Human Glioma Tissues Research Articles

Knockdown of SOX2OT inhibits the malignant biological behaviors of glioblastoma stem cells via up-regulating the expression of miR-194-5p and miR-122

BackgroundAccumulating evidence has highlighted the potential role of long non-coding RNAs (lncRNAs) in the biological behaviors of glioblastoma stem cells (GSCs). Here, we elucidated the function and possible molecular mechanisms of the effect of lncRNA-SOX2OT on the biological behaviors of GSCs.ResultsReal-time PCR demonstrated that SOX2OT expression was up-regulated in glioma tissues and GSCs. Knockdown of SOX2OT inhibited the proliferation, migration and invasion of GSCs, and promoted GSCs apoptosis. MiR-194-5p and miR-122 were down-regulated in human glioma tissues and GSCs, and miR-194-5p and miR-122 respectively exerted tumor-suppressive functions by inhibiting the proliferation, migration and invasion of GSCs, while promoting GSCs apoptosis. Knockdown of SOX2OT significantly increased the expression of miR-194-5p and miR-122 in GSCs. Dual-luciferase reporter assay revealed that SOX2OT bound to both miR-194-5p and miR-122. SOX3 and TDGF-1 were up-regulated in human glioma tissues and GSCs. Knockdown of SOX3 inhibited the proliferation, migration and invasion of GSCs, promoted GSCs apoptosis, and decreased TDGF-1 mRNA and protein expression through direct binding to the TDGF-1 promoter. Over-expression of miR-194-5p and miR-122 decreased the mRNA and protein expression of SOX3 by targeting its 3’UTR. Knockdown of TDGF-1 inhibited the proliferation, migration and invasion of GSCs, promoted GSCs apoptosis, and inhibited the JAK/STAT signaling pathway. Furthermore, SOX3 knockdown also inhibited the SOX2OT expression through direct binding to the SOX2OT promoter and formed a positive feedback loop.ConclusionThis study is the first to demonstrate that the SOX2OT-miR-194-5p/miR-122-SOX3-TDGF-1 pathway forms a positive feedback loop and regulates the biological behaviors of GSCs, and these findings might provide a novel strategy for glioma treatment.

Significance of perivascular tumour cells defined by CD109 expression in progression of glioma.

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol-anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower-grade glioma (World Health Organization grade II/III) by clinicopathological and whole-genome sequencing analysis of tissues from human glioma. The importance of CD109-positive perivascular tumour cells was confirmed not only in human lower-grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109-positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MAGEA6 promotes human glioma cell survival via targeting AMPKα1

Melanoma antigen A6 (MAGEA6)/TRIM28 complex is a cancer-specific ubiquitin ligase, which degradates tumor suppressor protein AMP-activated protein kinase (AMPK). We show that MAGEA6 is uniquely expressed in human glioma tissues and cells, which is correlated with AMPKα1 downregulation. It is yet absent in normal brain tissues and human astrocytes/neuronal cells. MAGEA6 knockdown by targeted-shRNA in glioma cells restored AMPKα1 expression, causing mTORC1 in-activation and cell death/apoptosis. Reversely, AMPKα1 knockdown or mutation ameliorated glioma cell death by MAGEA6 shRNA. In vivo, Glioma xenograft tumor growth in mice was largely inhibited following expressing MAGEA6 shRNA. AMPKα1 upregulation and mTORC1 inhibition were observed in MAGEA6 shRNA-bearing xenograft tissues. Collectively, MAGEA6 promotes glioma cell survival possibly via targeting AMPKα1.

MiR-320 inhibits the growth of glioma cells through downregulating PBX3

BackgroundMiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3.MethodsTwenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3′ UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated.ResultsMiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3′ UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation.ConclusionMiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.

Silencing Pre-B-cell leukemia homeobox 3 decreases the proliferation of human glioma cells in vitro and in vivo.

