| Part Ⅰ THE EXPRESSION OF SERUM MICRORNA-182 IN GLIOMA AND ITS CLINICAL APPLICATIONBackgroundGlioma is the most common human primary malignant brain tumors, comprising approximately 60% of all central nervous system tumors. It is one of the five tumors hard to cure, and characterized by a rapid infiltrative growth pattern making complete surgical resection impossible. Currently, despite the recent advances in tumor diagnosis and treatment, including surgery, radiotherapy and chemotherapy, glioma has still a high mortality and poor 5-year survival rate. The poor prognosis is due to the early local invasiveness as well as the lack of effective early diagnosis. The golden standard of glioma diagnosis is histological evaluation. However, its obstacle is still acquiring tissue owing to the special position of glioma. Therefore, it is extremely necessary to explore novel and highly sensitive molecular biomarkers with reliable clinical significance.MicroRNAs (miRNAs) are small non-coding RNAs (20-22 nucleotides), which negatively regulate the expression of gene by repressing the translation of target mRNAs. Accumulating evidence indicate that miRNAs are great significances in the crucial biological processes such as cellular proliferation, differentiation and tumorigenesis. The aberrant expression of miRNAs has been identified in many tumors, and its expression profiles are different among different types of tumor Moreover, circulating miRNAs have been reported to be convenient and non-invasive biomarkers with high stability, indicating great potential. Recently, several studies have explored the feasibility whether the abnormal single miRNAs could be used as potential diagnostic or prognostic biomarkers in glioma, such as miR-205, miR-128, and miR-210. However, the sensitivity and specificity is not good for clinical requirement. MiR-182 is an oncogene that is dysregulated in many human cancers, and its overexpression contributes to the growth, invasion and/or chemotherapeutic sensitivity of these tumors. Previous study reveals that miR-182 are significantly up-regulated in tissues, which provides us a hint that miR-182 may be a promising biomarker for early diagnosis and prognosis. Although circulating miR-182 has been found in the plasma or serum of some tumors, its expression and correlation with clinical features in glioma has not yet been determined.Hence, we detected the expression of plasma circulating miR-182 in glioma patients and healthy controls to evaluate its role of diagnosis and prognostic prediction. Furthermore, we analyzed the relationships among clinical data, clinicopathological variables, and diagnostic or prognostic value. Our results provide a new evidence that miR-182 can be a novel diagnostic and prognostic biomarker with a satisfactory sensitivity and specificity in patients with glioma.Objective1.To detect the expression level of miR-182 in the serum of patients with glioma.2.Analysis of the relationship between the expression level of serum miR-182 and the clinical characteristics of patients with glioma.3.Evaluate the value of serum miR-182 expression level in the early diagnosis of glioma patients.4.To study the significance of serum miR-182 expression level in evaluating the prognosis of patients with gliomas.Methods1. The serum miR-182 levels of 112 patients with malignant gliomas and 54 healthy controls were detected by qRT-PCR, and the differences between the serum miR-182 levels in patients with glioma and healthy controls were studied.2. To evaluate the correlation between the expression level of serum miR-182 and the clinical characteristics of patients with glioma, including gender, age, tumor size, KPS score and WHO pathological grading. In patients with gliomas, the level of miR-182 expression in the serum of patients with different pathological grade gliomas was compared.3. To further analyze the differences of serum miR-182 levels in patients with different levels and groups of patients with glioma. Area under the ROC curve (AUC) and ROC curves were used to assess the value of serum miR-182 expression levels in the early diagnosis of gliomas with different pathological grades and the evaluation of the degree of malignancy of gliomas.4. Follow up of all patients, the correlation between serum miR-182 level and total survival rate of patients with glioma was analyzed, and the disease free survival (DFS) and overall survival (OS) were analyzed. Kaplan-Meier analysis was used to evaluate the prognostic