Human Glioma Tissues Research Articles

Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma

Gliomas are the most prevalent primary brain tumors with immense clinical heterogeneity, poor prognosis and survival. The nucleotide-binding domain, and leucine-rich repeat containing receptors (NLRs) and absent-in-melanoma 2 (AIM2) are innate immune receptors crucial for initiation and progression of several cancers. There is a dearth of reports linking NLRs and AIM2 to glioma pathology. NLRs are expressed by cells of innate immunity, including monocytes, macrophages, dendritic cells, endothelial cells, and neutrophils, as well as cells of the adaptive immune system. NLRs are critical regulators of major inflammation, cell death, immune and cancer-associated pathways. We used a data-driven approach to identify NLRs, AIM2 and NLR-associated gene expression and methylation patterns in low grade glioma and glioblastoma, using The Cancer Genome Atlas (TCGA) patient datasets. Since TCGA data is obtained from tumor tissue, comprising of multiple cell populations including glioma cells, endothelial cells and tumor-associated microglia/macrophages we have used multiple cell lines and human brain tissues to identify cell-specific effects. TCGA data mining showed significant differential NLR regulation and strong correlation with survival in different grades of glioma. We report differential expression and methylation of NLRs in glioma, followed by NLRP12 identification as a candidate prognostic marker for glioma progression. We found that Nlrp12 deficient microglia show increased colony formation while Nlrp12 deficient glioma cells show decreased cellular proliferation. Immunohistochemistry of human glioma tissue shows increased NLRP12 expression. Interestingly, microglia show reduced migration towards Nlrp12 deficient glioma cells.

CDCA7L promotes glioma proliferation by targeting CCND1 and predicts an unfavorable prognosis.

Cell division cycle associated 7 like (CDCA7L) belongs to the JPO protein family, recently identified as a target gene of c-Myc and is frequently dysregulated in multiple cancers. However, to the best of our knowledge, no studies to date have been carried out to investigate the functions of CDCA7L in glioma. Thus, in this study, the expression level of CDCA7L and its association with the prognosis in glioma were detected through the TCGA database. The mRNA expression levels of CDCA7L in glioblastoma (GBM) tissues and normal brain tissues were detected by RT-qPCR and western blot analysis. To explore the role of CDCA7L in glioma, CDCA7L siRNA was constructed and transfected into U87 glioma cells. The expression levels of CDCA7L and cyclin D1 (CCND1) in glioma U87 cells following transfection with CDCA7L siRNA were measured by RT-qPCR and western blot analysis. CCK-8, colony formation, EdU and Transwell assays were used to measure the effects of CDCA7L on U87 cell proliferation, and flow cytometry was used to monitor the changes in the cell cycle following transfection with CDCA7L siRNA. Xenograft tumors were examined in vivo for the carcinogenic effects, as well as the mechanisms and prognostic value of CDCA7L in glioma tissues. The results revealed that CDCA7L was highly expressed in human GBM tissues, and a high expression of CDCA7L was associated with a poor prognosis of glioma patients through the TCGA database. We demonstrated that CDCA7L was highly expressed in human GBM tissues and 3 glioma cell lines. The downregulation CDCA7L expression significantly inhibited the proliferation and colony formation ability of U87 cells by blocking cell cycle progression in the G0/G1 phase. In addition, we found that the mRNA and protein levels of CCND1 were markedly decreased following transfection with CDCA7L siRNA compared with NC siRNA in vitro. The downregulation CDCA7L expression reduced the number of invading cells. Consistent with the results of the in vitro assays, the xenograft assay, immunohistochemistry (IHC) assay and western blot analysis demonstrated that, in response to CDCA7L inhibition, tumor growth was inhibited, Ki-67 and CCND1 expression levels were decreased in vivo. On the whole, the results of the current study indicate that CDCA7L is highly expressed in human glioma tissues and that a high CDCA7L expression predicts a poor prognosis of glioma patients. CDCA7L promotes glioma U87 cell growth through CCND1.