Among primary brain tumors, gliomas are the most common and most aggressive, with a poor prognosis and limited treatment options. Thus, it is essential to determine the mechanisms involved in glioma development to develop effective therapies for glioma patients. Pre-B-cell leukemia homeobox 3 (PBX3), a critical member of the PBX family, is frequently overexpressed in multiple human malignancies. However, the expression patterns and biological functions, as well as the involved molecular functions of PBX3 in human gliomas remain largely unknown. In this study, we demonstrate that PBX3 expression is increased in both human glioma tissues and cell lines compared with their normal counterparts. These results suggested that PBX3 might be involved in glioma progression. Thus, the role of PBX3 in glioma cell proliferation was investigated using genetic knockdown and overexpression methods. The results showed that PBX3 knockdown inhibited glioma cell proliferation and induced apoptosis, while PBX3 overexpression significantly promoted glioma cell proliferation. Mechanistically, we found that PBX3 promoted cell proliferation by modulating cell cycle progression. A xenograft LN229 model was used to confirm that PBX3 depletion decreased tumor growth in vivo. In summary, our findings reveal that PBX3 may be a potential therapeutic target in gliomas.

LncRNA-TP53TG1 Participated in the Stress Response Under Glucose Deprivation in Glioma.

Gliomas are the most common brain tumors of the center nervous system. And long non-coding RNAs (lncRNAs) are non-protein coding transcripts, which have been considered as one type of gene expression regulator for cancer development. In this study, we investigated the role of lncRNA-TP53TG1 in response to glucose deprivation in human gliomas. The expression levels of TP53TG1 in glioma tissues and cells were analyzed by qRT-PCR. In addition, the influence of TP53TG1 on glucose metabolism related genes at the mRNA level during both high and low glucose treatment was detected by qRT-PCR. MTT, clonogenicity assays, and flow cytometry were performed to detect the cell proliferation and cell apoptosis. Furthermore, the migration of glioma cells was examined by Transwell assays. The expression of TP53TG1 was significantly higher in human glioma tissues or cell lines compared with normal brain tissue or NHA. Moreover, TP53TG1 and some tumor glucose metabolism related genes, such as GRP78, LDHA, and IDH1 were up-regulated significantly in U87 and LN18 cells under glucose deprivation. In addition, knockdown of TP53TG1 decreased cell proliferation and migration and down-regulated GRP78 and IDH1 expression levels and up-regulated PKM2 levels in U87 cells under glucose deprivation. However, over-expression of TP53TG1 showed the opposite tendency. Moreover, the effects of TP53TG1 were more remarkable in low glucose than that in high glucose. Our data showed that TP53TG1 under glucose deprivation may promote cell proliferation and migration by influencing the expression of glucose metabolism related genes in glioma. J. Cell. Biochem. 118: 4897-4904, 2017. © 2017 Wiley Periodicals, Inc.

Nuclear factor-kappa B1 inhibits early apoptosis of glioma cells by promoting the expression of Bcl-2

Glioma is one of the most common types of adult primary brain tumors, and the underlying molecular mechanisms still remain unclear. Nuclear factor-kappa B1 (NF-κB1) is involved in a variety of malignancies and is widely expressed in malignant tumors. However, the expression of NF-κB1 in different grades of glioma, the correlation between NF-κB1 and Bcl-2 expressions in gliomas, and the research between NF-κB1 and early apoptosis of glioma cells have not been reported so far. In this study, the expression level of NF-κB1 in 31 human glioma tissues and six nonneoplastic brain tissues was determined using quantitative real-time polymerase chain reaction. Results showed that the expression of NF-κB1 in human glioma tissues and glioma cell lines, SHG44 and U87, was significantly higher compared to noncancerous brain tissues and that the expression increased with increasing degrees of tumor malignancy. Similar results were demonstrated with the expression of Bcl-2 in the same human glioma specimens. Flow cytometry results showed that inhibition of NF-κB1 expression significantly promoted apoptosis of SHG44 and U87 in human glioma cells. Western blot analysis further confirmed decreased expression of Bcl-2 protein after inhibition of NF-κB1 protein expression. Taken together, NF-κB1 overexpression inhibits early apoptosis of glioma cells and high expression of NF-κB1 promotes the expression of antiapoptotic gene Bcl-2. Therefore, our study results provide a theoretical basis for antiapoptotic mechanism of tumor cells in association with NF-κB1.

