Increased expression of microRNA-17 predicts poor prognosis in human glioma.

AIM: To investigate the clinical significance of microRNA-17 (miR-17) expression in human gliomas. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-17 in 108 glioma and 20 normal brain tissues. The associations of miR-17 expression with clinicopathological factors and prognosis of glioma patients were also statistically analyzed. RESULTS: Compared with normal brain tissues, miR-17 expression was significantly higher in glioma tissues (P < 0.001). In addition, the increased expression of miR-17 in glioma was significantly associated with advanced pathological grade (P = 0.006) and low Karnofsky performance score (KPS, P = 0.01). Moreover, Kaplan-Meier survival and Cox regression analyses showed that miR-17 overexpression (P = 0.008) and advanced pathological grade (P = 0.02) were independent factors predicting poor prognosis for gliomas. Furthermore, subgroup analyses showed that miR-17 expression was significantly associated with poor overall survival in glioma patients with high pathological grades (for grade III~IV: P < 0.001). CONCLUSIONS: Our data offer the convinced evidence that the increased expression of miR-17 may have potential value for predicting poor prognosis in glioma patients with high pathological grades, indicating that miR-17 may contribute to glioma progression and be a candidate therapeutic target for this disease.

MicroRNA-182 targets protein phosphatase 1 regulatory inhibitor subunit 1C in glioblastoma.

Glioblastoma (GBM) is an incurable cancer, with mean post-diagnosis survival time of 14-16 months. Metagenomic analysis by The Cancer Genome Atlas (TCGA) program has identified microRNA-182-5p (miR-182-5p or miR-182) as the only miRNA associated with favorable disease prognosis and temozolomide (TMZ) susceptibility. Previous reports have indicated that miR-182 down regulates expression of BCL2L12, c-MET, and HIF2A. However, other messenger RNA (mRNA) targets of miR-182 have not been validated which would explain its association with a favorable disease prognosis. In situ analysis revealed that protein phosphatase 1 regulatory inhibitor subunit 1C (PPP1R1C) is a putative target of miR-182. PPP1R1C protein and RNA expression as assessed by tissue microarray and quantitative real time PCR, respectively, was inversely correlated to miR-182 expression in glioblastoma patients and in the metastatic glioblastoma cell line U87-MG. Reporter assays using PPP1R1C 3' untranslated region (UTR) showed that miR-182 can interact with the wild-type but not a miR-182-5-seed mutant. Ectopic expression of miR-182 mimic in the U87-MG cell line significantly decreased proliferation as well as suppressed in vitro migration and invasion. Opposite observations were made when the non-malignant neuronal cell line HCN-2 was transfected with anti-miR-182 antagomir. The miR-182 mimic or siRNA targeting PPP1R1C induced TMZ susceptibility indicating that decreased susceptibility to TMZ in GBM patients might be attributed to high expression of PPP1R1C. Inverse correlation of PPP1R1C mRNA and miR-182 levels in 20 GBM patients confirmed the same. Cumulatively, our results indicate that loss of miR-182 leads to increased expression of PPP1R1C which in part explain disease progression and resistance to TMZ therapy.

Tetramethylpyrazine induces differentiation of human umbilical cord-derived mesenchymal stem cells into neuron-like cells in vitro.