value of serum miR-182 in patients with gliomas. Then using univariate and multivariate Cox regression analysis to determine the expression, of serum miR-182 levels are independent prognostic factors in patients with glioma, and explore the value of serum miR-182 on prognosis in patients with glioma.Results1. The expression of plasma circulating miR-182The studing showed that the level of circulating miR-182 in patients with glioma (mean±SD,2.57±1.95) was much higher than that of healthy controls (0.97±0.38) (P＜.001). The circulating miR-182 in grade IV was much higher than that of patients in garde Ⅰ (P<0.001), Ⅱ (P＜0.001) or Ⅲ (P＜0.05). The level in grade Ⅲ was higher than that in grade Ⅰ (P＜0.05).2. The correlation between circulating miR-182 and clinicopathological featuresWe evaluated the correlation of circulating miR-182 with clinicopathological parameters including gender, age, tumor size, KPS score and WHO grade. Plasma circulating miR-182 was statistically correlated with KPS score (P=0.025), whereas no significant correlation between miR-182 and gender, age, and tumor size was found (all at P>0.05). A significant correlation was observed between circulating miR-182 and WHO grade (r=0.786, P=0.006), indicating that the up-regulation of miR-182 might be correlated with clinical glioma progression.3. Predictive value of circulating miR-182 for gliomaThe level of circulating miR-182 in patients with high-grade (3.25±2.05) was higher than that of low-grade (1.38±0.94) or healthy controls (0.97±0.38) (both at P＜0.001). Interesting, no significant difference was catched between low-grade glioma and controls (P>0.05). ROC curve and area under ROC curve (AUC) were used to further estimate the prediction value of circulating miR-182 of glioma. It showed the AUC of all stage is 0.778 (95% CI,0.679-0.878). The cut-off value of circulating miR-182 in glioma patients was 1.56. The corresponding sensitivity and specificity were 58.5% and 85.2%. The predictive performance of circulating miR-182 was found in distinguishing high-grade glioma from healthy controls, and AUC was 0.815 (95% CI,0.718-0.913). We then evaluated the diagnostic performance for low-grade glioma. The AUC was 0.621 (95% CI,0.500-0.741), and was significantly lower than all stages or high-grade (both P<0.05), suggesting that miR-182 might be an unreliable biomarker for low-grade glioma.4. Correlation between circulating miR-182 level and prognosis in glioma patientsThe patients were categorized into low and high circulating miR-182 groups, based on the optimal cut-off value. The findings showed that the cumulative 5-year overall survival rate of disease-free survival (DFS) and/or overall survival (OS) with higher level of circulating miR-182 were shorter than that of patients with lower level (higher (32.786,95%CI:22.941-33.059) versus lower (44.923,95%CI:31.487-58.513)) for DFS (P=0.006), and higher (19.325,95%CI:9.620-20.380) versus lower (30.638, 95%CI:24.668-39.332) for OS (P=0.003). Moreover, univariate cox proportional hazard regression model analysis revealed a significantly relation between DFS and KPS score (P＜0.001), as well as WHO grade (P＜.001) or circulating miR-182 (P=0.009). OS was related to both KPS score (P＜0.001), WHO grade (P＜0.001), and circulating miR-182 (P=0.004). Subsequently, to determine whether circulation miR-182 was the independent prognostic factor of glioma patients, univariate and multivariate Cox regression analyses were employed. The results showed that circulating miR-182, as well as KPS score and WHO grade, was an independent prognostic indicator for DFS (P=0.034) or OS (P=0.013).Conclusions1.Serum miR-182 expression level was significantly increased in patients with glioma, and was closely related to WHO pathological grading in patients with brain glioma.2.Serum miR-182 has a certain value in early diagnosis of gliomas, and it may be a potential tumor marker.3.The level of serum miR-182 expression and the prognosis of patients with gliomas were significantly correlated, which had a certain effect on the prognosis of the patients.SignificanceThrough the research of this topic, the relationship between the expression level of-182 miR and the pathological grading in patients with glioma was defined, and it was suggested that the serum-182 miR level had a certain significance in the early diagnosis and prognosis of gliomas.Part II THE EFFECT AND UNDERLYING MECHANISM OF MICRORNA-182 ON PROLIFERATION IN GLIOMA CELLSBackgroundGlioma is one