FERMT3 contributes to glioblastoma cell proliferation and chemoresistance to temozolomide through integrin mediated Wnt signaling.

FERMT3, also known as kindlin-3, is one of three kindlin family members expressed in mammals. Kindlins are cytosolic, adaptor proteins that are important activators and regulators of integrin function. They have also been shown to play critical roles in the development and progression of various cancers. In the present study, we hypothesized that FERMT3 would enhance glioblastoma multiforme (GBM) cell survival. Indeed, expression level analyses showed significant FERMT3 upregulation in human glioma tissues as compared to normal brain tissues. The effect was particularly pronounced in high-grade gliomas. We then demonstrated that FERMT3 knockdown suppresses glioma cell proliferation and chemoresistance to temozolomide (TMZ). To determine the mechanism by which FERMT3 enhances glioma cell proliferation and chemoresistance, we examined the effects of FERMT3 on integrin activation and Wnt/β-catenin signaling. Through the use of western blot assays and TOPflash and FOPflash plasmid transfection into glioma cells lines, we demonstrated that FERMT3 regulates glioma cell activity through integrin-mediated Wnt/β-catenin signaling. These results suggest that FERMT3 activates integrin activity in high-grade gliomas to enhance glioma cell survival and chemoresistance. The present study thus indicates a potential role for FERMT3 as a genetic target in the treatment of GBM.

PCDH8 inhibits glioma cell proliferation by negatively regulating the AKT/GSK3β/β-catenin signaling pathway.

Protocadherin-8 (PCDH8), a member of the protocadherin superfamily of proteins, is frequently lost in numerous types of cancer. However, the role that PCDH8 serves in human glioma, and the molecular mechanisms underlying this, remain unclear. Data from the present study demonstrated that the expression levels of PCDH8 mRNA and protein were significantly decreased in human glioma tissue compared with normal brain tissue. This suggested that PCDH8 is associated with the development of glioma. Thus, the role of PCDH8 in glioma cell proliferation was investigated by silencing and overexpressing PCDH8 in U251 glioma cells. Overexpression of PCDH8 significantly inhibited glioma cell proliferation, while silencing of PCDH8 using small interfering RNA promoted glioma cell proliferation. Restoration of PCDH8 decreased phosphorylated (p)-Rac-α serine/threonine-protein kinase (AKT) [Threonine (T)308/Serine (S)473] and p-glycogen synthase kinase-3β (p-GSK3β) (S9) protein expression, thereby reducing the level of β-catenin when compared with the control. By contrast, silencing of PCDH8 increased levels of p-AKT (T308/S473) and p-GSK3β (S9), thereby increasing the level of β-catenin. In conclusion, the results of the present study suggested that PCDH8 suppressed glioma cell proliferation, and that the loss of PCDH8 may stimulate the proto-oncogene Wnt/β-catenin signaling pathway and therefore promote glioma cell proliferation.

MicroRNA-590-3p enhances the radioresistance in glioblastoma cells by targeting LRIG1.

microRNA (miR)-590 has been found to serve potential roles in cancer development; however, the expression and function of miR-590 in human gliomas remains to be elucidated. The present study aimed to investigate the expression of miR-590 in human glioma tissues and radioresistant human glioblastoma cells (U251R), and to determine the effect and related molecular mechanism of miR-590-3p on the radiosensitivity of U251R cells in vitro. The results from reverse transcription-quantitative polymerase chain reaction indicated that miR-590-3p was upregulated in human glioma tissues and radioresistant human glioblastoma cells, and that miR-590-3p expression was higher in high grade than in low grade gliomas. In vitro experiments revealed that the miR-590-3p inhibitor enhanced the radiosensitivity of U251R cells by suppressing cell viability, decreasing colony formation capacity and increasing cell apoptosis rate, as demonstrated by MTT, colony formation and flow cytometry analyses. A luciferase reporter assay demonstrated that leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) was a direct target of miR-590-3p. Furthermore, it was demonstrated that the effect of miR-590-3p suppression on cell viability, colony formation capacity and cell apoptosis rate was attenuated by the knockdown of LRIG1 in the U251R cells. In conclusion, the present study revealed that miR-590-3p was upregulated in human glioma tissues and radioresistant human glioblastoma cells, and miR-590-3p contributes to the radioresistance of human glioblastoma cells by directly targeting LRIG1. These findings may provide potential therapeutic strategies to prevent radioresistance in human gliomas.