The present study evaluated the ability and optimal concentration of tetramethylpyrazine (TMP) to induce human umbilical cord-derived mesenchymal stem cells (hUMSCs) to differentiate into neuron­like cells in vitro. Human umbilical cords from full-term caesarean section patients were used to obtain hUMSCs by collagenase digestion after removal of the umbilical artery and vein. The surface antigen expression profile of cultured hUMSCs was monitored by flow cytometry. After amplification, cells of the 5th passage were divided into experimental groups A­C treated with TMP at 4.67, 2.34 and 1.17 mg/ml, respectively, in low glucose­Dulbecco's Modified Eagle's Medium (L­DMEM) (induction medium), while group D (control) was exposed to L­DMEM culture medium only. Differentiation of hUMSCs into neuron­like cells and morphological changes were observed every 0.5 h with an inverted phase contrast microscope for 6 h. After the 6­h induction period, proportions of cells expressing neuronal markers neuron­specific enolase (NSE), neurofilament protein (NF­H) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The optimal concentration of TMP was selected on the basis of neuron­like cell positive rate. Western blotting and RT­polymerase chain reaction were applied to detect the expression of NSE, NF­H, and GFAP of the group of optimal concentration in each point­in­time. Results showed that most primary cells were adherent 12 h after seeding and first appeared as diamond or polygon shapes. Thereafter, they gradually grew into long spindle­shaped cells and finally in a radiating or swirling pattern. The cells maintained a strong proliferative capacity after continuous passage. Flow cytometry analysis of cultured hUMSCs at the 3rd, 5th and 10th passages expressed CD73, CD90 and CD105, but not CD11b, CD19, CD34, CD45 or human leukocyte antigen­DR. After 6 h of TMP treatment, typical neuron­like cells with many protrusions connected into a net­like pattern were observed in all experimental groups. These neuron­like cells were positive for NSE and NF­H, but negative for GFAP. Among the tested treatment groups, group A with TMP at 4.67 mg/ml had the highest expression of NSE and NF­H. By contrast, no change was found after induction in the control group. The mRNA expression of cells expressing neuronal markers as well as GAPDH was observed, with the relative NSE transcript levels of 0, 1.303±0.031, 1.558±0.025, 1.927±0.019 and 2.415±0.033 after 0, 1, 2, 4 and 6 h of treatment, respectively; the mRNA expression of NH­F was 0, 1.429±0.025, 1.551±0.024, 1.930±0.042 and 1.398±0.014 after 0, 1, 2, 4 and 6 h of treatment, respectively. There was no expression of GFAP before or after induction and all the groups showed high expression of GAPDH at each time point. Protein expression was also observed on cells expressing neuronal markers as well as GAPDH at each time point. The protein expression of NSE was 0, 0.717±0.097, 0.919±0.056, 1.097±0.143 and 1.157±0.055 in proper order; the protein expression of NH­F was 0, 0.780±0.103, 0.973±0.150, 1.053±0.107 and 0.753±0.094 in proper order. There was no expression of GFAP before or after induction, and all the groups showed high expression of GAPDH at each tested time point. Our results demonstrated that TMP can induce hUMSCs to differentiate into neuron­like cells effectively with the optimal concentration of 4.67 mg/ml. After induction, the NSE and NF-H of the neuron-like cells were positive, but the GFAP-2 was negative.

Cytoplasmic polyadenylation element-binding protein 4 is highly expressed in human glioma.

Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) is a highly conserved, sequence-specific RNA-binding protein that recruits translational repression or cytoplasmic polyadenylation machinery to target mRNAs. Recent studies have shown that CPEBs are expressed in somatic tissues and have essential functions supporting tumor growth, vascularization, and invasion. Overexpression of CPEB4 has been reported in pancreatic ductal adenocarcinoma and is associated with poor prognoses. However, whether CPEB4 plays a role in the tumorigenesis of gliomas is unknown. Here, we analyzed the expression of CPEB4 in gliomas. The expression profiles of CPEB4 mRNA and protein in nine normal brain tissues and 63 gliomas were detected using immunohistochemistry, real-time PCR, and western blotting. CPEB4-positive expression was significantly correlated with the pathological grade of glioma; abundant expression was observed in high-grade gliomas, whereas little or no expression was observed in normal astrocytes. Immunohistochemistry staining indicated that CPEB4 was mainly localized in the cytoplasm. In addition, CPEB4 was more highly expressed in U87 glioma cells than in U251 cells. CPEB4 expression significantly correlated with the grade in clinical gliomas. This study suggested that CPEB4 might play a role in the pathogenesis of glioma.

Monosialoteterahexosyl ganglioside induces the differentiation of human umbilical cord-derived mesenchymal stem cells into neuron-like cells.

