MircoRNA‐1275 promotes proliferation,invasion and migration of glioma cells via SERPINE1

This study was designed to explore the relationship between miR‐1275 and SERPINE1 and its effects on glioma cell proliferation, migration, invasion and apoptosis. Differentially expressed miRNAs and mRNAs in glioma tissues were screened out by bioinformatic analysis. Dual‐luciferase reporter gene assay was used to validate the targeted relationship between miR‐1275 and SERPINE1. qRT‐PCR was used to detect the expression of miR‐1275 and SERPINE1 in glioma tissues. The expressions of SERPINE1 and p53 pathway‐related proteins in glioma cells were detected by western blot. Glioma cell proliferation, apoptosis, migration and invasion were respectively detected by CCK‐8 assay, flow cytometry, wound healing assay and transwell assay. Tumour xenograft model was developed to study the influence of miR‐1275 and SERPINE1 on glioma growth in vivo. The results of microarray analysis, qRT‐PCR and western blot showed that miR‐1275 was low‐expressed while SERPINE1 was high‐expressed in glioma. Dual‐luciferase assay showed that miR‐1275 could bind to SERPINE1. Overexpression of miR‐1275 could promote the p53 pathway‐related proteins’ expression. Highly expressed miR‐1275 could repress the migration, proliferation and invasion of glioma cells while highly expressed SERPINE1 had inverse effects. Tumour xenograft showed that up‐regulated miR‐1275 or down‐regulated SERPINE1 could repress glioma growth in vivo. Up‐regulation of miR‐1275 activated p53 signalling pathway via regulating SERPINE1 and therefore suppressed glioma cell proliferation, invasion and migration, whereas promoted cell apoptosis.     

Scutellarin inhibits the glioma cell proliferation by downregulating BIRC5 to promote cell apoptosis

The expression changes of baculovirus inhibitor of apoptosis repeat-containing protein5 in brain glioma after administration of Scutellarin was detected. To explore the effort of scutellarin on anti-glioma by downregulating BIRC5.The effect of scutellarin on tumour growth and animal survival was detected by administering scutellarin to nude mice subcutaneous tumour formation and SD rats in situ tumour formation models. A significantly different gene BIRC5 was found by using the combination of TCGA databases and network pharmacology. And then qPCR was performed to detect the expression of BIRC5 in glioma tissues, cells and normal brain tissues and glial cells. CCK-8 was used to detect the IC50 of scutellarin on glioma cells. The wound healing assay, flow cytometry and MTT test were used to detect the effect of scutellarin on the apoptosis and proliferation of glioma cells. The expression of BIRC5 in glioma tissues was significantly higher than that in normal brain tissues. Scutellarin can significantly reduce tumour growth and improve animal's survival. After scutellarin was administered, the expression of BIRC5 in U251 cells was significantly reduced. And after same time, apoptosis increased and cell proliferation was inhibited. This original research showed that scutellarin can promote the apoptosis of glioma cells and inhibit the proliferation by downregulating the expression of BIRC5.     

Prefoldin 6 promotes glioma progression via the AKT signalling pathway

Gliomas are one of the most aggressive primary tumours, accounting for 81% of malignant brain tumours, and are associated with a significant mortality. Therefore, the elucidation of the molecular mechanism underlying glioma progression and identification of promising treatment targets are necessary. Here, the expression of prefoldin (PFDN) 6 in human glioma tissues and cell lines was evaluated using immunohistochemistry and quantitative polymerase chain reaction. Celigo and CCK-8 assays were performed for assessing cell viability. Flow cytometry was used to analyse apoptosis and cell cycle distribution. Wound-healing and transwell assays were performed to observe cell migration. Lastly, xenograft models were developed for the in vivo validation of the results, and a human phospho-kinase array was used to explore the downstream signalling pathways. PFDN6 was upregulated in gliomas, and PFDN6 overexpression was significantly correlated with a low survival rate, estimated glomerular filtration rate (EGFR) expression, and tumour grade and recurrence. Moreover, PFDN6 knockdown significantly attenuated cell proliferation and migration, induced apoptosis, and blocked cell cycle progression in the G2 phase, which was further confirmed in the in vivo experiments. Mechanistically, the effects of PFDN6 may be mediated via the AKT signalling pathway. In conclusion, we showed that PFDN6 promotes glioma development by activating AKT signalling and emphasised the potential of PFDN6 as a crucial target in glioma therapy.     

