Abstract: Long noncoding RNAs (lncRNAs), a class of ribonucleic molecules, participate in various cellular pro- cesses. They are highly expressed in several types of cancer and their expression was related to pathophysiological characteristics of tumor growth, therefore, they can be considered as a promising diagnostic tool and a convenient prognostic biomarker. SPRY4-IT1, belonging to a group of intron-retained lncRNAs, was reported to affect tumor development of many types of cancer. However, the expression and the role of SPRY4-IT1 in glioma are still unclear. Therefore, in this study, we examined for the first time the expression and role of SPRY4-IT1 in glioma cells. The results of our study showed that SPRY4-IT1 was up-regulated in human glioma tissues and cell lines. Knockdown of SPRY4-IT1 could inhibit gliomacell growth and migration. Moreover, knockdown of SPRY4-IT1 could inhibit epitheli- al-mesenchymal transition (EMT) phenotype in glioma cells. Based on these findings, SPRY4-IT1 may be used as a new target for diagnosis and treatment of glioma.
U251 and U87 cell lines, and a normal astroglial cell line, i.e. 1800 cell line, were used in our study. West- ern blot and RT-PCR analyses were performed. High expression of CELF1 mRNA and protein was ob- served in gliomacell lines, whereas low expression was found in the normal astroglial cell line (Figure 4, P < 0.005). The expression of CELF1 in U87 and U251 cells was higher than that in SHG44 cells. CELF1 siRNA was utilised to specifically knockdown CELF1 mRNA in U87 and U251 cells to decrease CELF1 ex- pression. Transfection of U87 and U251 gliomacell lines with an siRNA duplex for 2 d led to 83.8% and 68.6% reduction or so in both mRNA levels of CELF1, respectively, whereas con-siRNA showed no effect on the expression of CELF1 (Figures 5A and 5B). Western blot revealed consistent results for protein levels (Figures 5C and 5D).
Abstract: Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of gliomacell lines determined by MTT assay and colony formation as- say. ADGB knockdown also increased the apoptosis of gliomacell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro.
vector plasmids. Cell proliferation was assessed by MTT growth assay and colony formation assay. According to the MTT results, the proliferation rate of glioma cells overexpressing FZD7 was significantly higher than that of vector control cells; more FZD7-transfected cells were observed at 4 and 5 days after plating (*p < 0.05, Figure 2A and Supplementary Figure S2A). Colony formation assay in U-87MG cells showed that the numbers of colonies formed within vector control and FZD7 group were 66 ± 6 and 103 ± 5, respectively (*p < 0.01, Figure 2B). We also obtained similar results in U-251MG cells (Supplementary Figure S2B). To further test whether FZD7 is required for the proliferation of glioma cells, we silenced FZD7 in glioma cells using lentivirus-mediated shRNA interference. The MTT assay demonstrated that the number of FZD7-knockdown cells was significantly lower than the number of U-251MG cells transfected with scrambled control (Figure 2C). Inhibition of endogenous FZD7 resulted in reduced colony numbers compared to the scrambled control group revealed by colony formation assay (Figure 2D). The numbers of colonies formed in shRNA control, FZD7 shRNA-1 and FZD7 shRNA-2 group were 87 ± 10, 37 ± 3 and 53 ± 5, respectively (*p < 0.01, Figure 2D). Taken together, these results indicated that FZD7 promoted gliomacell proliferation in vitro.
