| Multiple myeloma(MM) is a malignant disorder characterized by the clonal proliferation of plasma cells in the bone marrow, secreting monoclonal immunoglobulin or fragments, which cause organ or tissue damage. It accounts for about 10% of all hematological cancers. It often occurs in the elderly. There is an increasing incidence of MM in China with the increasing aging population. The common clinical manifestations are bone pain, anemia, kidney dysfunction and low immunity and so on. Currently the main clinical treatment of MM are hormones, alkyl drugs, immunomodulatory agents and protease inhibitors, which primarily play roles in inhibiting hematopoietic stem cells, proteasome function, induction of apoptosis and regulating the bone marrow microenvironment as well as cytokines.Now that the pathogenesis of multiple myeloma is complex, previous studies focused on genetic mutations. The genomic DNA changes do not explain the complexity of multiple myeloma development process. More and more evidence suggests that epigenetic regulations, including non-coding RNA, especially small RNA(micro RNA, mi RNA), DNA methylation, histone modifications and other regulation, play important roles in the development of multiple myeloma. Mi RNA is about 21-23 nucleotides length, which target the m RNA 3 ’untranslated region(3’-UTR) and induced m RNA degradation or inhibited protein translation, as a result, which will control the protein in the balance of life. Recent studies providing evidence that micro RNAs play a pivotal role in MM biology may make important contributions to identifying molecules that may help to stratify MM patients better or become targets of new therapies.Mi R-145 is a new mi RNA molecule. Studies showed that it can participate in the regulation of cell invasion, metastasis, proliferation, differentiation and apoptosis process. It has been shown that mi R-145-5p can regulate apoptosis of multiple myeloma cells and play a therapeutic role, suggesting that mi R-145 is a potential therapeutic target to interfere with the invasion,proliferation, apoptosis and other biological processes of the disease. Mi R-145-3p expression levels and the possibility of being a diagnosis and prognosis marker are not particularly clear in MM. The regulation of apoptosis and autophagy has yet to be defined.In clinical treatment, induction of cell apoptosis is one of the important ways. Cytochrome c and mitochondrial protein Smac are released in the process of mitochondria-mediated apoptosis, which also interact with apoptosis protease activating factor-1 to form multimeric complexes and in conjunction with Caspase9 to form apoptotic bodies and activate Caspase cascade reaction, eventually leading to cell apoptosis. In addition to the mitochondria-mediated apoptosis, there is endoplasmic reticulum(endoplasmic reticulum stress, ERS) mediated apoptosis. Through activation of the unfolded protein response(UPR), ERS plays a role in the regulation of apoptosis. In which, Chop, apoptotic signaling kinase ASK1 / JNK, Bcl-2 and caspase-12 family of molecules are involved. While Chop and activation of JNK can also activate pro-apoptotic protein Bax so that the mitochondrial apoptotic pathway is activated, leading to cell death. In clinical treatment of multiple myeloma, Bcl-2 family proteins are deregulated in abnormal apoptotic pathway, leading to cell resistant to drugs such as dexamethasone(DEX). If the above problem can be resolved, it will effectively improve the treatment efficiency.In addition to regulation of apoptosis, regulation of autophagy is also one of the directions in the clinical treatment. In multiple myeloma, plasma cells proliferate malignantly and produce large amounts of immunoglobulin, causing endoplasmic reticulum stress and resulting in demand for degradation of misfolded protein. Autophagy is need to remove these proteins to survive, bone marrow cell rapid proliferate rapidly and synthesis immunoglobulin, which need more energy and autophagy, in this situation, autophagy can protect cells from death. When the stimulation is too much, excessive autophagy could lead to cell death. For example, excessive autophagy will occur when misfolded proteins are too much to degrade, which will result in accumulation of misfolded proteins. In this situation, autophagy will induce cell death. Therefore, regulating autophagy and autophagic cell death is one of the directions in MM clinical treatment.Histone deacetylase regulates many processes involved in tumor progression by removing the acetyl. HDAC4 is an important one. The dysfunction will induce tumor development. HDAC has become an important target for the clinical treatment including multiple myeloma. Currently study about histone acetylation focused on apoptosis, mitochondrial mediated apoptosis is one way of HDAC inhibitor functions. HDAC inhibitors activate caspase-9 and lead to cell apoptosis, It can also up-regulate Bcl-2 family of pro-apoptotic protein Bim and down-regulate anti-apoptotic protein Bcl-2. When HDAC was depleted in breast cancer cells, LC3-II increased over time. HDAC4 expression level and regulation of downstream molecules as well as autophagic cell death is not particularly clear in MM.Based on the above problem, in order to further define circulating mi RNA expression in multiple myeloma, whether its abnormal expression is associated with the diagnosis and prognosis, whether this mi RNA is involved in the express level of HDAC4 and cell apoptosis and autophagy in disease progression of MM, this paper explores to epigenetic regulation of circulation of non-coding RNA involved in multiple myeloma using quantitative PCR technology, bioinformatics analysis, classical molecular biology techniques, western blot, etc. in clinical samples and cells. The results are as following:Part ONE the expression and significance of circulating mi R-145-3p in MMIn recent years, certain mi RNA molecules are found in the peripheral blood, which is called circulating mi RNA. It can be sampled repeatedly, detected conveniently, in stable storage. In multiple myeloma, the mi R-21 level increased in MM patient