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.     

11 Long noncoding RNA UCA1 functions as miR-135a sponge to promote the epithelial to mesenchymal transition in glioma

The dysregulation of long noncoding (lncRNA) UCA1 may play an important role in tumor progression. However, the function in gliomas is unclear. Therefore, this experiment was designed to explore the pathogenesis of glioma based on lncRNA UCA1. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of lncRNA UCA1, miR-135a, and HOXD9 in gliomas tissues. The effect of lncRNA UCA1 and miR-135a on tumor cell proliferation and migration invasiveness was examined by CCK-8 and transwell assays. Target gene prediction and screening, luciferase reporter assay were used to verify downstream target genes of lncRNA UCA1. Expression of E-cadherin, N-cadherin, vimentin, and HOXD9 was detected by RT-qPCR and Western blotting. The tumor changes in mice were detected by in vivo experiments in nude mice. lncRNA UCA1 was highly expressed in glioma tissues and cell lines. lncRNA UCA1 expression was associated with significantly poor overall survival in gliomas. Moreover, lncRNA UCA1 significantly enhanced cell proliferation and migration, and promoted the occurrence of EMT. In addition, lncRNA UCA1 promoted the development of EMT by positively regulating HOXD9 expression as a miR-135a sponge. In vivo experiments indicated that UCA1 exerted its biological functions by modulating miR-135a and HOXD9. In conclusion, lncRNA UCA1 can induce the activation of HOXD9 by inhibiting the expression of miR-135a and promote the occurrence of EMT in glioma.     

Knockdown of DIXDC1 Inhibits the Proliferation and Migration of Human Glioma Cells

DIX domain containing 1 (DIXDC1), the human homolog of coiled-coil-DIX1 (Ccd1), is a positive regulator of Wnt signaling pathway. Recently, it was found to act as a candidate oncogene in colon cancer, non-small-cell lung cancer, and gastric cancer. In this study, we aimed to investigate the clinical significance of DIXDC1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that DIXDC1 was overexpressed in glioma tissues and glioma cell lines. The expression level of DIXDC1 was evidently linked to glioma pathological grade and Ki-67 expression. Kaplan–Meier curve showed that high expression of DIXDC1 may lead to poor outcome of glioma patients. Serum starvation and refeeding assay indicated that the expression of DIXDC1 was associated with cell cycle. To determine whether DIXDC1 could regulate the proliferation and migration of glioma cells, we transfected glioma cells with interfering RNA-targeting DIXDC1; investigated cell proliferation with Cell Counting Kit (CCK)-8, flow cytometry assays, and colony formation analyses; and investigated cell migration with wound healing assays and transwell assays. According to our data, knockdown of DIXDC1 significantly inhibited proliferation and migration of glioma cells. These data implied that DIXDC1 might participate in the development of glioma, suggesting that DIXDC1 can become a potential therapeutic strategy for glioma.     

MicroRNA-34a induces apoptosis in the human glioma cell line,A172, through enhanced ROS production and NOX2 expression

Background

MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma.

Methods

The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot.

Results

MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells.

Conclusion

MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.     

MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1

Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3′-untranslated regions (3′-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.     

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.     

C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation

The decreased antitumor immune response significantly contributes to the progression of glioma. To evaluate whether the antitumor immunity is restored by stable co-expression of IL-18 and Fas receptor, we retrovirally introduced these two genes into rat C6 glioma cells. We found that IL-18-transduced glioma cells secreted IL-18 and induced PBMC IFN-gamma production in vitro. We also found that Fas-transduced glioma cells were susceptible to Fas-mediated apoptosis. In vivo, we found that IL-18 expression and Fas expression synergistically inhibited C6 cell tumorigenesis with the glioma cells being subcutaneously injected in rat flank. Furthermore, we found that co-expression of IL-18 and Fas also produced a marked survival advantage with the rats being intracerebrally implanted with the glioma cells. Finally, we demonstrated that FasL-dependent PBMC cytotoxicity participated in the anti-glioma immunity induced by IL-18 and Fas expression. Taken together, these findings demonstrate that increasing IL-18 production in tumor microenvironment and prompting functional Fas receptor expression of tumor cells could enhance FasL-dependent cytotoxic antitumor immunity.     