Down-regulation of STIP1 regulate apoptosis and invasion of glioma cells via TRAP1/AKT signaling pathway

BackgroundIn recent years, many studies have confirmed that STIP1 (phosphorylation-induced protein 1) is involved in the development and progression of various tumors. However, its potential role in glioma progression and the underlying mechanisms of glioma development remain unclear. MethodsWe analyzed the expression of STIP1 in 35 human glioma tissue specimens of different grades, using 6 normal brain tissues for comparison. We transfected U87 and U251 cell lines with small interfering RNA (siRNA) to downregulate STIP1, and set up a negative control group and a blank group for comparison. The MTT assay was used to detect cell proliferation, and cell cycle progression and apoptosis were analyzed through flow cytometry. Transwell experiments were employed to detect the invasion and migration of STIP1-depleted and control U87 and U251 cells and western blotting was used to detect the expression of TRAP1/Akt pathway proteins. In addition, immunohistochemical analysis was used to reveal differences in expression and localization between transplanted tumor specimens of each group. ResultsWe observed a high expression of STIP1 in glioblastoma, MTT assay revealed a decreased cell proliferation rate in the STIP1-downregulated cells. Cell cycle analysis revealed an increased proportion of cells in G1 phase, as well as an increase in apoptosis, upon STIP1 downregulation. Western blotting showed that TRAP1, pAkt, and MMP2 expression was decreased upon STIP1 downregulation. In addition, TRAP1, ki-67, and MMP2 displayed a decreased expression in vivo. ConclusionsSTIP1 is highly expressed in glioblastoma compared to normal brain tissues. Downregulation of STIP1 in glioma cells reduces cell proliferation rate and invasion and increases cell apoptosis.

MiR-205 suppresses epithelial-mesenchymal transition and inhibits tumor growth of human glioma through down-regulation of HOXD9.

Epithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression. Hsa-miR-205 is considered one of the fundamental regulators of EMT. In the present study, we found that miR-205 was down-regulated in glioma tissues and human glioma cells U87 and U251. Meanwhile, miR-205 overexpression enhanced E-cadherin, reduced mesenchymal markers, and decreased cell proliferation, migration, and invasion in vitro. In vivo, miR-205 suppressed tumor growth. Additionally, HOXD9 was confirmed as a direct target of miR-205. Suppression of HOXD9 by miR-205 was demonstrated by luciferase reporter assay, quantitative real time-PCR analysis, and western blot. Moreover, we observed a negative correlation between miR-205 and HOXD9 in human glioma tissues. In summary, our findings demonstrated that miR-205 suppresses glioma tumor growth, invasion, and reverses EMT through down-regulating its target HOXD9.

MiR-25-3p promotes glioma cell proliferation and migration by targeting FBXW7 and DKK3.

MicroRNAs (miRs) serve important roles in glioma. However, the underlying molecular mechanism of miR-25 in glioma progression remains largely unknown; therefore, it was investigated in the present study. RT-qPCR analysis revealed that miR-25 expression levels were markedly increased in human glioma tissue and glioma cell lines compared with normal brain tissues and normal human astrocytes, respectively. miR-25 upregulation exhibited an association with glioma progression, and the knockdown of miR-25 significantly inhibited glioma cell proliferation and migration. F-box and WD repeat domain containing 7 (FBXW7) and dickkopf WNT signaling pathway inhibitor 3 (DKK3) were identified as target genes of miR-25. FBXW7 and DKK3 expression levels were significantly downregulated in glioma tissue samples compared with normal brain tissue, and their expression levels were negatively regulated by miR-25 expression in glioma cells. Furthermore, inhibition of FBXW7 and DKK3 expression suppressed the miR-25-induced effects on glioma cell proliferation and migration. The findings of the present study suggest that miR-25 may promote glioma cell proliferation and migration by inhibiting the expression of FBXW7 and DKK3. Therefore, miR-25 may serve as a promising molecular target for the treatment of glioma.

MiR‑342 inhibits glioma cell proliferation by targeting GPRC5A.

