| Glioma is one of the most common tumors in the central nervous system, being the most aggressive brain primary malignant tumor. At present, the prognosis of this disease is not ideal, even if the operation combined with chemotherapy or radiotherapy, recurrence rate is high, the averagesurvival period of only14months, the median survival time of malignant brain glioma cells the tumor is less than1years. Glioblastoma has become a difficult and hot research. Now think there may be genetic factors predisposing factors, biological factors, environmental factorsincluding the effect of various factors such as physical factors, chemical factors. Clinical on with pathological examination results of gliomadiagnosis and the corresponding classification, due to rely solely on themorphology, and according to the stereotactic pathological specimens from small, often based organization lacks the typical pathological changes, often can not according to the pathological change of accurate judgement condition, it brings many difficulties for the diagnosis,treatment and prognosis. Due to the complex etiology of glioma, so although the treatment of glioma has become increasingly diversified,from simple operation therapy before resection of the primary lesion and gradually change to the present in order to operation treatment ofintegrated application, including operation, chemotherapy, radiotherapy,TCM therapy, immunot herapy, gene therapy, psychological therapy etc.a treatment plan, but still have the glioma residual tumor remaining, thencontinue to proliferate, causing tumor recurrence, the ultimate prognosis is poor, short survival time. The glioma invasive growth of glioma therapybecome radical difficulties. At present, the existing various methods of treatment, cannot be completely cured glioma, and often due to normal tissue toxicity caused great damage to the body, not only the gliomadiagnosis treatment after five years survival rate has not improved significantly, and the serious influence the life quality of patients, so to seek the effective treatment of glioma, occurrence become and inhibition of glioma invasion characteristics of the problem in medical scienceresearch. With the biological target therapy means more and moreattention, at present is more focused on the molecular targeted therapy for cancer, but the mechanism is not clear at present molecular therapy. microRNA (miR), the highly conserved gene regulatory factor, appears to provide new ideas for the treatment of tumor, diagnosis. MicroRNA as abiomarker of tumor with tissue sensitivity, able to promote cancer genetumor growth or inhibit the growth of potentially malignant cells of tumor suppressor genes, play a role in affecting occurrence mode. the organism’s development, differentiation, proliferation, apoptosis, immune regulation and other physiological activity and malignant diseases on the precise control. The change of miR expression can be detected early,so the miR expression characteristics can be used for early cancer detection. The current research on miR and glioma still had manyblank. In previous studies, the relationship between miR-184andtumorigenesis, development closely.The FoxO family of forkhead transcription factors (including FoxO1, FoxO3a and FoxO4) is the maintarget protein AKT kinase, the shuttle in the nucleus of inside and outside, play an important role in proliferation, apoptosis,differentiation and resistance to oxidative stress in the body cells.FoxO1/3a phosphorylation by AKT can be transferred to the cytoplasmare ubiquitinated, and degradation by the proteasome pathway, leading to its protein levels and transcriptional activity was decreased.AKT in activated state can phosphorylation of downstream transcriptional factors, leading to nuclear transcription activity of FOXO protein, the loss of. The FOXO family molecules can direct transcriptional upregulation of p21cip1, p27kip1, or transcriptional downregulation of CyclinDl, inhibit the cell proliferation.Therefore, this study aims to investigate the effect and mechanism of miR-184in glioma development, and whether it is associated with the FOXO3.PART ONEThe relationship between different pathological grade and MiR-184expression in gliomaAim:To explore the relationship between different pathological grade and MiR-184expression in glioma.Methods: 20cases were collected with different pathological grading of pathological diagnosis and malignant degree of glioma tissue specimens and10cases with normal brain tissue after traumatic brain injury underwentdecompression of the specimens, the extraction of total micro RNA;expression using real-time quantitative PCR detection of miR-184gene in human brain glioma tissue and normal brain tissues, the correlation between miR-184expression and further comparison and different grade gliomas.Results:1miR-184is expressed in glioma and normal brain tissue, expression and miR-184of normal brain tissue, showed different levels or types of pathology of glioma miR-184table reached significantly increased, with statistical significance difference between the two groups (P<0.05)2The increased expression of miR-184with2different grades of gliomaand the expression of different. In low grade gliomas, the expression of miR-184in glioma although increased, compared with normal brain tissueexpression, but in the early days of low level such as differences in gradegliomas was not statistically significant (P>0.05), but increased with themalignant level, further up regulation of miR-184expression, in Ⅱ-Ⅳgrade of cerebral glioma, expression compared with normal brain tissue,there was a statistically significant difference (P<0.05; P<0.01).3miR-184expression in normal brain tissue were negative, scoring0; Igrade miR-184gliomas showed weak positive expression, score of1-2;the positive expression of miR-184grade Ⅱ gliomas, score3-4points; the expression of miR-184was strongly positive in grade Ⅲ gliomas, score6-8points; miR-184grade IV gliomas the tumor also showed strong positive expression, score6-9points; miR-184positive signals were observed in the cytoplasm were located in the cytoplasm, staining with the increase of the degree of glioma cases level is closely related to.With increasing pathological grade, staining also gradually deepened,and independent of the type of glioma cells.Conclusions:1miR-184in normal tissue and glioma were expressed, but the expression is up-regulated in gliomas. 