Abstract: Malignant gliomas are primary brain tumors with high rates of morbidity and mortality; they are the fourth most common cause of cancer death. Novel diagnostic and thera- peutic techniques based on nanomaterials provide promising options in the treatment of malignant gliomas. In order to evaluate the potential of FePt nanoparticles (NPs) for malignant glioma therapy, FePt NPs with different surface coatings and components were tunably synthesized using oleic acid/oleylamine (OA/OA) and cysteines (Cys) as the capping agents, respectively. The samples were characterized using X-ray diffraction, transmission electron microscopy (TEM), X-ray photon spectroscopy, Fourier transform infrared spectroscopy, atomic absorption spectrum, and zeta potential. The influence of the surface coatings and components of the FePt NPs on the proliferation of glioma cells was assessed through MTT assay and TEM observation using three typical gliomacell lines (glioma U251 cells, astrocytoma U87 cells, and neuroglioma H4 cells) as in vitro models. The results showed that the proliferation of glioma cells was significantly suppressed by lipophilic FePt-OA/OA NPs in a time- and/or dose-dependent manner, while no or low cytotoxic effects were detected in the case of hydrophilic FePt-Cys NPs. The IC 50 value of FePt-OA/OA NPs on the three gliomacell lines was approximately 5–10 µg mL −1 after
Abstract: Background: Long non-coding RNA (lncRNA) linc01116 was found to be abnormally expressed in many malignant tumor tissues and involved in cancer progression, but its expression and role in glioma tissue is still un- clear. This study was designed to investigate the expression of linc01116 in glioma tissues and the role of linc01116 in gliomacell migration and invasion. Methods: Linc01116 and miR-31 expression was measured in 135 cases of human glioma tissues and normal brain tissues using Real-time quantitative PCR (RT-qPCR). The function of linc01116 in glioma cells was determined by Transwell invasion assays and nude mice metastasis assay. Luciferase reporter system was used to confirm the connection between linc01116 and miR-31, or miR-31 and radixin. Results: Linc01116 is highly expressed in glioma tissue and cells, along with low expression of miR-31, and there was a neg- ative correlation between the expression of linc01116 and miR-31 in glioma tissue. In addition, the expression of linc01116 in glioma patients with metastasis was significantly higher than that in patients without metastasis, while miR-31 was significantly lower. In vitro and in vivo studies shown that linc01116 promoted invasion and migration of glioma cells. The luciferase gene reporter system had confirmed that linc01116 targeted miR-31 and miR-31 tar- geted radixin in U251 cells. Moreover, radixin was downregulated and decreased E-cadherin protein expression, but increased MMP-9 and vimentin protein expression in U251 cells. Conclusion: LncRNA linc01116 is highly expressed in glioma tissues, and it promotes gliomacell migration and invasion by modulation of radixin targeted by miR-31.
Compared with vimentin 466 , vimentin 354 lacks 112 residues at the N-terminus, representing the IF head (DNA binding) sequence, which is able to alter nuclear architecture and chromatin distribution. With 91% homology to vimentin 466 (Figure 2E), vimentin 354 has the same B-Box-type zinc finger, zinc-binding domain, RING-finger (Really Interesting New Gene) domain, involved in mediating protein-protein interactions, which have a wide range of functions such as signal transduction and development. Migration is closely linked to cell shape and to mechanics of the cytoskeleton. Other than the current model of viscous fluid-like cytoplasm and elastic membrane to be the major load-bearing elements, the idea that “hard-wired” transmembrane receptors, cytoskeleton filaments (including vimentin), and nuclear scaffolds are responsible for cell shape control had been proposed . Vimentin 354 may serve as the end connection molecule in the nucleus to hold the entire IF network, which is crucial for supporting the three-dimensional shape of the cell skeleton.
Abstract: The role of extracellular matrix proteins in glioma progression is largely unknown. In the current study, we screened different published GSE datasets and found that an extracellular matrix protein Lysyl Oxidase-Like 1 (LOXL1) is highly expressed in different cohorts of glioma patients. To confirm the results from datasets, we ex- amined the level of LOXL1 in 30 matched glioma tissues and we found that LOXL1 is highly expressed in glioma malignant tissues. We further investigated the biological functions of LOXL1 in glioma cells with both loss- and gain- function assays. These show that highly expressed LOXL1 promotes gliomacell proliferation and growth through regulating Wnt/β-catenin signaling. In conclusion, we report that thesecretory protein LOXL1 is highly expressed in malignantglioma tissues and promotes gliomacell proliferation through Wnt/β-catenin signaling.
are the markers for NF- κ B pathway activation, Western blot assay suggested that TRIM31 overexpression increased the nuclear translocation of p65 and the phosphorylation of I κ B α , and TRIM31 knockdown inhibited the nuclear translocation of p65 and the phosphorylation of I κ B α (Figure 4B). We also analyzed the expression of genes associated with tumor proliferation, invasion, and metastasis, such as BCL2L1, Snail, MMP3, IL8, MYC, MMP9, MMP13, IL1β , XIAP, CXCL5, MMP1, TWIST1, and CCND1. qPCR analysis suggested that TRIM31 overexpression increased their expression and TRIM31 knockdown inhibited their expression, supporting our findings (Figure 4C). To confirm whether TRIM31 promoted glioma proliferation and inva- sion through regulating NF- κ B activity, we inhibited NF- κ B activity through adding NF- κ B inhibitor JSH-23 (10 µ m, Selleck) in TRIM31-overexpressing cells. Colony formation assay and BrdU incorporation assay found that inhibition of NF- κ B activity inhibited TRIM31-overexpressing cell proliferation (Figure 4D and E). Transwell assay found inhibition of NF- κ B activity in TRIM31 overexpressing inhibited cell invasion (Figure 4F). These results suggested TRIM31 promoted gliomacell proliferation through activat- ing NF- κ B activity.