Accumulating evidence suggests that microRNAs (miRNAs) play a key role in the biological behaviors and progression of glioma. However, the function and bio-molecular mechanisms of miR-342 in glioma remain largely unclear. In the present study, reverse transcription quantitative-polymerase chain reaction and western blotting were performed to determine the mRNA and protein expression levels of the factors investigated. MTT assay was performed to examine the proliferation rates. Luciferase reporter assay was performed to test the binding between miRNA-342 and its putative target. Data indicated that miR-342 expression was markedly decreased in human glioma tissues and cell line U87, and reduced miR-342 expression significantly promoted cell proliferation. In order to explore the mechanisms, G-protein coupled receptor family C group 5 member A (GPRC5A) was identified as a target of miR-342 and depletion of GPRC5A suppressed cell proliferation. Our findings demonstrated that miR-342 regulates the cell proliferation of glioma by targeting GPRC5A, which indicates that miR-342 is a target of interest regarding the treatment of refractory glioma, and it may provide a promising prognostic and therapeutic strategy for glioma treatment.

Spalt-Like Transcription Factor 1 (SALL1) Gene Expression Inhibits Cell Proliferation and Cell Migration of Human Glioma Cells Through the Wnt/β-Catenin Signaling Pathway.

BackgroundThe spalt-like transcription factor 1 (SALL1) gene is a member of the Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) and has been shown to modulate the onset and progression of human tumors. This study aimed to investigate the regulatory effects and mechanisms of SALL1 gene expression in human glioblastoma and glioma cells and tissue samples from patients with cerebral glioma.Material/MethodsThe human glioblastoma cell lines, LN229, U87-MG, U-251, U343, and the Hs683 glioma cell line were studied. The cell counting kit-8 (CCK-8) assay, cell cycle assay, wound-healing assay, transwell assay, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate cell proliferation, cell migration, and the cell cycle and expression of SALL1. Expression of SALL1 mRNA in 120 samples of cerebral glioma and 20 samples of normal brain were studied. Overall survival data from patients with cerebral glioma were analyzed.ResultsSALL1 expression was down-regulated in human glioblastoma and glioma cells and in cerebral glioma tissues. Down-regulation of SALL1 expression was associated with reduced overall survival. Overexpression of SALL1 was associated with inhibition of cell proliferation associated with cell cycle arrest at the G0/G1 phase. SALL1 inhibited cell migration by preventing epithelial-mesenchymal transition (EMT) and down-regulating the expression of stem cell markers. Reduced levels of β-catenin and downregulation of c-Myc and cyclin D1 and upregulation of p21and p27 expression were associated with SALL1 expression.ConclusionsIn human glioblastoma cells and cerebral glioma tissues, SALL1 acted as a tumor suppressor gene by inhibiting Wnt/β-catenin signaling.

MicroRNA-4500 inhibits human glioma cell progression by targeting IGF2BP1

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) overexpression promotes glioma cell progression. The aim of the current study is to silence IGF2BP1 in glioma cells by the microRNA (miRNA) strategy. The bio-informatic analyses identified that microRNA-4500 (miR-4500) putatively targets 3′-UTR (3′-untranslated region) of IGF2BP1. In A172 cells and primary human glioma cells ectopic overexpression of the wild-type miR-4500 (but not the mutant form) downregulated IGF2BP1 and its target genes (Gli1, IGF2 and c-Myc). Functional studies show that ectopic miR-4500 overexpression inhibited glioma cell growth, survival, proliferation, migration and invasion. Conversely, in A172 cells miR-4500 inhibition, by a lentiviral construct, increased expression of IGF2BP1 and its targets, promoting cell survival, proliferation and migration. Furthermore, IGF2BP1 knockout by the CRISPR/Cas9 method inhibited A172 cell progression. Significantly, miR-4500 overexpression or miR-4500 inhibition was ineffective in IGF2BP1 knockout A172 cells. At last, we show that miR-4500 levels are downregulated in human glioma tissues, correlating with IGF2BP1 upregulation. Together, we conclude that miR-4500 inhibits human glioma cell progression by targeting IGF2BP1.