2miR-184was mainly distributed in cytoplasm of glial cells in the.3miR-184expression in glioma cases classification are related, has nothing to do with the gliomas pathology type.PART TWORegulation of MiR-184on the proliferation and invasion of glioma cellsAim:To explore the regulation of MiR-184on the proliferation and invasion of glioma cells.Methods:Real time PCR was used to detect the expression of MiR-184in glioma cell lines U87, T98G, A-172, LN18, T98G, LN229cells in U87and T98G; will be divided into three groups, including control group, MiR-184mimic group (transfected MiR-184simulated body), MiR-184inhibitor group(transfected with MiR-184inhibitor). Effect of MiR-184MTT detectionmethod on the proliferation of U87and T98G glioma cells; cloning experiments MiR-184plate detecting influence on the formation of theU87and T98G glioma cell cloning; on invasion of human U87and T98G glioma cell by cell scratch assay and cell invasion assay for detection of miR-184.Results:1As the histological examination results were similar, miR-184is expressed in both normal brain cells and glioma cell lines. But the increase in the expression of T98G, U87, A-172, LN229, LN18cells ofglioma cell line, compared with the normal brain cells, NHA cells, miR-184increased significantly2when transfected into miR-184simulation after the body, so that the excessive expression of miR-184, visible both in U87glioma cells orT98G cells, could significantly promote the proliferation of tumor cells);conversely, when the ability of transfected miR-184inhibitor was transfected into glioma cells, artificially suppressed miR-184expression,visible in the two cell lines tumor cell proliferation was inhibited. 3When transfected into miR-184simulation after the body, so that the excessive expression of miR-184, visible both in U87glioma cells orT98G cells, could significantly promote the clone formation of tumor cells,compared with the blank control group, with significant difference (P<0.05); on the contrary, when transfected with miR-184inhibitor was transfected into glioma expression of human cells, inhibit miR-184, visible in the two cell lines, clonal formation of tumor cells was inhibited,compared with the blank control group, with significant difference.4When transfected into miR-184simulation after the body, so that the excessive expression of miR-184, visible both in U87glioma cells orT98G cells, could significantly promote the migration of tumor cells,compared with the blank control group, miR-184scratch changman cells;on the contrary, when transfected with miR-184inhibitor was transfected into glioma cells, expression of artificially suppressed miR-184, visible in the two cell lines, the migration ability of tumor cells was inhibited,compared with the blank control group, miR-184cells significantly reduce the scratch area.5Blank control cells was(98.2±6.1); miR-184mimics control cells (102.3±5.8); miR-184mimics transfected cells (115.3±11.8); miR-184inhibitorsin control cells (96.6±5.3); miR-184inhibitor transfection cells (35.9±4.1), compared with the blank control group, miR-184in mimics transfected cells was significantly increased, with significant difference (P<0.05); inhibitor of miR-184transfected cells was significantly reduced,the differences were statistically significant (P<0.05); miR-184mimicsthe control group and miR-184inhibitors in control group compared with the blank control group, the difference was not statistically significant (P>0.05). When transfected into miR-184simulation after the body, so thatthe excessive expression of miR-184, visible both in U87glioma cells orT98G cells, could significantly promote tumor cell invasion ability; on the contrary, when transfected with miR-184inhibitor was transfected intoglioma cells, inhibition of miR-184expression of human, visible in the twocell line, tumor cells the invasion ability was restrainedConclusions:1Increased expression of1MiR-184can be seen in glioma cells. 2The increased expression of MiR-184can promote the proliferation ofglioma cells.3The increased expression of miR-184can promote the glioma cellmigration, invasion and role.PART THREE The mechnisms of MiR-184on glioma cellsAim:To explore the mechnisms of MiR-184on glioma cells.Methods:U87and T98G were each divided into five groups, including control group,MiR-184mimic transfection group, MiR-184mimic group, control group,MiR-184inhibitor MiR-184inhibitor transfection group. Targeted regulation of miR-184and FOXO3gene detection using luciferase activity in Experiment3’UTR. Using Western cyclinD1, p27, Blot were detectedFOXO3protein expression level changesResults:1transfection of miR-184inhibitor, containing the wild type FOXO33’UTR reporter plasmid luciferase activity was significantly increased(P<0.05).2transfected miR-184simulated body, containing the wild type FOXO33’UTR reporter plasmid luciferase activity was decreased (P<0.05).3transfection of miR-184simulated body, U87, T98G Cell Cyclin D1(CyclinD1) gene was significantly increased (P＜0.05), and the expression of cyclin kinase inhibitor p27and FOXO3gene expression was significantly down regulated (P＜0.05).4transfection of miR-184inhibitor, U87, T98G Cell Cyclin D1(CyclinDl)gene was significantly reduced (P<0.05), and the cell cycle inhibitor p27and FOXO3gene expression was significantly increased (P<0.05). 5transfected miR-184simulated body, consistent with the geneexpression in T98G cells, U87, cyclin D1(CyclinDl) protein was increased significantly (P＜0.05), and the cell cycle inhibitory proteins p27and FOXO3protein expression were significantly lowered (P<0.05).6transfection of miR-184inhibitor, consistent with the gene expression in T98G cells, U87, cyclin D1(CyclinD1) protein was significantly reduced(P<0.05), and the cell cycle inhibitor p27and FOXO3protein expression were significantly increased (P<0.05).Conclusions:1miR-184by adjusting the FOXO3, transcriptional upregulation of CyclinD1, transcriptional downregulation of p27, promote the proliferationof glioma cell invasion.2This is the first study to reveal the important role of miR-184in gliomaproliferation, invasion, and analyses the mechanism, for the understanding of malignant glioma occurrence, development mechanism,and provide the theoretical basis and strategies as well as newrepression. |
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