To explore the relevance of miR-126 and gliomacell growth, miR-126 mimic or miR-NC was transfected into U87 cells. The intracellular level of miR-126 was significantly higher in U87 cells after transfecting with miR-126 mimic compared with the miR-NC group (Figure 2A). Then, cell proliferation rate was measured using CCK-8 assays. It was found that restored expression of miR-126 led to significantly decrease of proliferation in U87 cells (Figure 2B). As proliferation directly linked to cell cycle distribution, we investigate the effect of miR- 126 on cell cycle progression. As expected, the percentage of S phase cells decreased, and the percentage of G1 phase cells increased in U87 cells transfected with miR-126 mimic compared to the miR-NC group (Figure 2C). Furthermore, to reveal the biological role of miR-126 on migration and invasion, transwell assay were performed. We find that restored expression of miR-126 could significantly in- hibit migration (Figure 2D) and invasion (Figure 2E) in U87 cells. In addition, to reveal miR-126 on apoptosis in U87 cells, PE Annexin-V stain- ing was used. As shown in Figure 2F, transfec- tion with miR-126 increased cells apoptosis compared to miR-NC and control group. These findings indicated that ectopic expression of miR-126 inhibited cell proliferation, migration
Abstract: MicroRNAs, small non-coding RNAs (21-23 nt in length), are known for regulating carcinogenesis and tumor progression in glioma. MicroRNA-874 (miR-874) has shown play an important role in many human cancers as tumor suppressors. Our previous studies found that miR-874 reduced in glioma tissue and overexpression of miR-874 inhibits gliomacell proliferation and induces apoptosis. However, the biological functions of miR-874 in chemotherapy sensitivity enhancing effect have not been elucidated completely. The present study evaluated the biological function and mechanism of miR-874 in chemotherapy sensitivity enhancing effect in glioma. Our results revealed that the cell proliferation and migration were decreased, cell apoptosis was induced, and the mitochondrial membrane potential was also declined in glioma cells treated with miR-874 with temozolomide (TMZ). And miR-874 with TMZ can reduce the expression level of migration related protein MMP-2 and MMP-9, down-regulate the expres- sion level of Bcl-2 and induce the expression level of Bax. More importantly, miR-874 with TMZ reduced the expres- sion level of STAT3 protein and inhibited STAT3 phosphorylation. Our results demonstrated that overexpression of miR-874 potentiates chemosensitivity of glioblastoma to TMZ by the oncogenic STAT3 pathway, which might provide novel strategies for clinical treatment.
To determine whether the c(RGDfC) polyionic complex micelles could inhibit glioma cells in vivo, Wistar rats were implanted with the C6 gliomacell line and treated with c(RGDfC) polyionic complex micelles or vehicle. A mass with high density found within the contrast-enhanced computed tomographic image of the rat brain (Figure 5A) indicated successful implantation of glioma. c(RGDfC) polyionic complex micelles (300 µ mol/mL, 1 mL per rat) were given to rats with glioma via the tail vein, setting phosphate-buffered saline (0.01 mol/L, 1 mL per rat) for rats with glioma and c(RGDfC) polyionic complex micelles for sham operation (stereotactic injection of Dulbecco’s Modified Eagle’s Medium without glioma cells) as controls. When the c(RGDfC) polyionic complex micelles were applied to rats with glioma, there was a statistically significant increase in
the rescue experiments were performed. The results dis- played that miR-152-3p mimic inhibited cell prolifera- tion, migration, invasion and glycolysis in glioma cells, while SLC2A1 knockdown abolished the effect of miR- 152-3p mimic on glioma cells. The results reveal that LINC00174 promotes gliomacell proliferation, migra- tion, invasion and glycolysis through regulating miR- 152-3p/SLC2A1 axes. Furthermore, the expressions of SLC2A1, E-cadherin, N-cadherin, Vimentin, Cleaved caspase-3, Cleaved caspase-9, Bcl-2, and Bax were identi- fied by western blot analysis. E-cadherin, N-cadherin, and Vimentin are important factors participating in the epithelial-mesenchymal transition (EMT), which pro- motes the migration and invasion of cells . As well known, Cleaved caspase-3, Cleaved caspase-9, Bcl-2, and Bax play crucial role in cell apoptosis . LINC00174 knockdown evidently regulated the protein expression, while miR-152-3p inhibitor effectively abolished the ef- fect of LINC00174 knockdown on protein expression. The results indicate that LINC00174 adjust cellular ac- tivities by regulating these proteins.