Downregulation of AIF-2 Inhibits Proliferation, Migration, and Invasion of Human Glioma Cells via Mitochondrial Dysfunction.

Glioma remains the leading cause of brain tumor-related death worldwide. Apoptosis inducing factor (AIF) is a family of mitochondrial oxidoreductases that play important roles in mitochondrial metabolism and redox control. AIF-1 has been demonstrated to exert cell-killing effect via apoptosis in cancer cells, whereas the role of AIF-2 in cancer cells has not been determined. This study aimed to investigate the role of AIF-2 in human glioma cells. We found that AIF-2 was upregulated in human glioma tissues and cell lines, especially in U251 cells. Downregulation of AIF-2 using specific siRNA (Si-AIF-2) significantly reduced cell proliferation, induced G1 cell cycle arrest and differently regulated the expression of cell cycle regulator proteins in U251 cells. In addition, the results of Matrigel invasion assay and live-cell tracking assay showed that knockdown of AIF-2 inhibited cell invasion and migration. The results of immunocytochemistry indicated that knockdown of AIF-2 significantly attenuated the nuclear translocation of AIF-1, which was confirmed by western blot analysis. Furthermore, downregulation of AIF-2 resulted in mitochondrial dysfunction in U251 cells, as evidenced by reduced mitochondrial membrane potential (MMP), mitochondrial complex I activity, and mitochondrial Ca2+ buffering capacity. In conclusion, we found that AIF-2 plays a key role in promoting cell proliferation, invasion, and migration via regulating AIF-1-related mitochondrial cascades. Downregulation of the candidate oncogene AIF-2 might constitute a strategy to kill human glioma cells.

Sirtuin 1 knockdown inhibits glioma cell proliferation and potentiates temozolomide toxicity via facilitation of reactive oxygen species generation.

Malignant glioma is one of the most common types of primary malignancies in the human central nervous system. Temozolomide (TMZ) is the most commonly used drug in clinical therapy of glioma; however, chemoresistance makes glioma difficult to cure and relapse likely. Sirtuin 1 (SIRT1) serves important roles in cell proliferation, differentiation and metabolism, but the role of SIRT1 in human glioma remains largely unexplored. In the present study, SIRT1 expression was assessed in human glioma tissues and cells. RNA interference and SIRT1 inhibitor were used to determine the effect of SIRT1 on glioma growth inhibition and glioma cell chemoresistance in vitro and in vivo. The levels of reactive oxygen species (ROS) in glioma cells were detected with the dihydroethidium probe following SIRT1 inhibition. The results demonstrated that SIRT1 was overexpressed in glioma tissues and cells, and patients with higher SIRT1 expression exhibited poorer prognosis. SIRT1 inhibition inhibited the proliferation of U87 and U251 cells. In addition, SIRT1 knockdown and SIRT1 inhibitor could significantly sensitize glioma cells to TMZ treatment in vitro and in vivo. The expression of Ki67 and p53 was demonstrated to be regulated by SIRT1. Finally, SIRT1 could regulate intracellular ROS generation in TMZ. In summary, SIRT1 was essential for glioma tumorigenesis and glioma cell chemoresistance. SIRT1 inhibition increased the sensitivity of glioma cells for TMZ via the facilitation of intracellular ROS generation, which suggested that SIRT1 may serve as a target for clinical therapy of glioma.

Long noncoding RNA LINC00515 promotes cell proliferation and inhibits apoptosis by sponging miR-16 and activating PRMT5 expression in human glioma.

