| BackgroundGlioma, a kind of malignant tumor, is one of the most harmful cancers to human health in 21st Century. Its incidence rate ranks first in intracranial tumors, characterized by genetic instability, histopathological changes and unpredictable clinical behavior. Gliomas are classified by the World Health Organization (WHO) into four grades, based on pathological and morphological classification. And the higher the grade, the higher the degree of malignancy. High grade gliomas such as glioblastoma, often infiltrates the surrounding brain tissue, so surgical resection alone is difficult to cure, often combined with Postoperative chemotherapy, radiotherapy, gene therapy, immune therapy, the prognosis is often poor, and the median survival time is short. In recent years, the early diagnosis, the mechanism of the occurrence and development, and the target of molecular biological therapy has become the main target of the study.Amount of studies have confirmed that microRNA (miRNAs) shows abnormal expression in kinds of cancers and is closely related to the development and progression of multiple tumors, in which the glioma is included. miRNAs is an endogenous small-molecule non-encoding RNA (approximately 17-20 nucleotides) that govern gene expression in a post-transcriptional manner by binding to the target mRNAs, thereby repressing their translation or inducing their degradation, which is highly conserved during evolution. Primary miRNAs (pri-miRNA) are transcribed in the nucleus by polymerase ?. pri-miRNA is then cleaved into a short about 70 nucleotides hairpin precursor (pre-miRNA) by Drosha nucleases. This nuclear processing is followed by the transport of pre-miRNA from the nucleus into the cytoplasm via exportin-5 and then by the further processing for mature miRNA by Dicer complexes. Mature miRNA is incorporated into an effector complex known as an RNA-in-duced silencing complex (RISC), which binds to mRNA and can affect the translation and stability of mRNA. Expression of the target mRNA is regulated, either by mRNA cleavage or by translational repression, depending on the complementarity of "seed" sequences. MiRNAs have emerged to play important roles in many physiological and pathophysiological processes such as the embryonic development, organogenesis, tumourigenesis and other human diseases such as arrhythmia, ischaemic heart disease, cardiac hypertrophy, viral hepatitis and diabetes. Many reports have revealed that miRNAs play crucial roles in tumorigenesis, angiogenesis, invasion, and apoptosis in various types of tumor.More than 700 miRNAs are predicted in human genome, though physiological role of the majority of these miRNAs remains unknown. Interest in miR-146a, a miRNA important in the negative regulation of acute responses during activation of innate immune, is increased in recent years. miR-146a is located in chromosome 5 LOC285628 gene with its mature sequences at exon 2. Rencent recerch found that the expression of the mature miR-146a is increased in THP-1 cells when stimulated with the microbial components lipopolysaccharide (LPS) and cytokines including IL-lb and TNF-alpha (TNF-a). Another report shows that human T cell leukemia virus type I (HTLV-I, and T lymphocytic leukemia), EB virus infection are also have connection with miR-146a. Its role in the pathogenesis of many inflammatory diseases and tumors has become a hot spot, which may be related to the depression of the target gene. Recent studies have found that the expression of miR-146a in autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and psoriasis is also changing. The initial study focused on miR-146a and tumourigenesis comes from the study by He et al, who reported that miR-146a is overexpressed in unaffected part of thyroid glands of patients with papillary thyroid carcinoma (PTC). Subsequently, the report about the relationship between the occurrence and development of children with acute lymphoblastic leukemia and acute myeloid leukemia, lymphoma, nasopharyngeal carcinoma and other tumors with the abnormal expression of miRNA is increasing. Aberrant increase in miR-146a expression is also observed in several primary muscle disorders. In short, the role of miR-146a in the development of these diseases is related the post translational inhibition of the target gene, such as IRAK1, IRAK2, TRAF6, RIG-I, IRF-5, STAT-1, PTC1 and so on. Studies have confirmed that miR-146a content in patients with glioma is lower than that in benign neuroglia tissue and the expression quantity in glioma cells is lower than that in normal nerve cells with low miR-146a expression and high WHO grade. It has shown that miR-146a has close relation with the development and progression of the gloma, but the specific mechanism is unknown.Macrophage migration inhibitory factor (MIF) was first described as a proinflammatory T-cell-derived cytokine. It is a 12.5-kDa protein expressed in a wide variety of cells including endothelial cells, eosinophils, epithelial cells, lymphocytes, macrophages, and neutrophils. It is unique in structure, consisting of 115 amino acids with two antiparallel alpha-helices and six beta-pleated sheets. MIF exist as a homo trimer and that the carboxy terminus is key to the molecular enzymatic activity. It has been implicated in a variety of diseases including atherosclerosis, acute respiratory distress syndrome (ARDS), and rheumatoid arthritis. As a pro-inflammatory molecule, MIF plays a role in activation of TNF alpha, IL-1 and PGE2 and promotes monocyte and macrophage activation. MiR-146a and MIF are abnormal expression in gliomas, we speculate that MIF may be a downstream target protein miR-146a.Therefore, on the basis of the above, this paper aims to explore the effect of miR-146a on the proliferation of glioma cells by targeting MIF expression. This paper will be divided into three parts. RT-PCR and immunohistochemical