Knockdown of PHLDA2 promotes apoptosis and autophagy of glioma cells through the AKT/mTOR pathway.

Pleckstrin homology like domain family A member 2 (PHLDA2) is an imprinted gene expressed in placenta and has been shown to be associated with tumor progression. However, the effect of PHLDA2 on glioma cell growth has not been reported yet. Data based on TCGA database showed that PHLDA2 was up-regulated in glioma tissues. Moreover, PHLDA2 was also elevated in glioma cells. Functional assays showed that siRNA-mediated knockdown of PHLDA2 reduced cell viability of glioma cells and suppressed the cell proliferation. Cell apoptosis of glioma cells was promoted by silencing of PHLDA2 with increased Bax and decreased Bcl-2. Silencing of PHLDA2 reduced protein expression of p62, enhanced LC3 and Beclin1 to promote autophagy. Phosphorylated AKT and mTOR were down-regulated in glioma cells by interference of PHLDA2. In conclusion, downregulation of PHLDA2 inhibited glioma cell proliferation, and promoted cell apoptosis and autophagy through inactivation of AKT/mTOR signaling.

Downregulation of ZMYND11 induced by miR-196a-5p promotes the progression and growth of GBM.

ZMYND11 (zinc finger MYND-type containing 11) has been widely regarded to be involved in a variety of cancers as a potential suppressor. However, the biological role and mechanism of ZMYND11 in glioblastoma multiform (GBM) remain unknown. In this study, we found that ZMYND11 expression was remarkably decreased in GBM tissues from 20 cases and cell line (U87) compared to normal brain tissue from 10 cases (P < 0.001). Furthermore, we explored that ZMYND11 upregulation significantly suppressed U87 cells proliferation and invasion, induced cell cycle arrest and apoptosis in vitro. Subsequently, we identified increased ZMYND11 inhibited the tumor growth using tumor cells xenograft experiment on rude mice. Moreover, we explored that ZMYND11 was a new direct and functional target of miR-196a-5p in U87 via luciferase reporter assay. In addition, we confirmed the negative correlation between miR-196a-5p and ZMYND11 in GBM tissue and U87 cells by changing the expression level of miR-196a-5p with lentivirus and plasmid vector. Furthermore, we demonstrated that decreased ZMYND11 could reverse suppressive effect of downregulated miR-196a-5p on U87 by rescue experiment. Taken together, ZMYND11 was demonstrated to be a potential and extremely promising suppressor of GBM, while miRNA-196a-5p was quite an important target of treatment of GBM.

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.

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.

Downregulation of miR-95-3p inhibits proliferation, and invasion promoting apoptosis of glioma cells by targeting CELF2.

Gliomas are the most common and aggressive types of tumors in human brain, of which the prognosis remains dismal because of their biological behavior. The involvement of miRNAs in tumorigenesis of various kinds of cancers drives us to explore new miRNAs related to gliomas. We measured expression level of miR­95­3p by qRT-PCR in human glioma and non-neoplasm brain tissues and found that higher level of miR­95­3p in glioma tissues of higher grade. Biological functions of miR­95­3p on glioma cells were investigated by MTT assay, flow cytometry and transwell assay. We discovered the cell lines transfected with miR­95­3p ASO (antisense oligonucleotide) had retarded proliferation and invasion but enhanced apoptosis ability. We searched on-line tool Targetscan and selected CELF (CUGBP- and ETR-3-like family 2) as a putative target. Luciferase reporter was employed to confirm the binding sites in 3'UTR region of CELF2 for miR­95­3p. The correlation between expression of CELF2 and miR­95­3p was determined by western blotting and qRT-PCR both in cell lines and human samples. Results showed CELF2 was a direct target of miR­95­3p and expression levels of CELF2 and miR­95­3p were negatively correlated. Finally, CELF2 largely abrogated the effects of miR­95­3p on proliferation, invasion and apoptosis of glioma cells in rescue experiments, which verified the role of CELF2 in miR­95­3p regulating glioma biological behavior. In conclusion, our data suggest the expression level of miR­95­3p is positively related to glioma grade and downregulation of miR­95­3p affects proliferation, invasion and apoptosis of glioma cells by targeting CELF2. We identified miR­95­3p as a putative therapeutic target and CELF2 as a potential tumor suppressor.

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.

Concomitant microRNA-31 downregulation and radixin upregulation predicts advanced tumor progression and unfavorable prognosis in patients with gliomas.

PURPOSE: To clarify the clinical significance of microRNA-31 (miR-31) and radixin (RDX) in human glioma. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to characterize the expression patterns of miR-31 and RDX mRNA in 108 glioma and 20 normal brain tissues. The associations of miR-31 and RDX mRNA expressions with clinicopathologic factors and prognosis of glioma patients were also statistically analyzed. RESULTS: The expression levels of miR-31 in glioma tissues were significantly lower than those in normal brain tissues (P<0.001), while RDX mRNA was significantly overexpressed in glioma tissues compared with normal brain tissues (P<0.001). There was a negative correlation between miR-31 and RDX mRNA expression in glioma tissues (r=-0.69, P=0.01). Additionally, concomitant miR-31 downregulation and RDX upregulation (miR-31-low/RDX-high) was significantly associated with advanced pathological grade (P=0.001) and low Karnofsky performance score (P=0.01). Moreover, Kaplan-Meier survival and Cox regression analyses showed that the glioma patients with miR-31-low/RDX-high expression had poorest overall survival (P<0.001) and conjoined expression of miR-31-low/RDX-high was an independent prognostic indicator of glioma (P=0.01). Furthermore, subgroup analyses showed that miR-31-low/RDX-high expression was significantly associated with poor overall survival in glioma patients with high pathological grades (for grade III-IV: P<0.001). CONCLUSIONS: Our findings have implications concerning the importance of concomitant miR-31 downregulation and RDX upregulation in tumor progression and poor prognosis of patients with gliomas. A combined detection of miR-31/RDX expression may benefit us in predicting clinical outcomes of glioma patients with high pathological grades.

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.