Introduction: In recent years, an increasing amount of literature has demonstrated the functional role of long non-coding RNA (lncRNA) in human diseases. LINC00515 is a newly defined lncRNA and is reported to act as an oncogene in multiple myeloma. However, the function of LINC00515 in glioma is still uncertain.Materials and methods: We examined the expression levels of LINC00515 in human glioma tissues and cell lines using real-time PCR analysis. In addition, we confirmed the distribution of LINC00515 in glioma cells and suppressed LINC00515 expression with siRNAs. CCK-8, colony formation assay and apoptosis analysis were used to study the function of LINC00515 in glioma progression. Then, we used bioinformatics prediction and subsequent experiments to reveal the underlying molecular mechanism.Results: We found that LINC00515 was up-regulated in glioma tissues and cell lines. LINC00515 was mainly located in the cytoplasm in glioma cells. Knockdown of LINC00515 led to decreased proliferation and increased apoptosis of glioma cells. Mechanistically, our data indicated that there was a LINC00515/miR-16/PRMT5 regulatory axis in glioma. LINC00515 could activate PRMT5 expression and promote glioma progression by acting as a sponge of miR-16.Conclusion: LINC00515 expression is elevated in human glioma and promotes growth and inhibits apoptosis of glioma cells. The regulatory cascade LINC00515/miR-16/PRMT5 plays a critical role in glioma progression.

Overexpression of miR-770 indicates a favorable prognosis and suppresses tumorigenesis by modulating PI3K-AKT pathway in glioma.

Previous studies showed that miR-770 expression was deregulated in many tumors. However, the effect of miR-770 function on glioma remains as a mystery. The present study aimed to explore its expression, cellular function and clinic features in glioma. We analyzed RNA sequencing data to explore abnormally expressed miRNAs in glioma. Glioma tissue specimens and their matched normal tissues were collected to test miR-770 expression using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis. The correlation between miR-770 and the clinicopathological factors and the prognostic value of miR-770 was statistically analyzed. We then investigated alterations in a series of cancer-related phenotypes, including cell viability, apoptosis, colony formation and metastasis capacities. Western blot analysis was performed to examine the expression changes of EMT-related proteins and PI3K/Akt signaling pathway proteins. We identified a novel glioma-related miRNA miR-770, which was significantly down-regulated in human glioma tissues. The results of RT-PCR further showed that miR-770 expression was significantly down-regulated in both glioma tissues and cell lines. Furthermore, decreased miR-770 expression was significantly associated with advanced WHO grade, KPS score and shorter five-year overall survival. Then, functional assays indicated that overexpression of miR-770 suppressed proliferation, migration, invasion and EMT pathway, and induced the apoptosis of glioma cells in vitro. Moreover, we further illustrated that the up-regulation of miR-770 suppressed the PI3K-AKT signaling pathway. Our present findings firstly reported the roles and mechanisms associated with miR-770 in glioma progression, highlighting miR-770 as a potential therapeutic target for glioma patients.

CHD7 promotes glioblastoma cell motility and invasiveness through transcriptional modulation of an invasion signature

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.

Effects of SIRT1 silencing on viability, invasion and metastasis of human glioma cell lines.

Silent information regulator 1 (SIRT1), a member of the sirtuin family, is involved in the development of various types of tumor. Previous studies have revealed that SIRT1 has dual functions, as a promoter and an inhibitor, in certain tumors. However, the role of SIRT1 in invasion and metastasis of glioma cells and its associated signaling pathway remain unclear. The aim of the present study was to determine the effects of SIRT1 on these processes and on the epithelial-mesenchymal transition (EMT) in human glioma and adjacent tissues, and in the human glioma cell lines U87 and U251. SIRT1 expression in tissues was investigated using the reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. The U87 and U251 cell lines were divided into control and SIRT1-small interfering RNA (siRNA) groups. The Cell Counting Kit-8, cell invasion assays were used to evaluate the effects of SIRT1 silencing on cell viability, invasion and EMT. Results indicated that SIRT1 was highly expressed in glioma tissues compared with in adjacent brain tissues. In addition, SIRT1-siRNA significantly inhibited the viability and invasion of U87 and U251 cells. Furthermore, EMT analysis revealed that the expression levels of the mesenchymal markers fibronectin and vimentin were significantly lower in the SIRT1-siRNA group compared with in the control group. Conversely, expression levels of the epithelial markers epithelial cadherin and β-catenin were significantly higher in the SIRT1-siRNA group compared with in the control group. In conclusion, the results of the present study indicated that SIRT1 was positively associated with viability and invasion of U87 cells, potentially through EMT. These results suggested that SIRT1 may serve a crucial role in the proliferation and development of glioma.