methods were used to detect the expression of miR-146a and MIF at different levels of glioma tissues and cell lines from the gene level and protein level. Relationship between MIF and miR-146a was detected with the luciferase reporter gene. Cell jamming technology was adopted, by which, miR-146a mimics and miR-146a negative control (NC) were transferred into cell line of neurogliocytoma for detecting the expressions of MIF mRNA and proteins. MTT method was used to detect cell viability and cloning experiments to detect the proliferative potential of cells.PART I The expression and clinical significance of MIF and miR-146a in gliomasObjectives:To investigate the expression of miR-146a and MIF in gliomas and the correlation analysis and clinical significance of different pathological grades.Methods:Twenty cases of glioma tissue samples and 20 cases of corresponding non-tumor normal tissue samples which were removed surgically were collected between July 2014 to June 2015 in Laiwu City People Hospital. Clinical staging was performed according to the 2007 WHO staging criteria for intracranial tumors. Stage as follows: 14 cases in Stage ?-?,6 cases in Stage ?-?. The cases above had received chemoradiotherapy before operation and were pathologically confirmed. Among them, 9 male patients,11 female patients, aged 31-56 years old, each sample was divided into two parts, one was used to RT-PCR detection, and another was used to MIF expression by immunohistochemistry.Results:1.RT-PCR results showed that the expression of miR-146a in glioma tissues was significantly lower than that in corresponding non-tumor normal tissues with the ratio of (0.68 ± 0.05) vs. (0.23 ± 0.00), which showed significant difference (P< 0.05), and the expression level was decreased with the increase of tumor grade.2. RT-PCR results showed that the expression of MIF in glioma tissues was significantly lower than that in corresponding non-tumor normal tissues.3. Immunohistochemical results showed that when compared positive staining of golden area with corresponding non-tumor normal tissues, MIF positive expression in glioma tissues was higher (0.89 ± 0.02) vs. (5.49 ± 0.48), which showed significant difference (P< 0.01).4. RT-PCR results showed that when the expression of miR-146a increased, the expression of MIF decreased, whereas, when miR-146a expression was lower, the expression of MIF increased.Conclusions:1. MiR-146a and MIF were both expressed in normal brain tissue and glioma tissues, but the expression of miR-146a was down regulated and the expression of MIF was up-regulated in gliomas.2. MIF was mainly distributed in the cytoplasm, and was expressed in normal brain tissue and glioma tissues.3. The expression of miR-146a and MIF in gliomas was correlated with the degree of malignancy of gliomas.PART ? Effection of miR-146a overexpression on invasion and proliferation of glioma cellsObjectives:To investigate the effection of miR-146a overexpression on the growth of glioma cells.Methods:U251, U87, C6 were cultured in vitro. The expression of miR-146a was detected by Q-PCR in three glioma cell lines and normal astrocytes (RA). According to the expression of miR-146a in three cell lines, U87 cell lines were selected to carry on the study. It was divided into two groups, one was transfected with miR-146a micmic group and the other was miR-146a negative control group (NC). MTT and colony forming experiment method were used to detect cell viability and cloning experiments to detect the proliferative potential of cells.Results:1. Q-PCR detection results showed that the relative expressions of miR-146a in glioblastoma cell lines U251, U87 and C6 were significantly lower than those in normal astrocytes(RA),0.20 ± 0.02,0.24 ± 0.04,0.51 ± 0.05,0.52 ± 0.02) vs. (0.75 ± 0.02), which showed significant difference (P< 0.05).2. after transfecting the miR-146a mimics with U87 cell, MTT experiment found that cell viability in transfection group was significantly decreased when compared with that in control group (P= 0.0074).3. after transfecting the miR-146a mimics with U87 cell, cloning experiment found that cell cloning ability in transfection group was significantly decreased when compared with that in control group (68.34 ± 8.23) vs. (35.49 ± 3.65), (P= 0.00032).Conclusions:1.MiR-146a was expressed in normal astrocytes and three types of glioma cell lines, but was down regulated in three glioma cell lines, and the expression was moderate in U87 cell line.2.The up-regulation of miR-146a can decrease the proliferation of glioma cells.PART ? Mechanism of miR-146a targeting MIF on glioma cellsObjectives:To investigate the mechanism of miR-146a targeting MIF on glioma cells.Methods:plasmid co-transfection in glioma cells. Grouping as follow:miR-146a mimics + Wt MIF, miR-146a mimicsNC + Wt MIF, miR-146a mimics + Mut MIF, miR-146a mimics NC+Mut MIF. Dual-luciferase detection system was used to detect the transfected luciferase activity. RT-PCR and Western blot were used to detect the changes of MIF mRNA and protein expression levels in U87 cells transfected with mimic miR-146a and NC, respectively.Results:1. The fluorescence signal intensity in cotransfection group of miR-146a mimics and wild-type vector pGL3-MIF 3'UTR-Wt was obviously weaker than that in other transfection group, which showed significant difference (P< 0.01), Whereas for mutant-type vector pGL3-MIF 3'UTR-Mut, fluorescence intensity in different groups had no any difference (P> 0.05).2. After miR-146a mimics and NC were transfected into U87 cell, MIF protein and mRNA expression quantity were significantly decreased (P=0.0045, P= 0.0041).Conclusions:MiR-146a can be combined with the 3 'UTR of MIF gene to control the transcription, which provides the theory of the mechanism of MIF transcription. It may provide a new target for the treatment of gliomas. |
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