of the most common malignant tumor in the central nervous system. The current treatment with integrated treatment mode of chemotherapy resection and postoperative glioma, but the prognosis is still poor. The survival of patients is generally about 14 months. The cause of poor prognosis of glioma is a series of malignant biological behavior of the tumor cells, including the proliferation of brain tissue surrounding the invasion and chemotherapy drug resistance. It is note that the excessive proliferation of tumor cells is an important factor leading to rapid recurrence of glioma after operation. How to effectively inhibit the excessive proliferation of glioma cells, as well as prolonging the survival of patients and improving the quality of life, which is a major problem in the field of glioma treatment. The key is to explore the molecular mechanism of excessive proliferation of glioma cells, and then to carry out effective interference and suppression, which is help for us to find a new target for glioma treatment.A number of studies have indicated that miRNAs may be involved in the proliferation, invasion and apoptosis of tumor cells, which is regulated by the UTR 3’region of the target mRNA to regulate the expression of the target gene. The abnormal expression of miRNAs has been proved to have a close correlation with the development of glioma. MiR-663 can inhibit the proliferation, migration and invasion of glioma cells by regulating its target gene TGF-β1. The abnormal expression of miR-506 can regulate its target gene IGF2BP1 to inhibit the proliferation of glioma cells. MiR-182 is an important miRNAs molecule, which is overexpressed in tumor cells, and promotes the growth and proliferation of tumor cells. Recent studies have found that miR-182 can promote proliferation by regulating its downstream target gene in some malignant tumors, such as ovarian cancer. The first part of this study has confirmed the high expression of miR-182 in serum of patients with glioma, and patients with WHO pathological grade and the clinical features of various closely related. However, there is no study on the expression of miR-182 in human glioma cells. The effect of abnormal expression on the proliferation of glioma cells and its mechanism is still unclear.In this study, we analyzed the expression of miR-182 in human glioma cell line and its effect on cell proliferation. Then we find and verify thetarget gene of miR-182, and explore the molecular biological mechanism in glioma cell proliferation, in order to provide a new target for the treatment of glioma.Objective1. To detect the expression of miR-182 in human glioma cell lines and normal astrocytes in U251, U118 and LN319.2. The effects of miR-182 on the proliferation of human glioma cell line were analyzed by the inhibition and/or overexpression miR-182 respectively.3. To search and verify a downstream target gene of miR-182, and to study the effect of inhibiting or overexpression of miR-182 on the level of downstream target gene.4. The effects of downstream target genes on the proliferation of human glioma cell.5. Interference or overexpression of miR-182 or downstream target genes, to explore the role of miR-182 in regulating the proliferation of glioma cells by regulating downstream target genes.Methods1. The expression of miR-182 in glioma cell lines and normal stellate cells.Cultured glioma cell lines U251, U118, LN319 and normal cells, then the expression of miR-182 in three glioma cell lines and normal cells were detected by qRT-PCR.2. Interference and overexpression of miR-182 respectively, to observe its effect on the proliferation of glioma cellsThe transient transfection of liposome was used to carry out transfection of U251 glioma cells, and the interference and overexpression of miR-182. Transfection efficiency was detected by qRT-PCR. WST-1 was to use to detect the proliferation of glioma cell lines after 24 hours,48 hours,72 hours and 96 hours.3. To search for and verify the target gene of miR-182, and study the influence of interference and over expression of miR-182 on target gene transcription and protein expression level.Search for a potential target gene of miR-182 using bioinformatics databases such as miRBase. The luciferase reporter gene system was used to validate the target gene. qRT-PCR and blot Western were used to detect the effect of miR-182 on the transcriptional and protein expression level of target genes.4. To study the effect of inhibiting and over expressing