miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells

Background

Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.

Methods

We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting.

Results

Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100β-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins.

Conclusion

microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.     

The human Fn14 receptor gene is up-regulated in migrating glioma cells in vitro and overexpressed in advanced glial tumors

Glioblastoma multiforme comprises the majority of human brain tumors. Patients with glioblastoma multiforme have poor survival rates, with an average life expectancy of <1 year. To assess possible mechanisms and to potentially target invasive glioma cells, we previously measured the gene expression profiles of glioma cells under migration-activated or passive states. One of the genes identified was Fn14, which encodes a cell surface receptor for the tumor necrosis factor superfamily member named TWEAK. In this study, we show that Fn14 gene expression is induced in migration-activated glioma cells in vitro and significantly increases according to tumor grade in vivo (P < 0.01), with highest levels in glioblastoma tissue specimens. The in situ expression pattern of Fn14 mRNA and protein was confined to primary glioma cells and the vascular endothelium, with no detection in adjacent normal brain. Conversely, TWEAK mRNA levels are low in glioblastoma samples relative to normal brain tissue. In addition, activation of the Fn14 receptor by addition of recombinant TWEAK resulted in increased glioma cell migration in vitro. These results suggest a positive role for TWEAK and Fn14 in glioma progression and indicate that Fn14 gene expression may serve as a marker for invasive glioma cells.     

MicroRNA‐137‐mediated Src oncogenic signaling promotes cancer progression

The tyrosine kinase c‐Src is frequently overexpressed and activated in a wide variety of human cancers. However, the molecular mechanisms responsible for the upregulation of c‐Src remain elusive. To examine whether microRNA‐mediated c‐Src upregulation promotes cancer progression, we screened miRNAs with complementarity to the 3′‐UTR of c‐Src mRNA. Among these miRNAs, down‐regulation of miR‐137 was tightly associated with c‐Src‐mediated tumor progression of human colon cancer cells/tissues. Re‐expression of miR‐137 in human colon cancer cells suppressed tumor growth and caused the disruption of focal contacts, suppression of cell adhesion, and invasion, although restoration of c‐Src in miR‐137‐treated cells could not fully rescue the tumor‐suppressive effect of miR‐137. We found that miR‐137 targets AKT2 and paxillin also and miR‐137‐mediated regulation of c‐Src /AKT2 is crucial for controlling tumor growth, whereas that of c‐Src/paxillin contributes to malignancy. miR‐137 suppressed Src‐related oncogenic signaling and changed the expression of miRNAs that are regulated by Src activation. miR‐137 controls the expression of c‐Src/AKT2/paxillin and synergistically suppresses Src oncogenic signaling evoked from focal adhesions. In various human cancers that harbor c‐Src upregulation, the dysfunction of this novel mechanism would serve as a critical trigger for tumor progression.     

洛伐他汀对胶质瘤干细胞增殖和凋亡的影响

BACKGROUND:Increasing evidence has shown that lovastatin with less toxicity to normal cells has crucial effects on proliferation, apoptosis and differentiation of various cancer cells. However, its roles in glioma stem cells remain unclear. OBJECTIVE:To explore the effect of lovastatin on proliferation and apoptosis of glioma stem cells. METHODS:Flow cytometric sorting was used to separate glioma stem cells from human glioblastoma cell line U87. Effects of lovastatin on the proliferation and apoptosis of glioma stem cells were determined by MTT and flow cytometry, respectively. Furthermore, expression levels of Ki67, Bax and Bcl-2 in glioma stem cells treated with lovastatin were detected using western blot analysis. RESULTS AND CONCLUSION:The CD133-positive glioma stem cells were sorted from human glioblastoma cell line U87 with a positive percentage of 85%. MTT assay showed that lovastatin inhibited the proliferation of glioma stem cells in dose (5, 10, 20 μmol/L)- and time (24, 48, 72, 96 hours)-dependent manners. Flow cytometry analysis showed that 10 μmol/L lovastatin (48 hours) induced apoptosis in glioma stem cells. In addition, the expression level of Ki67 was decreased by lovastatin treatment in a dose-dependent manner, and the Bcl-2 and Bax expression levels were reduced and increased by 10 μmol/L lovastatin treatment, respectively. In conclusion, lovastatin can inhibit cell proliferation and induce apoptosis of glioma stem cells, and lovastatin may be a potential drug for treatment of brain tumors.     