In the present study, human umbilical cord-derived mesenchymal stem cells (hUMSCs) were investigated for their potential to be induced to differentiate in vitro into neuron-like cells by monosialoteterahexosyl ganglioside (GM1). Mononuclear cells obtained from umbilical cords from women with full-term pregnancies whose babies were delivered by cesarean section were cultivated in vitro and their surface antigen expression profiles were monitored. Following amplification, the cells were divided into 5 groups, of which 4 (groups A-D) were treated with GM1 at doses of 50, 100, 150 and 200 µg/ml, respectively. The control (group E) was treated with the vehicle only. The ability of GM1 to induce the differentiation of the hUMSCs into neuron-like cells was monitored for 6 h. The expression levels of microtubule-associated protein-2 (MAP-2), neurofilament protein (NF-H) and glial fibrillary acidic protein (GFAP) were measured by immunohistochemistry. Following exposure to GM1, the hUMSCs first appeared to have a diamond or polygonal shape and gradually grew into long spindle-shaped cells, finally exhibiting a radiating or swirling pattern. The cells maintained a strong proliferative capacity after continuous passage. Flow cytometry revealed that the hUMSCs expressed CD73, CD90 and CD105 up to passage 10, but not CD11b, CD19, CD34, CD45 or HLA-DR. Treatment with GM1 for 6 h led to the appearance of neuron-like cells with oval-shaped cell bodies and protruding neurites. These neuron-like cells were positive for MAP-2 and NF-H, but negative for GFAP expression. No changes in the expression of these markers were observed in the control group. Thus, the findings of the present study demonstrate that GM1 effectively induces hUMSCs to differentiate into neuron-like cells.

Decreased expression of microRNA-206 correlates with poor clinical outcome in patients with malignant astrocytomas.

MicroRNA-206 (miR-206) has been proved to function as a tumor suppressor in several types of human malignant cancers. More recently, it has been demonstrated that the ectopic expression of miR-206 significantly inhibited the proliferation and promoted apoptosis at the early stages in glioma cell U343. In order to investigate the clinical significance of miR-206 expression in human astrocytoma, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-206 in 108 astrocytoma and 20 normal brain tissues. As the results, the expression levels of miR-206 in astrocytoma tissues were significantly lower than those in normal brain tissues (P < 0.001). Additionally, the decreased expression of miR-206 in astrocytoma was significantly associated with advanced pathological grade (P = 0.008), low Karnofsky performance score (KPS, P = 0.02), and large tumor size (P = 0.01). Moreover, Kaplan-Meier survival and Cox regression analyses showed that low miR-206 expression (P < 0.001) and advanced pathological grade (P = 0.02) were independent factors predicting poor prognosis for astrocytomas. In conclusion, this is the first report of the differential expression of miR-206 in human astrocytoma tissues. MiR-206 could be a valuable marker of astrocytoma progression and low miR-206 expression is associated with poor overall survival in patients with malignant astrocytomas.

Combined aberrant expression of microRNA-214 and UBC9 is an independent unfavorable prognostic factor for patients with gliomas.

MicroRNA-214 (miR-214) plays an important role in tumor cell proliferation, migration and invasion, as well as tumor angiogenesis. Ubiquitin-conjugating enzyme 9 (UBC9) is implicated in regulating several critical cancer-related pathways. Recent study has demonstrated that miR-214 reduction may facilitate UBC9 expression and may be involved in the regulation of glioma cell proliferation. The aim of this study was to clarify the clinical significance of miR-214 and UBC9 in human glioma, which has not been fully elucidated. Quantitative real-time polymerase chain reaction analysis was used to characterize the expression patterns of miR-214 and UBC9 mRNA in 108 glioma and 20 normal brain tissues. The associations of miR-214 and UBC9 mRNA expressions with clinicopathological factors and prognosis of glioma patients were also statistically analyzed. Compared with normal brain tissues, the expression levels of miR-214 and UBC9 mRNA in glioma tissues were significantly downregulated and upregulated, respectively (both P < 0.001). There was a negative correlation between miR-214 and UBC9 mRNA expression in glioma tissues (r = -0.61, P = 0.01). Additionally, the combined miR-214 downregulation and UBC9 upregulation (miR-214-low/UBC9-high) was significantly associated with advanced pathological grade (P = 0.008). Moreover, Kaplan-Meier survival and Cox regression analyses showed that the glioma patients with miR-214-low/UBC9-high expression had poorest overall survival (P < 0.001) and conjoined expression of miR-214-low/UBC9-high was an independent prognostic indicator of glioma (P = 0.01). Furthermore, subgroup analyses showed that miR-214-low/UBC9-high expression was significantly associated with poor overall survival in glioma patients with high pathological grades (for grade III-IV: P < 0.001). This prospective study offers the convincing evidence for the first time that miR-214 and its target gene UBC9 may contribute to the development and the clinical outcome of glioma, and are valuable prognostic factors for glioma patients. A combined detection of miR-214/UBC9 expression may benefit us in predicting the prognosis of patients with advanced gliomas.