Background: Newcastle disease virus (NDV) is an avian paramyxovirus, which selectively exerts oncolytic effects in cancer cells. Mesenchymal stem cells (MSCs) have been reported to affect tumor growth and deliver anti-tumor agents to experimental glioblastoma (GBM). Here, we explored the effects of NDV-infected MSCs derived from different sources, on glioma cells and glioma stem cells (GSCs) and the mechanisms involved in their effects. Methods: The gliomacell lines (A172 and U87) and primary GSCs that were generated from GBM tumors were used in this study. MSCs derived from bone marrow, adipose tissue or umbilical cord were infected with NDV (MTH-68/H). The ability of these cells to deliver the virus to gliomacell lines and GSCs and the effects of NDV- infected MSCs on cell death and on the stemness and self-renewal of GSCs were examined. The mechanisms involved in the cytotoxic effects of the NDV-infected MSCs and their influence on the radiation sensitivity of GSCs were examined as well.
Purpose: Cancer stem cells have recently been thought to be closely related to tu- mor development and reoccurrence. It may be a promising way to cure malignant glioma by using glioma stem cell-targeted dendritic cells as a tumor vaccine. In this study, we explored whether pulsing dendritic cells with antigens of glioma stem cells was a potent way to induce specific cytotoxic T lymphocytes and anti-tumor immunity. Materials and Methods: Cancer stem cells were cultured from gliomacell line U251. Lysate of glioma stem cells was obtained by the repeated freezing and thawing method. Dendritic cells (DCs) were induced and cultured from the murine bone marrow cells, the biological characteristics were detected by electron microscope and flow cytometry. The DC vaccine was obtained by mixing DCs with lysate of glioma stem cells. The DC vaccine was charactirizated through the mixed lymphocyte responses and cell killing experiment in vitro. Level of interferon-γ (IFN-γ) in the supernatant was checked by ELISA. Results: After stim- ulation of lysate of glioma stem cell, expression of surface molecules of DC was up-regulated, including CD80, CD86, CD11C and MHC-II. DCs pulsed with lysate of glioma stem cells were more effective than the control group in stimulating origi- nal glioma cells-specific cytotoxic T lymphocytes responses, killing glioma cells and boosting the secretion of IFN-γ in vitro. Conclusion: The results demonstrated DCs loaded with antigens derived from glioma stem cells can effectively stimulate naive T cells to form specific cytotoxic T cells, kill glioma cells cultured in vitro.
Li et al discovered through reverse transcription- polymerase chain reaction (RT-PCR) that HOXD9 was highly expressed in cervical cancer cells, but not in normal cervical cells. 12 In esophageal squamous cell carcinoma (ESCC), Liu 10 detected the expression of HOXD9 protein in samples through IHC and found that over 60% of ESCC cells were stained for HOXD9 protein to varying degrees. Japanese scholars ﬁ rst discovered through PCR and IHC that HOXD9 is highly expressed in gliomas. They investigated the HOXD9 function in gliomacell lines, and their results suggested that silencing HOXD9 could effectively suppress the proliferation of U87 glioma cells and promote cell cycle arrest and apoptosis. 11 A similar phenomenon was observed in glioma stem cells. In a study on liver cancer, HOXD9 was highly expressed in invasive hepatocellular carcinoma cells. In vitro experiments demonstrated that the over-expression of HOXD9 could remarkably enhance the migratory and invasive capacities of liver cancer cells and promote their epithelial – mesenchymal transition. 18 Concerning the mechanisms, the interaction of HOXD9 with the promoter region of ZEB1 to promote its transcription has been sug- gested. Bao et al 19 discovered through a bioinformatic ana- lysis that the HOXD gene family is speci ﬁ cally upregulated in human lung squamous carcinoma, including HOXD9. In another glioma study, 20 it was discovered that miR-205 could downregulate HOXD9, suppress epithelial – mesenchymal transition in tumor cells, and inhibit growth of human glioma. However, the relationship between HOXD9 and GC has not
In order to understand the effect of miR-132 on gliomacell invasion and migration, we used miR-132 mimics and inhibitors to infect human glioma U87, SHG44, and A172 cells. Seventy-two hours after transfection with miR-132 or scrambled-miRNA, U87, SHG44, and A172 cells were seeded into the upper chamber, and then cells that invaded through the extracellular matrix (ECM) after 48 hours were imaged and counted (Figure 2A and B). In both cell lines, miR-132 could decrease the number of cells that invaded compared to controls. Taken together, these data indicate that miR-132 may be as a regulatory molecule concern cell migration and invasion in vitro.
