Regulation Of Act1 To The BAFF Signal Pathway In Multiple Myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasma cells in the bone marrow (BM), which induces multi-organic injuries. Large numbers of monoclonal gammaglobulin are secreted to the blood, urine and other fluid, so MM is also a kind of immunoproliferative disease. Accumulation of malignant plasma cells in the bone marrow led to osteolytic bone disease and some other diseases induced from the afunctional gammaglobulin, such as infection, anaemia, thrombocytopenia, renal failure and so on.Many studies indicated that the BM microenvironment played an important role in the generation of MM. The bone marrow stromal cells (BMSC) secreted a great deal of cytokines which were very important to the growth, proliferation and living of the myeloma cells. Interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1) are the popular cytokines. Recently,B-cell-activating factor of the tumor necrosis factor (TNF) family(BAFF) was identified as a critical factor in normal B-cell development and homeostasis too. BAFF was cloned in 1999 by Mukhopadhyay firstly which acted as a new member of TNF family, and it had three receptors named BCMA, TACI and BAFFR respectively. This molecule was expressed mainly by macrophages and dentritic cells and provides a key survival signal for the maturation of peripheral B cells, in addition, it was nearly correlative with autoimmune diseases, Hodgkin's lymphoma and some other B-cell diseases. Now the mechanism how BAFF act in multiple myeloma is a pop problem. It has been reported that BAFF can activate the ERK1/2 and PI3-K/AKT accesses and then the NFκB, also, it can up-regulate Bcl-2 and Bcl-x proteins who can restrain apoptosis. All these functions sustain the proliferation and living of MM cells together.Act1(NFκB activator 1)is a new molecule which is identified as an activator in the NFκB signal pathway and it is known as the name of CIKS (Connection to IKK-complex and SAPK) too, the name showed that Act1 was included in both NFκB and SAPK/JNK signal passages. Act1 reacted with TRAFs and had dual effects in IL-17 and CD-40L/BAFFR. It activated the IL-17 signal in one hand and was a negative regulator in CD-40L/BAFFR signal pathway in the other hand. The negative regulation of Act1 to CD-40L/BAFFR signal Has been proved by gene-knockout mice, but the essential mechanism is not known. BAFF can promote the proliferation and living of MM cells and Act1 can regulate the BAFFR signal in B cells negatively, but are there Act1 protein in MM cells and what is the relationship between the two molecules in MM?Based on the understanding of the studies above, we plan to find the function of Act1 to the proliferation and living in MM cells. Mainly our research will focus on how the BAFF signal is affected by Act1 and the level of Act1 gene expression in the peripheral blood of MM patients at the same time to supply a new idea to the therapy of MM.Part one. Expression of BAFF and Act1 gene in peripheral blood of MM patientsBAFF can sustain the proliferation and living of B cells, and Act1 is a negative regulator in CD-40L/BAFFR signal passage of B cells, but these conclusions were gained from the mice. What is the situation of the two molecules in human MM cells? We collect the PBMCs of MM patients and extract the mRNA then put up reverse transcription, followed by amplification of the gene with RTQ-PCR. Through these experiments we can know the expression of BAFF and Act1 genes in MM patients, and provide a precondition to the following research.We can see that the mRNA of BAFF and Act1 are all increased in MM patients contrasted to the healthy persons from our experiments, the former shared 4.72 and 3.18 times to the later respectively. The results indicate the importance of BAFF and Act1 in the generation and development of MM.Part two. Research on the negative regulation of Act1 to BAFF signal pathway in MM cellsAct1 has the function of negative regulation to BAFF signal in B cell, and the mice who were knocked out the Act1 gene behave as the B cells which were stimulated with BAFF, such as hypergammaglobulinemia and the generation of autoantibodies. However, what is the expression of Act1 in human MM cells, and if it is of negative regulation to BAFF signal pathway in MM cells? To settle the problem, we culture the U266 Cell Strain and extract the total protein of the cells to perform western blot analysis in finding out the protein expression of Act1 in MM. whereafter we transfer the siRNA of Act1 into the U266 cells and stimulate the cells with o.1μg/ml BAFF, then we extract the total protein of the cells at 0, 5, 10, 25 minutes to perform the western blot assay on the p-IκB. At the same time the Act1 gene were synthesized and transferred to the cells to overexpress the Act1 protein and the cells were deal with by the same methods.From the results we found that the p-IκB increased more highly in the cells transferred with siRNA than in the untreated cells, but the cells overexpressed the Act1 changed very mildly. The level of p-IκB culminated at 5 minutes after the BAFF stimulation in the cells transferred with siRNA, and the molecules disappeared at 25 minutes when they were stimulated. This result proved the negative regulation of Act1 to BAFF signal pathway in MM cells.Part three. The mechanism of Act1 in BAFF signal passageThrough the experiments in part two, we proved the negative regulated function of Act1 to BAFF, but the mechanism is not clear and possibly it is relative with TRAF3 and NIK molecules, because the two molecules are both involved in the BAFF signal passage. So in this part we also used the U266 cells which were transferred with siRNA to Act1 gene and stimulated by BAFF, then the TRAF3 and NIK were analyzed by western blot to find the relationship between the two molecules and the BAFF signal. By this experiment we hope to know the role of the two molecules in the regulative action of Act1, and we want to find if there is direct contact between them by Immunoprecipitation(IP).Results showed that TRAF3 was lower in the cells transferred whit siRNA than in the unperformed cells, but the NIK was increased in the later. The results of IP showed there was not direct contact between TRAF3 and NIK, they both reacted with Act1 in signal passage. So the Act1 molecule is a bridge in the passage of cells'activation.Part four. Detection of the functions in MM cellsThe MM cells lacked Act1 acted as the behavior of fortified BAFF signal passage, so the functions of multiplication, excretion and expression could change in this state. We evaluated the level of the Bcl-2 protein which acted as a protein resisted apoptosis in MM cells lacked Act1 by western blot, and performed the MTT multiplication test and detection of IgG at the same time. Based on the experiments we found higher levels of Bcl-2 protein in Act1 lacked cells , but the IgG had no obvious difference between Act1 lacked cells and normal MM cells. On the other hand, the Act1 lacked cells showed stronger multiplication than the cells untreated.