Sequential expression of miR‐182 and miR‐503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma

Genetic changes in colon cancer are known to parallel the tissue abnormalities associated with the disease, namely adenoma and adenocarcinoma. The role of microRNA dysregulation in dysplastic progression, however, is not well understood. Here, we show that miR‐182 and miR‐503 undergo sequential up‐regulation and drive the progression of colon adenoma to adenocarcinoma by cooperatively down‐regulating the tumour suppressor FBXW7. We identified that increased expression of miR‐182 is a feature of adenomas. A subsequent increase in miR‐503 expression works cooperatively with miR‐182 to induce transformation of an adenoma to adenocarcinoma. We show that introducing miR‐503 into AAC1 cells, which are derived from a benign adenoma, confers tumourigenic potential. We also demonstrated that blocking both miR‐182 and miR‐503 in HCT116 colon cancer cells resulted in increased FBXW7 expression and significantly reduced tumour size in xenograft models. We confirmed relevance of these results in patients by examining the expression levels of miR‐182 and miR‐503 in over 200 colon cancer patients with 12 year survival outcome data. Decreased patient survival was correlated with elevated expression of both miRNAs, suggesting that elevated levels of both miR‐182 and miR‐503 define a novel prognostic biomarker for colon cancer patients. In conclusion, we show that a sequential expression of miR‐182 and miR‐503 in benign adenoma cooperatively regulates the tumour suppressor FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma and miR‐182 and miR‐503 may prove to be novel therapeutic targets. Array data are available at: http://www.oncomir.umn.edu/ Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Brain angiogenesis inhibitor 1 is differentially expressed in normal brain and glioblastoma independently of p53 expression

Brain angiogenesis inhibitors (BAI) are putative transmembrane proteins containing an extracellular domain with thrombospondin type-1 repeats which can exhibit anti-angiogenic activity. BAI1 mRNA is expressed mainly in the brain, while BAI2 and BAI3 mRNAs are more widely expressed. We hypothesized that the BAI family might have anti-tumoral properties and studied the expression of BAI1 protein in normal human brain and in glioblastoma multiforme. We generated an anti-BAI1 antibody and showed that BAI1 was widely expressed in normal brain but was absent in 28 glioma cell lines and in the majority of human glioblastoma investigated. BAI1 expression did not correlate with TP53 status and we did not confirm previous findings that p53 regulates BAI1 mRNA expression in glioma cells. The finding that expression of BAI proteins may be lost during tumor formation is of special interest as restoration of their function in tumors may be of therapeutic benefit.     

miR‐221/222 cluster expression improves clinical stratification of non‐muscle invasive bladder cancer (TaT1) patients' risk for short‐term relapse and progression

Clinical heterogeneity of bladder cancer prognosis requires the identification of bladder tumors' molecular profile to improve the prediction value of the established and clinically used markers. In this study, we have analyzed miR‐221/222 cluster expression in bladder tumors and its clinical significance for patients' prognosis and disease outcome. The study included 387 tissue specimens. Following extraction, total RNA was polyadenylated at 3′‐end and reversed transcribed. SYBR‐Green based qPCR assays were performed for the quantification of miR‐221/222 expression. Extensive statistical analysis was completed for the evaluation of miR‐221/222 cluster's clinical significance. The expression of miR‐221/222 is significantly downregulated in tumors compared to normal urothelium, while ROC curve and logistic regression analysis highlighted cluster's discriminatory ability. However, miR‐222 levels were increased in muscle‐invasive (T2‐T4) compared to superficial tumors (TaT1), and in high compared to low‐grade tumors. Kaplan‐Meier survival curves and Cox regression analysis revealed the stronger risk of TaT1 patients overexpressing miR‐222 for disease short‐term relapse and progression following treatment. Moreover, multivariate Cox models highlighted the independent prognostic value of miR‐222 overexpression for TaT1 patients' poor prognosis. Finally, the analysis of miR‐222 expression improved significantly the positive prediction strength of the clinically used prognostic markers of tumor stage, grade, EORTC risk‐stratification and recurrence at the first follow‐up cystoscopy for TaT1 patients' outcome, and resulted to higher clinical net benefit following decision curve analysis. In conclusion, the expression of miR‐221/222 cluster is deregulated in bladder tumors and miR‐222 overexpression results to a superior positive prediction of TaT1 patients' short‐term relapse and progression.     

