Fraxetin exerts anticancer effect in glioma by suppressing MiR-21-3p.

Fraxetin (FXT) exerts anticancer function in multiple cancers, but its function on glioma was ill-defined. This article expounded the mechanism by which FXT exerts an anticancer effect in glioma. The effect of gradient concentration of FXT on the viability of glioma cell lines was determined by cell counting kit 8. Effects of FXT on proliferation, apoptosis, and cell cycle in glioma cell lines were determined by colony formation assay, flow cytometry, and Hoechst 33342 staining. Expressions of apoptosis-related gene, cycle-related gene, and glioma-related miRNAs after FXT (25 and 50 µmol/L) treatment were determined by quantitative reverse transcription polymerase chain reaction and western blot as needed. After miR-21-3p overexpression, cell viability and apoptosis of glioma cell lines treated with FXT (50 µmol/L) were tested again. Although 1 µmol/L FXT had no significant effect on cell viability, 5, 10, 25, and 50 µmol/L FXT suppressed cell viability in a concentration-dependent manner. FXT inhibited proliferation, promoted apoptosis, and induced cell cycle arrest in G0/G1 phase in glioma cell lines. These effects may be achieved by elevated expressions of Bax and cleaved caspase-3 and diminished expressions of Bcl-2, Bcl-XL, cyclin E1, cyclin D1, and cyclin-dependent kinase-6. FXT attenuated the contents of miR-21-3p and miR-455-3p, and escalated the contents of miR-124-3p and miR-7-5p. The regulation of FXT on cell viability, proliferation and apoptosis was reversed by miR-21-3p overexpression. FXT suppressed the development of glioma cells by downregulating miR-21-3p.

Activated protein C overexpression suppresses the pyroptosis of subarachnoid hemorrhage model cells by regulating the NLRP3 inflammasome pathway.

Subarachnoid hemorrhage (SAH) is a condition with a high associated mortality rate that is caused by hemorrhagic stroke. Activated protein C (APC) serves a neuroprotective role in central nervous system diseases. However, its role in SAH remains unclear. The present study aimed to investigate the role of APC and its regulatory mechanism in SAH. The SAH rat model was constructed through internal carotid artery puncture, while the SAH cell model was established via the application of oxygenated hemoglobin. ELISA was performed to detect the level of cytokines, and flow cytometry was used to determine the population of pyroptotic cells. Reverse transcription-quantitative PCR and western blotting were used to examine the relative mRNA and protein levels of APC. APC was silenced using specific APC short hairpin RNA. Neurological functions of rats were estimated using modified Garcia scoring and the balance beam test, while SAH was estimated using modified Sugawara's scoring. The results demonstrated that the expression of APC was significantly decreased, whereas the expression of NLR family pyrin domain-containing 3 (NLRP3) was increased in the SAH rat model in a time-dependent manner. The application of APC recombinant protein 3K3A-APC could significantly ameliorate SAH and improve neurological functions. In addition, 3K3A-APC could inhibit pyroptosis in a dose-dependent manner in the SAH cell model. Moreover, the NLRP3 inhibitor BAY11-7082 could reverse the upregulation of pyroptosis induced by APC-knockdown. Overall, the present study revealed that APC could ameliorate SAH-induced early brain injury by suppressing pyroptosis via inhibition of the NLRP3 inflammasome, which could provide a novel strategy for the treatment of SAH.

FBXW7 induces apoptosis in glioblastoma cells by regulating HDAC7.