Silencing of the MEG3 gene promoted anti-cancer activity and drug sensitivity in glioma

Aberrant expression of MEG3 has been shown in various cancers. The purpose of this study is to evaluate the effect of MEG3 on glioma cells and the use of potential chemotherapeutics in glioma by modulating MEG3 expression. Cell viability, migration and chemosensitivity were assayed. Cell death was evaluated in MEG3 overexpressing and MEG3 suppressed cells. MEG3 expression was compared in patient-derived glioma cells concerning IDH1 mutation and WHO grades. Silencing of MEG3 inhibited cell proliferation and reduced cell migration while overexpression of MEG3 promoted proliferation in glioma cells. MEG3 inhibition improved the chemosensitivity of glioma cells to 5-fluorouracil (5FU) but not to navitoclax. On the other hand, there is no significant effect of MEG3 expression on temozolamide (TMZ) treatment which is a standard chemotherapeutic agent in glioma. Suppression of the MEG3 gene in patient-derived oligodendroglioma cells also showed the same effect whereas glioblastoma cell proliferation and chemosensitivity were not affected by MEG3 inhibition. Further, as a possible cell death mechanism of action apoptosis was investigated. Although MEG3 is a widely known tumour suppressor gene and its loss is associated with several cancer types, here we reported that MEG3 inhibition can be used for improving the efficiency of known chemotherapeutic drug sensitivity. We propose that the level of MEG3 should be evaluated in the treatment of different glioma subtypes that are resistant to effective drugs to increase the potential effective drug applications.     

microRNA-199a-5p suppresses glioma progression by inhibiting MAGT1

microRNAs (miRNAs) can function as a tumor suppressor or oncogenic genes in human cancers. Alternation expression of miR-199a-5p has been revealed in several human cancers. However, its expression pattern and biological roles in glioma remain unclear. Expression levels of miR-199a-5p in glioma were evaluated at first. The effects of miR-199a-5p expression on cell proliferation, migration, and invasion were investigated using the MTT assay, wound-healing assay, and transwell invasion assay. The expression of miR-199a-5p was found to be reduced in glioma cell lines. Overexpression of miR-199a-5p inhibits glioma cell proliferation, migration, and invasion in vitro. Furthermore, the target of miR-199a-5p was predicted by TargetScan and validated by luciferase activity reporter assay. We found magnesium transporter 1 (MAGT1) was a direct target of miR-199a-5p. Overexpression of MAGT1 reversed the effects of miR-199a-5p on glioma cell behaviors. Taken together, our study revealed that miR-199a-5p and MAGT1 have the potential to be used as a biomarker for glioma.     

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.     

Overexpression of the long non-coding RNA MEG3 impairs in vitro glioma cell proliferation

Gliomas are the most common type of primary brain tumor in the central nervous system of adults. Maternally Expressed Gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a non-coding RNA (ncRNA) associated with tumorigenesis. However, little is known about whether and how MEG3 regulates glioma development. In the present study we assayed the expression of MEG3 in glioma tissue samples by real-time polymerase chain reaction assay, and defined the biological functions and target genes by CCK-8 assay, flow cytometry, and RNA immunoprecipitation. We first demonstrated that MEG3 expression was markedly decreased in glioma tissues compared with adjacent normal tissues. Moreover, ectopic expression of MEG3 inhibited cell proliferation and promoted cell apoptosis in U251 and U87 MG human glioma cell lines. We further verified that MEG3 was associated with p53 and that this association was required for p53 activation. These data suggest an important role of MEG3 in the molecular etiology of glioma and implicate the potential application of MEG3 in glioma therapy.     

G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。