Involvement of Hif-1 in desferrioxamine-induced invasion of glioblastoma cells

Glioblastoma multiforme are highly invasive brain tumors. Experimental approaches focus on unravelling the mechanisms of invasion, this being a major reason for the poor prognosis of these tumors. Our previous results hinted towards involvement of the iron metabolism in invasion. In this study, we examined the effect of iron depletion on the invasive phenotype of glioblastoma cells. Transwell Matrigel invasion assays were used to monitor iron-dependent invasion of human glioblastoma cell lines U373MG and DBTRG05MG. Intracellular iron concentrations were modulated by applying desferrioxamine (DFO) and ferric ammonium citrate (FAC). We detected enhanced invasion of glioblastoma cells upon DFO-induced iron depletion. Treatment of cells with FAC strongly inhibited invasion. DFO treatment resulted in hypoxia-inducible factor 1 (Hif-1)-mediated induction of urokinase plasminogen activator receptor and matrix metalloproteinase 2. Further, RNA interference-mediated repression of urokinase plasminogen activator receptor inhibited DFO-induced invasion. Our data demonstrate a direct effect of DFO on Hif-1 expression resulting in activation of factors associated with ECM degradation and invasion of glioma cells. These findings caution on utilization of DFO and other iron chelators in the treatment of tumors with invasive potential.     

hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair

Homologous recombinational repair (HR) is one of the major repair systems for DNA double‐strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage‐induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.     

Molecular mechanism of SSFA2 deletion inhibiting cell proliferation and promoting cell apoptosis in glioma

Gliomas are the most common primary brain malignant tumors in humans. Glioblastoma multiforme(GBM) is the most malignant intracranial tumor with a relatively poor prognosis. There promote us to find effective anti-cancer therapies to reduce cancer mortality. By using bioinformatic analysis, we found SSFA2 as a gene with elevated expression in the glioma tissues. We detected the expression of SSFA2 in glioma tissues and in the glioma cell lines, as well as in normal brain tissues. SSFA2 expression was higher in glioma tissues, especially in glioblastoma multiforme than normal brain tissues. Subsequently, we found that down-regulate SSFA2 in glioma cell lines can regulate the cell cycle to reduce the proliferation ability and induce the early apoptosis rate in shSSFA2 cells relative to control cells. Moreover, we found that down-regulate SSFA2 in glioma cell line U87(shSSFA2-U87) inhibited the growth effectiveness compared to the control cell line U87. These result reveals us that SSFA2 may act as oncogene to promote the progression of glioma. For further research specific mechanisms of SSFA2 in gliomas, we used the gene chip to detect the downstream gene in U87. We found that 30 genes also may be as target gene of SSFA2, and we testify the protein expression by western-blot. The result reveal that IL1A, IL1B and CDK6 as target gene of SSFA2 to regulate the progression of glioma. These finding suggest that SSFA2 could be a new therapeutic target for gliomas.     

CD133 expression and cancer stem cells predict prognosis in high-grade oligodendroglial tumors

High-grade oligodendroglial tumors, that is, anaplastic oligodendroglial tumors and glioblastomas with oligodendroglial component, differ significantly in terms of prognosis and response to chemotherapy. Differentiation might be difficult because the histological differences are vague and reliable markers are not established. We correlated the presence of putative cancer stem cells (CSC) in high-grade oligodendroglial tumors (WHO grades III and IV) with clinical outcome. Tumors with favorable prognosis neither contained CSC nor did they show CD133 expression. Tumor cells resembled lineage-restricted progenitor cells with limited proliferative capacity and differentiation profile. In contrast, CD133 expression and stem cell-like tumor cells characterized tumors with poor prognosis. They showed neurosphere-like growth, differentiated into cells of all neural lineages, and were tumorigenic in nude mice. In our series, CSC and expression of CD133 predicted the clinical course of disease better than the histological grading. To confirm these results, we retrospectively analyzed 36 high-grade oligodendroglial tumors. Again, CD133 expression indicated shorter survival and predicted clinical outcome more reliable than the histological assessment. Our data show that detection of CSC and expression of CD133 is predictive of prognosis in high-grade oligodendroglial tumors. The presence or absence of CD133(+) CSC might explain the crucial biological difference between WHO grade III and IV oligodendroglial tumors.     

4-1BBL分子在脑胶质瘤组织中的表达及其临床意义

目的:探讨共刺激分子4-1BBL(CD137L)在胶质瘤组织中的表达及其临床意义。方法:免疫组化方法检测4-1BBL在82例脑胶质瘤组织中的表达,并分析其与病理分级、患者年龄、性别及生存时间的关系。结果:正常3例脑组织标本未见4-1BBL表达。82例胶质瘤标本31例4-1BBL阳性,阳性表达率为37.8%。统计学提示组织标本中4-1BBL表达与病理级别无明显相关性,与年龄有关(P0.05),4-1BBL阳性患者生存期更长(P0.05)。结论:4-1BBL在部分胶质瘤组织中有表达,其表达对判断胶质瘤患者的预后有一定的参考价值。