CADM2 inhibits human glioma proliferation, migration and invasion.

Malignant glioma is one of the most common malignant tumors in the brain parenchyma with a poor prognosis. Cell adhesion molecules (CADMs) immunoglobulin super family is involved in the maintenance of cell adhesion, polarity and tumor suppression. However, the role and mechanisms of CADM2 in human glioma have yet to be elucidated. Therefore, the present study evaluated the expression level of CADM2 and demonstrated that CADM2 was markedly downregulated in human glioma tissues compared with normal brain tissue and glioma cell lines, and the CADM2 expression level was significantly decreased in high‑grade glioma tissues. Overexpression of CADM2 inhibited the proliferation of glioma cell proliferation in vitro and in vivo. CADM2 also inhibited the migration and invasion of U87 and U251 cells. Furthermore, overexpression of CADM2 induced a significant decrease in the expression of G1/S transition key regulators, cyclin D1, cyclin E, cyclin‑dependent kinase (CDK)2 and CDK4. Additionally, CADM2 expression was associated with alterations in epithelial‑mesenchymal transition (EMT) markers, including E‑cadherin and β‑catenin. Taken together, the results of the present study demonstrated that CADM2 inhibits glioma tumorigenesis by regulating the cell cycle and the EMT process, suggesting that CADM2 may be a novel potential therapeutic target in human glioma.

Reciprocal regulation of integrin β4 and KLF4 promotes gliomagenesis through maintaining cancer stem cell traits

BackgroundThe dismal prognosis of patients with glioma is largely attributed to cancer stem cells that display pivotal roles in tumour initiation, progression, metastasis, resistance to therapy, and relapse. Therefore, understanding how these populations of cells maintain their stem-like properties is critical in developing effective glioma therapeutics.MethodsRNA sequencing analysis was used to identify genes potentially involved in regulating glioma stem cells (GSCs). Integrin β4 (ITGB4) expression was validated by quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) staining. The role of ITGB4 was investigated by flow cytometry, mammosphere formation, transwell, colony formation, and in vivo tumorigenesis assays. The reciprocal regulation between Integrin β4 and KLF4 was investigated by chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, immunoprecipitation, and in vivo ubiquitylation assays.ResultsIn this study, we found that ITGB4 expression was increased in GSCs and human glioma tissues. Upregulation of ITGB4 was correlated with glioma grades. Inhibition of ITGB4 in glioma cells decreased the self-renewal abilities of GSCs and suppressed the malignant behaviours of glioma cells in vitro and in vivo. Further mechanistic studies revealed that KLF4, an important transcription factor, directly binds to the promoter of ITGB4, facilitating its transcription and contributing to increased ITGB4 expression in glioma. Interestingly, this increased expression enabled ITGB4 to bind KLF4, thus attenuating its interaction with its E3 ligase, the von Hippel-Lindau (VHL) protein, which subsequently decreases KLF4 ubiquitination and leads to its accumulation.ConclusionsCollectively, our data indicate the existence of a positive feedback loop between KLF4 and ITGB4 that promotes GSC self-renewal and gliomagenesis, suggesting that ITGB4 may be a valuable therapeutic target for glioma.

The Prognostic and Therapeutic Value of PD-L1 in Glioma.