plasma and is related with other clinical indexes. Circulating mi R-148 a, mi R-99 b, mi R-221 expression levels increase, which have correlations with t(4:14) translocation and 13 q deletion. These suggest that it is possible for circulating mi RNA as biomarkers.The main purpose of this part is to study the mi RNA expression in plasma and potential applications of the screening mi RNA in clinical diagnosis and prognosis. Our previous study using Taq Man micro RNA microarray screened MM patients(in ISS 2/3 stage 3 patients) and 3 normal controls. The result show that there are 25 abnormal expressed mi RNAs between the two groups(fold change> 2, p <0.05), of which 23 increased, while 2 decreased. Mi R-145-3p expression level was significantly lower in MM patients. We further verified this result by q PCR in 30 multiple myeloma patients.We further test the expression of mi R-145-3p in MM cell lines LP-1, U266, PRMI-8226, H929, KM3, normal bone marrow CD138 + plasma cells as control. The result suggests that the level of mi R-145-3p is low(high in KM3 cell). We further discuss the correlation between this mi RNA and clinical indexes, including serum albumin, β2-microglobulin, serum creatinine, serum Ca2+, immunoglobulin, Progression Free Survival and other clinical indexes. It was found that mi R-145-3p was negatively correlated with β2-MG, serum creatinine, serum Ca2+(r =-0.429, p = 0.02; r =-0.370, p = 0.044; r=-0.644, p<0.01) and was positively correlated with serum albumin(r=0.419, p=0.021)respectively, High mi R-145-3p patient have longer progression-free survival. These results suggest that mi R-145-3p has a certain diagnostic value, but also may be involved in the biological processes of multiple myeloma cells.Part TWO effects of mi R-145-3p on apoptosis and autophagy in multiple myelomaStudies have shown that mi RNA may be involved in the development of MM, mi R-21 regulates myeloma cell growth and drug resistance by targeting SPRY2 and MAPK / ERK signal pathway. Mi R-148 a controls cell proliferation and apoptosis by targeting CDKN1 B and PTEN. In the previous study, we found that the level of mi R-145-3p decreased in the plasma of patients and was related with poor prognosis. However, the molecular mechanisms involved in the pathogenesis of MM are unknown.Therefore, this section aim to overexpress mi R-145-3p by constructing mi R-145-3p mimic to explore the role of mi R-145-3p in multiple myeloma cells by transfection, flow cytometry, CCK-8 kit, quantitative PCR, western blot electrophoresis techniques and so on. We found that:Mi R-145-3p mimic significantly inhibit cell proliferation and this inhibition is time-dependent; in addition, it can also induce cell apoptosis verified by an increase of Annexin-V positive cells and high expression of Caspase3-, Apaf-1 as well as low expression of Bcl-2 in m RNA and protein level. It also showed that mi R-145-3p overexpression can induce autophagy, with high LC3-Ⅱ/ LC3-Ⅰconversion, increased Beclin-1 expression, decreased P62 expression by Western blot. We further make sure that cell apoptosis decrease after autophagy inhibitor hydroxychloroquine inhibiting autophagy.These results suggest that mi R-145-3p overexpression can inhibit cell proliferation and induce apoptosis and autophagy and possibly induced apoptosis by inducing autophagy.Part THREE regulation mechanism of mi R-145-3p on multiple myeloma cell apoptosis and autophagy.We have determined that expression of mi R-145-3p is low, positively correlated with prognosis in MM patients. Mi R-145-3p overexpression can inhibit cell proliferation and induce cell death and autophagy and possibly induced apoptosis by inducing autophagy, but the regulation mechanisms of mi R-145-3p on apoptosis and autophagy is still unclear.In this section, we further explore the mechanisms of mi R-145-3p. We use Targetscan, mi Rwalk bioinformatics software, quantitative PCR, western blot, luciferase reporter system, si RNA, focusing on the target of mi R-145-3p, the interaction between them, the downstream activating transcription factor 4, ATF4 expression, the expression of key apoptotic signaling pathways p53-p21, the autophagy pathway expression of key molecules ULK1, PI3KC3 and LAMP2, to investigate the regulatory role of mi R-145-3p.It was found that HDAC4 is one of the targets of mi R-145-3p, which was elevated in bone marrow in multiple myeloma patients. It is negatively correlated with mi R-145-3p level. The transfection experiments showed that overexpression of mi R-145-3p can reduce m RNA and protein levels of HDAC4, luciferase reporter system confirmed that mi R-145-3p has the direct binding on 3’UTR of HDAC4.Si RNA experimental show that it can also induce apoptosis and autophagy as overexpression of mi R-145-3p does. By analyzing the downstream molecules, we found that mi R-145-3p can lead to the increased expression of the transcription factor ATF4, suggesting that by targeting HDAC4, mi R-145-3p possibly increase ATF4 expression and play a regulatory role in apoptosis and autophagy-related genes.The detection of key molecule in apoptosis and autophagy pathway show that overexpression of mi R-145-3p can upregulate P53-independing p21 expression in apoptotic pathway and up-regulate autophagy key molecule ULK1(Ser638) and LAMP2 levels, while having no significant effect on the expression of PI3KC3.In summary, by adopting microarray, quantitative PCR, this study confirmed that mi R-145-3p expression was low in multiple myeloma patients and there were correlations with clinical indexes such as serum albumin, β2-MG and so on. By targeting HDAC4, mi R-145-3p is able to upregulate the ATF4 expression to induce apoptosis and autophagy in MM. Besides that, mi R-145-3p probably induced apoptosis by inducing autophagy. In this process, p53 independent-P21 and ULK1 as well as LAMP2 signaling pathways are involved in mi R-145-3p induced MM cell apoptosis and autophagy process. The result may illustrate the development of MM and provide a new theoretical basis for mi R-145-3p / HDAC4 as therapeutic target. |
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