Epigenetically controlled Six3 expression regulates glioblastoma cell proliferation and invasion alongside modulating the activation levels of WNT pathway members.

Glioma is the most common primary brain tumor in adults. Six3 is a human homologue of the highly conserved sine oculis gene family and essential transcription regulatory factor in process of eye and fetal forebrain development. However, little is known about the role of Six3 in human tumorigenesis. The aim of this study is to investigate the methylation/expression of Six3 and reveal its function and action mechanism in glioma. Our results showed that Six3 was down-regulated in human glioma tissues and human glioma SHG-44, U251, SF126 and U373-MG cells compared with the normal tissues. And the down-regulation of Six3 was associated with the methylation of its promoter. Glioma U251 cells lacked endogenous Six3. Treatment with demethylating agent (5-aza-2'-deoxycytidine) or exogenous expression of Six3 restored Six3 production and resulted in suppression of cell cycle G1/S transition, proliferation and invasion and down-regulation of the expression of Wnt1, p-GSK3-β, β-catenin and cyclin D1 in glioma U251 cells. However, knockdown of Six3 in SHG-44 cells, which have relative higher baseline level of Six3, resulted in an opposite action. These results demonstrate that Six3 silence or loss in glioma is induced by its promoter hypermethylation and Six3 down-regulation contributes to proliferation and invasion of glioma. And this process is involved in activation of Wnt/β-catenin pathway. Six3 play a suppressor role in the initiation and progression of human glioma and potentially serve as a target for the diagnosis and treatment of human glioma.

MicroRNA-211 expression is down-regulated and associated with poor prognosis in human glioma.

Accumulating evidence has supported the role of microRNAs in the initiation and development of malignant tumors. MicroRNA-211 (miR-211), which was reported to involve in diverse physiological activities in several cancers, was investigated for its expression in human glioma and adjacent normal brain tissues, as well as its correlation with patient prognosis. Glioma tissues and adjacent normal brain tissues were obtained from 82 patients who underwent surgical resection, and quantitative real-time polymerase chain reaction was performed to assess the expression level of miR-211. Here, we found that miR-211 was significantly decreased in glioma tissues compared with adjacent normal brain tissues (glioma, 3.52 ± 0.14 vs. normal, 4.96 ± 0.17, p < 0.001), and inversely associated with ascending WHO classification (grade III-IV, 3.16 ± 0.21 vs. grade I-II, 4.22 ± 0.26, p < 0.001). Then, the correlation of miR-211 with clinicopathological factors was investigated by Pearson's Chi square test, indicating that miR-211 might be a potential biomarker to predict the malignant status of glioma. Further, Kaplan-Meier curves with log-rank analysis were carried out to determine the relationship between miR-211 expression level and the overall survival rate of glioma patients. Our data showed that there was a close correlation between down-regulated miR-211 and shorter survival time in 82 patients (p = 0.026). Finally, the multivariate Cox regression analysis indicated that WHO grade (HR = 2.437, 95% CI 1.251-4.966, p = 0.007), KPS (HR = 2.215, 95% CI 1.168-4.259, p = 0.016), and miR-211 expression level (HR = 3.614, 95% CI 2.152-6.748, p < 0.001) were considered as independent risk factors for glioma prognosis. These results suggested that lower miR-211 expression might be a marker for poor prognosis of glioma patients.