Glioma is the most common type of primary brain tumors. After standard treatment regimen (surgical section, radiotherapy and chemotherapy), the average survival time remains merely around 14 months for glioblastoma (grade IV glioma). Recent immune therapy targeting to the immune inhibitory checkpoint axis, i.e., programmed cell death protein 1 (PD-1) and its ligand PD-L1 (i.e., CD274 or B7-H1), has achieved breakthrough in many cancers but still not in glioma. PD-L1 is considered a major prognostic biomarker for immune therapy in many cancers, with anti-PD-1 or anti-PD-L1 antibodies being used. However, the expression and subcellular distribution of PD-L1 in glioma cells exhibits great variance in different studies, severely impairing PD-L1's value as therapeutic and prognostic biomarker in glioma. The role of PD-L1 in modulating immune therapy is complicated. In addition, endogenous PD-L1 plays tumorigenic roles in glioma development. In this review, we summarize PD-L1 mRNA expression and protein levels detected by using different methods and antibodies in human glioma tissues in all literatures, and we evaluate the prognostic value of PD-L1 in glioma. We also summarize the relationships between PD-L1 and immune cell infiltration in glioma. The mechanisms regulating PD-L1 expression and the oncogenic roles of endogenous PD-L1 are discussed. Further, the therapeutic results of using anti-PD-1/PD-L1 antibodies or PD-L1 knockdown are summarized and evaluated. In summary, current results support that PD-L1 is not only a prognostic biomarker of immune therapy, but also a potential therapeutic target for glioma.

Chemokine (C-C motif) ligand 18 is highly expressed in glioma tissues and promotes invasion of glioblastoma cells.

The objective of the study is to evaluate levels of chemokine (C-C motif) ligand 18 (CCL18) in human glioma tissues and effects of CCL18 on U251 glioma cells. By using the real-time reverse transcription polymerase chain reaction and immunochemically histological staining, we determined the mRNA and protein levels of CCL18 in tissues of 60 patients with World Health Organization (WHO) Grades II, III and IV glioma and the normal brain. Cultured U251 glioma cells were incubated with CCL18 and then subjected to transwell. The scratch wound-healing and cell count kit (CCK-8) assays were performed to detect the possible effects of CCL18 on the cell invasion, migration, and proliferation. In the tissues of the normal brain (n = 10), glioma Grade II (n = 26), III (n = 18), and IV (n = 16), CCL18 mRNA expression levels were 1.00 ± 0.09, 6.02 ± 1.26, 26.35 ± 3.98, and 112.21 ± 13.25 fold, respectively (P < 0.01); the percentage of CCL18-positive glioma cells was 0%, 58.8%, 70.0%, and 100% in the normal brain, glioma WHO Grade II, III, and IV, respectively (P < 0.01). Different concentrations of CCL18 (0, 5, and 10 ng/ml) enhanced the of U251 glioma cell invasion in 24 h transwell assays [from 43.5 ± 8.3 to 202.0 ± 18.5 and 279.7 ± 18.6 cells (P < 0.01)], increased the cell migration quantified by comparing the areas of the scratch (pixel) [at 12 h, 498.4 ± 75.3, 381.3 ± 21.4, and 347.7 ± 14.2; at 24 h, 299.5 ± 15.3, 284.6 ± 7.8, and 237.3 ± 20.6 (P < 0.05)], and significantly increased the cell growth in CCK-8 assay [from 1.000 ± 0.019-1.260 ± 0.094 and 2.070 ± 0.138 fold in CCL18, respectively (n = 20/each group) (P < 0.01)]. We have found that CCL18 is highly expressed in glioma tissues and enhances the invasion, migration, and proliferation of U251 glioma cells. Therefore, CCL18 may be a potential biomarker for detecting and grading human glioma.

The miR-26a/AP-2α/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma.