the target gene on the proliferation of human glioma cells.The method of transient transfection of liposome was used to carry out the transfection of human glioma cells, respectively. qRT-PCR and Western blot are used to verify the efficiency.The proliferation ability of cells was detected by WST-1 method, and its effect on the proliferation of glioma cell line was studied.5. To investigate the role and underlying mechanism of miR-182 through the regulation of FOXO3 in regulating glioma cells proliferation.To interference and overexpression of miR-182 or FOXO3, WST-1 was used for detection of various proliferation of glioma cells and to explore the molecular mechanism of miR-182 and its downstream target gene FOXO3 in glioma cell proliferation in glioma cells.Results1. The expression of miRNA-182 in glioma cell linesThe results showed that the expression of miRNA-182 in the three glioma cells (LN319 (0.1±0.05), U118 (0.08±0.03), U251 (0.24±0.05)) were significantly higher than normal astrocytes (0.06±0.01) (all P<0.01). The expression of three cell lines is also different, and we selected the U251 cells, which have the highest expression, to do the next function test.2. The influence of miRNA-182 on the proliferation of glioma cellsLiposomes transient transfection methods successfully transfected mimics and inhibitor of miRNA-182 into glioma cell line U251. Compared with the control group, miRNA-182 mimics significantly promoted the proliferation of glioma cells (P<0.01), while siRNA-miRNA-182 reduced the proliferation of glioma cell (P<0.01). After culture 48 hours, the similar trends were also observed. Therefore, we selected the cell to culture for 48 hours for the next experiments.3. To find and verily the one of target genes of miRNA-182FOXO3 was found as one of the potential target genes by searching miRBase and other database, and the forecast showed that 3’-UTR region of FOXO3 contains complementary binding sequences with miRNA-182. Subsequently, the results demonstrate that the cotransfected with miRNA-182 mimics luciferase reporter vector containing WT FOXO3 3’-UTR, cellular luciferase activity was significantly decreased (P＜0.01), results suggest that over-expression of miRNA-182 could significantly inhibit wild-type reporter plasmid luciferase activity was confirmed FOXO3 is one of its target genes regulated important downstream targets. Moreover, our results showed that over-expression of miRNA-182 could significantly down regulation the expression of FOXO3 at the levels of mRNA (44.4%■6.96%, P=0.004) and protein (38.3%±5.94%,P=0.004). while siRNA-miRNA-182 had a upregulation with FOXO3 mRNA (2.08±0.41,P=0.002), and the corresponding protein levels also raised (2.09 ± 0.38, P= 0.004).4. The effect of FOXO3 on the proliferation of human glioma cell line U251.We found a satisfying efficiency of FOXO3 on the interference and over-expression. After 48 hours transfection, interference FOXO3 can significantly increase the proliferation of glioma cells (1.40±0.04 times,P=0.006). And 48 hours after over-expression FOXO3, compared with control group, the proliferation of glioma cells significantly decreased, (23.8%±2.44%,P=0.005).5. The effect of interference and over-expression of miRNA-182 and FOXO3 on the proliferation of glioma cellsThe results suggest that glioma cell proliferation induced by over-expression of miRNA-182 can be down regulation by over-expressed FOXO3(P<0.001). The combined effects of the over-expression of miRNA-182 and siRNA-FOXO3 could make a upregulation of cell proliferation(P=0.018). Inhibition of glioma cell proliferation induced by interfere with miRNA-182 can be down regulation by siRNA-FOXO3(P=0.008). The combined effects of the over-expression of FOXO3 and interfere with miRNA-182 could significantly reduce cell proliferation (P=0.006).Conclusions1. miR-182 was highly expressed in human glioma cell line, which could significantly increase the proliferation of glioma cells.2. FOXO3 is one of the target genes of miR-182, which is involved in regulating the proliferation of glioma cells.3. miR-182 promotes the proliferation of glioma cells by down-regulating FOXO3.SignificanceThrough the research of this topic, it is clear that miR-182 regulates the downstream target gene FOXO3 to play a role in the proliferation of glioma cells. The expression of miR-182 and the expression of its target gene FOXO3 may provide a new target for glioma treatment. |
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