Upregulation of long noncoding RNA HOXA-AS3 promotes tumor progression and predicts poor prognosis in glioma.

Long noncoding RNAs (lncRNAs) have recently emerged as new potentially promising therapeutic targets in many cancers. However, their prognostic value and biological functions associated with glioma remain to be elucidated. Here, High-throughput RNAseq was performed to detect the expression profiles of lncRNAs in 325 human glioma tissues. It was shown that a novel lncRNA HOXA-AS3 was one of the most significantly upregulated lncRNAs in glioma tissues. Quantitative PCR further verified the increased expression of HOXA-AS3 in patient samples and glioma cell lines. Uni and Multivariate Cox regression analysis revealed that HOXA-AS3 was an independent prognostic factor in glioma patients. Gene set enrichment analysis indicated that the gene sets correlated with HOXA-AS3 expression were involved in cell cycle progression and E2F targets. Functionally, HOXA-AS3 silencing resulted in proliferation arrest by altering cell cycle progression and promoting cell apoptosis, and impaired cell migration in glioma cells. Furthermore, the growth-inhibiting effect of HOXA-AS3 knockdown was also demonstrated in Xenograft mouse model. Our results highlight the important role of HOXA-AS3 in glioma progression, and indicate that HOXA-AS3 may be served as a valuable prognostic biomarker for glioma.

Phase I trial of dimethyl fumarate, temozolomide, and radiation therapy in glioblastoma multiforme.

2060 Background: Evidence is increasing for altered immune responses in malignant gliomas. Tumor-associated microglia/macrophages infiltrate human glioma tissue and produce cytokines that promote glioma growth, invasion and angiogenesis. Dimethyl fumarate (DMF), approved for relapsing-remitting multiple sclerosis, is toxic to in vitro activated microglial cells. Based on pre-clinical data demonstrating synergism with radiation (RT) and temozolomide (TMZ), we conducted a phase I study of DMF in patients with newly-diagnosed glioblastoma (GBM) in combination with RT and TMZ. Methods: Using a standard 3+3 dose escalation design (3 dose levels: 120 mg bid, 240 mg bid and 240 mg tid), newly-diagnosed GBM patients received daily DMF with RT (60 Gy) and concurrent TMZ 75 mg/m2 daily, followed by adjuvant DMF (continuously) and TMZ for up 6 maintenance cycles (150-200 mg/m2 on days 1-5 of each 28 day cycle). The maximum tolerated dose (MTD) was defined as the dose with ≤ 1/6 dose-limiting toxicities (DLT). The MTD was determined by evaluation of DLTs during the first 6 weeks of therapy. Results: Twelve patients were treated at the three dose levels, and no DLT was identified. There were no unexpected toxicities. The most common toxicities were lymphopenia (11 grade 3/4 events) and thrombocytopenia (2 grade 3/4 events). The only grade 3/4 non-heme toxicity was a grade 3 hemorrhoid event. Of the 12 evaluable patients, one remains on active treatment in maintenance phase. Three patients completed all treatment (concurrent and maintenance) with stable disease. Two patients had a partial response (RANO criteria) but then experienced disease progression during maintenance. Five patients had disease progression during study treatment and one patient chose to withdraw from the study during maintenance. Conclusions: These data suggest that DMF may be safely combined with RT and TMZ in GBM patients. A phase II study is under consideration. Clinical trial information: NCT02337426.

PAQR3 inhibits the proliferation, migration and invasion in human glioma cells

Progestin and AdipoQ Receptor 3 (PAQR3), a member of the PAQR family, is down-regulated in several types of cancers and has been closely associated with tumor progression and development. However, little is known about the functions of PAQR3 in the tumorigenesis of human glioma. Therefore, in this report, we investigated the role of PAQR3 in human glioma. Our results showed that the expression of PAQR3 was significantly reduced in human glioma tissues and cell lines. PAQR3 overexpression inhibited the proliferation of glioma cells in vitro and attenuated tumor xenograft growth in vivo. In addition, PAQR3 overexpression suppressed the migration and invasion of glioma cells, as well as prevented the EMT process. Mechanistic studies demonstrated that PAQR3 overexpression significantly down-regulated the levels of phosphorylated PI3K and Akt in U251 cells. In conclusion, these results demonstrated that PAQR3 inhibited the proliferation, migration and invasion in glioma cells, at least in part, through the inactivation of PI3K/Akt signaling pathway. Therefore, PAQR3 may be a therapeutic target for the treatment of glioma.