Aberrant expression of transcription factor AP-2α has been functionally associated with various cancers, but its clinical significance and molecular mechanisms in human glioma are largely elusive.Methods: AP-2α expression was analyzed in human glioma tissues by immunohistochemistry (IHC) and in glioma cell lines by Western blot. The effects of AP-2α on glioma cell proliferation, migration, invasion and tumor formation were evaluated by the 3-(4,5-dimethyNCthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) and transwell assays in vitro and in nude mouse models in vivo. The influence of AP-2α on glioma cell stemness was analyzed by sphere-formation, self-renewal and limiting dilution assays in vitro and in intracranial mouse models in vivo. The effects of AP-2α on temozolomide (TMZ) resistance were detected by the MTT assay, cell apoptosis, real-time PCR analysis, western blotting and mouse experiments. The correlation between AP-2α expression and the expression of miR-26a, Nanog was determined by luciferase reporter assays, electrophoretic mobility shift assay (EMSA) and expression analysis.Results: AP-2α expression was downregulated in 58.5% of glioma tissues and in 4 glioma cell lines. AP-2α overexpression not only reduced the proliferation, migration and invasion of glioma cell lines but also suppressed the sphere-formation and self-renewal abilities of glioma stem cells in vitro. Moreover, AP-2α overexpression inhibited subcutaneous and intracranial xenograft tumor growth in vivo. Furthermore, AP-2α enhanced the sensitivity of glioma cells to TMZ. Finally, AP-2α directly bound to the regulatory region of the Nanog gene, reduced Nanog, Sox2 and CD133 expression. Meanwhile, AP-2α indirectly downregulated Nanog expression by inhibiting the interleukin 6/janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway, consequently decreasing O6-methylguanine methyltransferase (MGMT) and programmed death-ligand 1 (PD-L1) expression. In addition, miR-26a decreased AP-2α expression by binding to the 3' untranslated region (UTR) of AP-2α and reversed the tumor suppressive role of AP-2α in glioma, which was rescued by a miR-26a inhibitor. TMZ and the miR-26a inhibitor synergistically suppressed intracranial GSC growth.Conclusion: These results suggest that AP-2α reduces the stemness and TMZ resistance of glioma by inhibiting the Nanog/Sox2/CD133 axis and IL6/STAT3 signaling pathways. Therefore, AP-2α and miR-26a inhibition might represent a new target for developing new therapeutic strategies in TMZ resistance and recurrent glioma patients.

LncRNA small nucleolar RNA host gene 20 predicts poor prognosis in glioma and promotes cell proliferation by silencing P21.

BackgroundIn multiple cancers, long non-coding RNA small nucleolar RNA host gene 20 (lncRNA SNHG20) is generally dysregulated. In the present study, both the biological role and clinicopathological value of lncRNA SNHG20 in glioma are explored.MethodsReal-time PCR was employed to determine lncRNA SNHG20 expression in glioma patients. The prognostic role of expression of lncRNA SNHG20 was evaluated in a retrospective cohort study. In addition, the association between lncRNA SNHG20 expression and the clinicopathological features of glioma patients, such as tumor recurrence, survival status, follow-up time, WHO grade, resection extent, tumor location, Karnofsky performance scale score, cystic change, tumor size, gender and age, was discussed. By constructing and transfecting siRNAs that targeted lncRNA SNHG20 into the glioma U87 cells, the effects of lncRNA SNHG20 on the proliferation and cell cycle of U87 cells were assessed through cell counting kit-8, colony formation and cell cycle assays, respectively. In addition, Western blot and real-time PCR measured the expression levels of P21 and CCNA1 in U87 cells after being transfected with SNHG20 siRNA.ResultsOur results suggested the high expression of lncRNA SNHG20 in human glioma tissues compared with normal brain tissues, which was related to recurrence-free survival and poor overall survival in glioma patients. According to the existing retrospective cohort study, high lncRNA SNHG20 expression was associated with tumor size, extent of resection, WHO grade, follow-up time, survival status and recurrence. Besides, knocking down the expression of lncRNA SNHG20 could inhibit the proliferation and colony formation abilities of glioma U87 cells through cell cycle arrest. Consequently, the expression of CCNA1 was inhibited, and the expression of P21 was up-regulated in U87 cells.ConclusionA high lncRNA SNHG20 expression level predicts the poor prognosis for glioma patients. Moreover, lncRNA SNHG20 can promote glioma proliferation through silencing P21 and thus lncRNA SNHG20 is an independent potential prognostic biomarker for glioma patients.