (E.coli) by CaCl2method;1.3To transform pcDNA3.1-hdacl plasimid into competence bacterium (E.coli), harvest large number of pcDNA3.1-hdacl plasimid, purify and check it by the enzyme digestion;1.4To transfect the TNBC cell with pcDNA3.1-hdacl plasimid, detect the protein expression level with the western bloting;1.5To examine the change of TNBC cell cycle and the expression of four protein (cylin A2, cylin D2, P21,14-3-3sigma) after transfection of pcDNA3.1-hdacl plasimid by using Flow cytometry (FCM).1.6To examine the change of the four protein expression cylin A2, cylin D2, P21, and14-3-3sigma by using the western bloting.2. Effect of histone deacetylase inhibitor on triple negative breast cancer cell proliferation (Negative intervention)2.1To observe the change of TNBC cell proliferation after being treated by histone deacetylase inhibitor SAHA under inverted microscope.2.2To calculate the change of TNBC cell cycle after being treated histone deacetylase inhibitor SAHA by MTS method.2.3To examine the change of TNBC cell cycle and four protein after treatment of histone deacetylase inhibitor SAHA by using Flow cytometry (FCM).2.4To examine the change of four protein after treatment of histone deacetylase inhibitor SAHA by using western blotting. 【Results】1. Effect of histone deacetylase1(HDAC1) on triple negative breast cancer cell proliferation (Positive intervention)1.1Construction and identification of histone deacetylase1(HDAC1) plasimid (pcDNA3.1-hdacl):After successfully constructing of histone deacetylase1(HDAC1) plasimid (pcDNA3.1-hdac1), we confirmed that hdacl DNA fragment is1449bp (cutting position spot is BamH I/EcoR I)by the enzyme digestion; Western bloting showed HDAC1protein expression in TNBC cell lines after transfection of HDAC1plasimid.1.2Effect of histone deacetylase1(HDAC1) on TNBC MDA-MB-468cell cycle: before transfection of HDAC1plasimid into cells: Flow cytometry(FCM)showed: G1phase cell proportion (60.2±0.3)%, S-phase cell proportion (13.7±0.2)%.after transfection of HDAC1plasimid into cells, Flow cytometry(FCM)showed: G1phase cell proportion dropped(45.7±0.2)%, S-phase cell proportion increased (36.1±0.3)%,; Compared between the two cell lines, P<0.05.1.3Study of the influence of histone deacetylase1(HDAC1) on four proteins. FCM showed:after transfection of HDAC1plasimid into TNBC cells, the protein CyclinA2increased over20%; the protein CyclinD2,P21,14-3-3σ decreased; compared between transfected and control group, P<0.05. The western blotting also showed the same result.2. Effect of histone deacetylase inhibitor (HDACi) on TNBC cell proliferation.(Negative intervention)2.1MTS showed after24h treatment of SAHA, the cell demonstrated growth inhibition and cell apoptosis, the greatest inhibition happened at72h after treatment; FCM showed after adding histone deacetylase inhibitor SAHA on MDA-MB-468cell48h, S-phase cell proportion decreased, the expression of CyclinA2decreased, the expression of Cyclin Dl,P21,14-3-3σ increased, also the cell apoptosis increased. Compared to control group, P<0.05. The western blotting also showed the same result. 【Conclusions】1. The histone deacetylase1(HDAC1) gene express in TNBC cell line, its expression can promote cancer cell growth;2. SAHA is an efficient inhibitor for the proliferation of TNBC cells and can promote their cell apoptosis. Its inhibition is related on time. Tumor suppressor gene P21,14-3-3σ and Cyclin A2,cyclinDl participated in the regulation of SAHA on the cell proliferation.