Aptamer-conjugated PEGylated quantum dots targeting epidermal growth factor receptor variant III for fluorescence imaging of glioma.

The extent of resection is a significant prognostic factor in glioma patients. However, the maximum safe resection level is difficult to determine due to the inherent infiltrative character of tumors. Recently, fluorescence-guided surgery has emerged as a new technique that allows safe resection of glioma. In this study, we constructed a new kind of quantum dot (QD)-labeled aptamer (QD-Apt) nanoprobe by conjugating aptamer 32 (A32) to the QDs surface, which can specially bind to the tumors. A32 is a single-stranded DNA capable of binding to the epidermal growth factor receptor variant III (EGFRvIII) specially distributed on the surface of glioma cells. To detect the expression of EGFRvIII in human brain tissues, 120 specimens, including 110 glioma tissues and 10 normal brain tissues, were examined by immunohistochemistry, and the results showed that the rate of positive expression of EGFRvIII in the glioma tissues was 41.82%, and 0.00% in normal brain tissues. Besides, the physiochemical properties of QD-Apt nanoparticles (NPs) were thoroughly characterized. Biocompatibility of the NPs was evaluated, and the results suggested that the QD-Apt was nontoxic in vivo and vitro. Furthermore, the use of the QD-Apt in labeling glioma cell lines and human brain glioma tissues, and target gliomas in situ was also investigated. We found that not only could QD-Apt specially bind to the U87-EGFRvIII glioma cells but also bind to human glioma tissues in vitro. Fluorescence imaging in vivo with orthotopic glioma model mice bearing U87-EGFRvIII showed that QD-Apt could penetrate the blood–brain barrier and then selectively accumulate in the tumors through binding to EGFRvIII, and consequently, generate a strong fluorescence, which contributed to the margins of gliomas that were visualized clearly, and thus, help the surgeons realize the maximum safe resection of glioma. In addition, QD-Apt can also be applied in preoperative diagnosis and postoperative examination of glioma. Therefore, these achievements facilitate the use of tumor-targeted fluorescence imaging in the diagnosis, surgical resection, and postoperative examination of glioma.

G protein-coupled receptor kinase 6 is overexpressed in glioma and promotes glioma cell proliferation.

The expression and potential biological functions of G protein-coupled receptor kinase 6 (GRK6) in human glioma are tested in this study. We show that protein and mRNA expression of GRK6 in human glioma tissues was significantly higher than that in the normal brain tissues. Further immunohistochemistry assay analyzing total 118 human glioma tissues showed that GRK6 over-expression was correlated with glioma pathologic grade and patients’ Karnofsky performance status (KPS) score. At the molecular level, in the GRK6-low H4 glioma cells, forced over-expression of GRK6 promoted cell proliferation. Reversely, siRNA-mediated knockdown of GRK6 in the U251MG (GRK6-high) cells led to proliferation inhibition and cell cycle arrest. Intriguingly, GRK6 could also be an important temozolomide resistance factor. Temozolomide-induced cytotoxicity was prominent only in GRK6-low H4 glioma cells. On the other hand, knockdown of GRK6 by targeted siRNA sensitized U251MG cells (GRK6-high) to temozolomide. Thus, GRK6 over-expression in glioma is important for cell proliferation and temozolomide resistance.

Oncogenic effects of evolutionarily conserved noncoding RNA ECONEXIN on gliomagenesis.