Abstract: Glioma is a common brain tumor which is highly invasive, responds poorly to therapy, and has a poor prognosis. There is growing evidence that an abnormal expression of many genes is related to glioma and leads to gliomacell growth and metastasis. Phospholipase C beta 1 (PLCB1) plays critical roles in intracellular transduction and regulating signal activation, which are important to tumorigenesis. Therefore, it could bind to miRNA as a target gene. The purpose of our study was to confirm that PLCB1 plays a critical role in suppressing glioma progression. We found that the expression of miR-423-5p was reduced, but the expression of PLCB1 was increased, in glioma tissues and cells. To explore whether miR-423-5p affects PLCB1, a bioinformatics approach suggested that miR- 423-5p can directly target PLCB1. Moreover, we observed, using luciferase reporter assays, that miR-423-5p could target PLCB1 3’-UTR. Functionally, the overexpression of miR-423-5p could attenuate the proliferation, invasion, and migration and promote the apoptosis of glioma cells. Furthermore, we found that miR-423-5p could enhance p- ERK expression in glioma cells. Taken together, we deduced that miR-423-5p inhibited proliferation and metastasis by targeting PLCB1, and it also promotes apoptosis in glioma cells. These results suggest that miR-423-5p directly targets PLCB1 3’-UTR and could inhibit cell invasion and migration through the ERK-dependent pathway in glioma, and the miR-423-5p/PLCB1 axis may be a potential target for new potential therapeutic strategies to treat glioma.
Abstract: Purpose: Malignant glioma is the most common brain tumor and one of the most devastating human diseases. The goal do this study was to identify drugs that inhibit the proliferation of malignant brain glioma both in vivo and in vitro. Method: A commercial kinase inhibitor library containing 464 drugs was screened. Using Western blotting, quantitative Polymerase Chain Reaction (qPCR) and Enzyme-linked immunosorbent assay (ELISA), the in- hibitory effects of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitors on the growth of malignant brain glioma cells was tested. An in vivo glioma tumor-bearing mouse model was used to investigate the effect of NF-κB inhibitors in vivo. Results: Three NF-κB inhibitors, namely caffeic acid phenethyl ester (CAPE), JSH- 23, and sodium 4-aminosalicylate (S4-A)-significantly suppressed proliferation of three malignant brain gliomacell lines and significantly suppressed primary malignant gliomacell proliferation. Pharmacological NF-κB inhibition led to decreases in phospho-p65 levels and in TNF-α expression at both the transcriptional and translational levels. In vivo experiments showed that CAPE exerted strong antitumor activity in an in situ tumor-formation mouse model. The NF-κB pathway activation may be a crucial step in the growth of malignant brain glioma cells. Conclusion: This research identifies a potential new treatment for malignant brain glioma.
The brain microenvironment has emerged as an important component in malignant progression of human glioma. However, astrocytes, the most abundant glial cells in the glioma microenvironment, have as yet a poorly defined role in the development of this disease, particularly with regard to invasion. Here, we co-cultured human astrocytes with human gliomacell lines, U251 and A172, in an in vitro transwell system in order to ascertain their influence on migration and invasion of gliomas. mRNA and protein expression assays were subsequently used to identify candidate proteins mediating this activity. Astrocytes significantly increased migration and invasion of both U251 and A172 cells in migration and invasion (plus matrigel) assays. Membrane type 1 matrix metalloproteinase (MMP14) originating from glioma cells was identified in qRT-PCR as the most highly up-regulated member of the MMP family of genes (~ 3 fold, p < 0.05) in this system. A cytokine array and ELISA were used to identify interleukin-6 (IL-6) as a highly increased factor in media collected from astrocytes, especially under co-culture conditions. IL-6 was also the key cytokine inducing cytomembrane MMP14 expression, the active form of MMP14, in glioma cells. Knockdown of MMP14 with siRNA led to decreased migration and invasion. Taken together, our results indicated that cytomembrane MMP14 was induced by IL-6 secreted from astrocytes, thereby enhancing the migration and invasion of glioma cells through activation of MMP2. Therefore, this IL-6 and MMP14 axis between astrocytes and glioma cells may become a potential target for treatment of glioma patients.