Expression and clinical significance of microRNA-326 in human glioma miR-326 expression in glioma.

As a suppressor of Hedgehog signaling pathway, microRNA-326 (miR-326) has been demonstrated to control the development of cerebellar neuronal progenitor and tumor cells. More recently, it has been reported that miR-326 was down-regulated in glioblastoma tissues and might regulate the metabolic activity of glioma and glioma stem cells, suggesting the involvement of miR-326 in tumorigenesis and progression of gliomas. However, the role of miR-326 in human glioma has not been clearly understood. Therefore, the aim of this study was to investigate the clinical significance of miR-326 expression in human glioma. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-326 in 108 glioma and 20 normal brain tissues. The associations of miR-326 expression with clinicopathological factors and prognosis of glioma patients were also statistically analyzed. The expression levels of miR-326 in glioma tissues were significantly lower than those in normal brain tissues (P < 0.001). Additionally, the decreased miR-326 expression in glioma was significantly associated with advanced pathological grade (P = 0.01) and low Karnofsky performance score (KPS, P = 0.03). Moreover, Kaplan-Meier survival and Cox regression analyses showed that low expression of miR-326 (P = 0.01) and advanced pathological grade (P = 0.02) were independent factors predicting poor prognosis for gliomas. Furthermore, subgroup analyses showed that miR-326 expression was significantly associated with poor overall survival in glioma patients with high pathological grades (for grade III-IV: P < 0.001). Down-regulation of miR-326 may have potential value for predicting clinical outcomes in glioma patients with high pathological grades, suggesting that miR-326 is an important candidate tumor suppressor, and its down-regulated expression may contribute to glioma progression.

Over-expression of wild-type p53-induced phosphatase 1 confers poor prognosis of patients with gliomas.

Wild-type p53-induced phosphatase 1 (Wip1) is a member of the protein phosphatase 2C family, which is characterized by distinctive oncogenic properties. Overexpression of Wip1 is observed in certain types of human tumors that are associated with significantly poor prognosis. This study aimed to detect the expression of Wip1 in gliomas and to analyze its prognostic value in the patients. Wip1 mRNA and protein expression profiles in 81 gliomas and 15 normal brain tissues were detected using RT-PCR, Western blot and immunohistochemistry. The specimens were stained with proliferating cell nuclear antigen (PCNA) and p53 and evaluated using immunohistochemistry. Detailed clinical and demographic information of patients were retrospectively collected until 5years post-operation. Kaplan-Meier survival and Cox's regression analyses were performed to evaluate the prognosis of patients. Wip1-positive expression was observed in the majority of glioma tissues, whereas no Wip1 expression was detected in the normal brain tissues. Wip1-positive expression significantly correlated with glioma histological grade. The PCNA index was higher in the Wip1-positive group compared to that in the Wip1-negative group. A univariate analysis and log-rank test indicated that statistically significant association between Wip1 expression and the lower overall survival rate in the patients with glioma. A multivariate analysis also indicated a statistically significant association between increased Wip1 expression and lower overall survival rate. Our results suggest that Wip1 may be related to pathological diagnosis and prognosis evaluation for malignant gliomas.