Accumulating studies have demonstrated the importance of long noncoding RNAs (lncRNAs) during oncogenic transformation. However, because most lncRNAs are currently uncharacterized, the identification of novel oncogenic lncRNAs is difficult. Given that intergenic lncRNA have substantially less sequence conservation patterns than protein-coding genes across species, evolutionary conserved intergenic lncRNAs are likely to be functional. The current study identified a novel intergenic lncRNA, LINC00461 (ECONEXIN) using a combined approach consisting of searching lncRNAs by evolutionary conservation and validating their expression in a glioma mouse model. ECONEXIN was the most highly conserved intergenic lncRNA containing 83.0% homology with the mouse ortholog (C130071C03Rik) for a region over 2500 bp in length within its exon 3. Expressions of ECONEXIN and C130071C03Rik were significantly upregulated in both human and mouse glioma tissues. Moreover, the expression of C130071C03Rik was upregulated even in precancerous conditions and markedly increased during glioma progression. Functional analysis of ECONEXIN in glioma cell lines, U87 and U251, showed it was dominantly located in the cytoplasm and interacted with miR-411-5p via two binding sites within ECONEXIN. Inhibition of ECONEXIN upregulated miR-411-5p together with the downregulation of its target, Topoisomerase 2 alpha (TOP2A), in glioma cell lines, resulting in decreased cell proliferation. Our data demonstrated that ECONEXIN is a potential oncogene that regulates TOP2A by sponging miR-411-5p in glioma. In addition, our investigative approaches to identify conserved lncRNA and their molecular characterization by validation in mouse tumor models may be useful to functionally annotate novel lncRNAs, especially cancer-associated lncRNAs.

Knockdown of Long Non-Coding RNA KCNQ1OT1 Restrained Glioma Cells' Malignancy by Activating miR-370/CCNE2 Axis.

Accumulating evidence has highlighted the potential role of long non-coding RNAs (lncRNAs) as biomarkers and therapeutic targets in solid tumors. Here, we elucidated the function and possible molecular mechanisms of lncRNA KCNQ1OT1 in human glioma U87 and U251 cells. Quantitative Real-Time polymerase chain reaction (qRT-PCR) demonstrated that KCNQ1OT1 expression was up-regulated in glioma tissues and cells. Knockdown of KCNQ1OT1 exerted tumor-suppressive function in glioma cells. Moreover, a binding region was confirmed between KCNQ1OT1 and miR-370 by dual-luciferase assays. qRT-PCR showed that miR-370 was down-regulated in human glioma tissue and cells. In addition, restoration of miR-370 exerted tumor-suppressive function via inhibiting cell proliferation, migration and invasion, while promoting the apoptosis of human glioma cells. Knockdown of KCNQ1OT1 decreased the expression level of Cyclin E2 (CCNE2) by binding to miR-370. Further, miR-370 bound to CCNE2 3′UTR region and decreased the expression of CCNE2. These results provided a comprehensive analysis of KCNQ1OT1-miR-370-CCNE2 axis in human glioma cells and might provide a novel strategy for glioma treatment.

High expression of B7-H6 in human glioma tissues promotes tumor progression.

B7-H6, a new member of B7-family ligand, also known as NCR3LG1, plays an important role in NK cells mediated immune responses. Many studies have shown that it is highly expressed in various human cancers, and its expression levels are significantly associated with cancer patients’ clinicopathological parameters and postoperative prognoses. But, still the exact role of B7-H6 expression in human glioma remains elusive. In the present study, we have characterized the B7-H6 expression in the human glioma tissues as well as glioma cell lines, U87 and U251. We observed that B7-H6 was highly expressed in the human glioma tissues, and its expression was significantly associated with cancer progression. By using the RNA interference technology, we successfully ablated B7-H6 expression in human glioma cell lines to further study its contribution towards various biological features of this malignancy. Our study identified that the B7-H6 knockdown in U87 and U251 glioma cells significantly suppressed cell proliferation, migration, invasion, and enhanced apoptosis along with induction of cell cycle arrest. It thus suggested that B7-H6 play an important role in the regulation of the biological behavior of these glioma cells. However, the detailed mechanism of B7-H6 mediated regulation of glioma cancer cell transformation and its prognostic value merits further investigation.