Glioblastoma is an aggressive type of brain cancer with an extremely poor prognosis. Additionally, the F-box WD repeat-containing protein 7 (FBXW7) is a component of the ubiquitin-proteasome system that has been widely implicated in human cancers. In this study, we investigated the role and mechanism of FBXW7 in glioblastoma. FBXW7 expression was analyzed in normal and glioblastoma tissue samples using The Cancer Genome Atlas Glioblastoma Multiforme (TCGA-GBM) database. Then, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine mRNA expression, whereas, western blot analysis was conducted to determine protein levels of the samples. Furthermore, cell apoptosis was assessed using the Annexin V staining method, followed by flow cytometry analysis. Immunoprecipitation (IP) assay was conducted as well to test protein-protein interactions. Lastly, protein expression in tissues was examined by conducting immunohistochemistry (IHC). Results showed that the glioblastoma tissue samples displayed an FBXW7 downregulation compared with normal tissues. In vitro, the overexpression of FBXW7 in glioblastoma cells induced apoptosis, whereas, its knockdown displayed the opposite effect. Mechanistically, FBXW7 interacted with HDAC7 to promote HDAC7 ubiquitination, however, the overexpression of HDAC7 in glioblastoma cells blocked FBXW7-induced apoptosis. Finally, FBXW7 and HDAC7 displayed an inverse correlation in glioblastoma tissues in vivo. Therefore, our data demonstrated an important function of FBXW7 in promoting glioblastoma apoptosis by interacting with HDAC7 and promoting HDAC7 ubiquitination.

A novel tailored immune gene pairs signature for overall survival prediction in lower-grade gliomas.

Lower-grade gliomas (LGGs) have a good prognosis with a wide range of overall survival (OS) outcomes. An accurate prognostic system can better predict survival time. An RNA-Sequencing (RNA-seq) prognostic signature showed a better predictive power than clinical predictor models. A signature constructed using gene pairs can transcend changes from biological heterogeneity, technical biases, and different measurement platforms. RNA-seq coupled with corresponding clinical information were extracted from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). Immune-related gene pairs (IRGPs) were used to establish a prognostic signature through univariate and multivariate Cox proportional hazards regression. Weighted gene co-expression network analysis (WGCNA) was used to evaluate module eigengenes correlating with immune cell infiltration and to construct gene co-expression networks. Samples in the training and testing cohorts were dichotomized into high- and low-risk groups. Risk score was identified as an independent predictor, and exhibited a closed relationship with prognosis. WGCNA presented a gene set that was positively correlated with age, WHO grade, isocitrate dehydrogenase (IDH) mutation status, 1p/19 codeletion, risk score, and immune cell infiltrations (CD4 T cells, B cells, dendritic cells, and macrophages). A nomogram comprising of age, WHO grade, 1p/19q codeletion, and three gene pairs (BIRC5|SSTR2, BMP2|TNFRSF12A, and NRG3|TGFB2) was established as a tool for predicting OS. The IPGPs signature, which is associated with immune cell infiltration, is a novel tailored tool for individual-level prediction.

Calycosin down-regulates c-Met to suppress development of glioblastomas.

The antitumor effect of calycosin has been widely studied, but the targets of calycosin against glioblastomas are still unclear. In this study we focused on revealing c-Met as a potential target of calycosin suppressing glioblastomas. In this study, suppressed-cell proliferation and cell invasion together with induced-cell apoptosis appeared in calycosin-treated U251 and U87 cells. Under treatment of calycosin, the mRNA expression levels of Dtk, c-Met, Lyn and PYK2 were observed in U87 cells. Meanwhile a western blot assay showed that c-Met together with matrix metalloproteinases-9 (MMP9) and phosphorylation of the serine/threonine kinase AKT (p-AKT) was significantly down-regulated by calycosin. Furthermore, overexpressed c-Met in U87 enhanced the expression level of MMP9 and p-AKT and also improved cell invasion. Additionally, the expression levels of c-Met, MMP9 and p-AKT were inhibited by calycosin in c-Met overexpressed cells. However, an AKT inhibitor (LY294002) only effected on MMP9 and p-AKT, not on c-Met. These data collectively indicated that calycosin possibility targeting on c-Met and exert an anti-tumor role via MMP9 and AKT.

Inhibition of Proliferation by Knockdown of Transmembrane (TMEM) 168 in Glioblastoma Cells via Suppression of Wnt/ß-Catenin Pathway.

Human glioblastoma multiforme (GBM) accounts for the majority of human brain gliomas. Several TMEM proteins, such as TMEM 45A, TMEM 97, and TMEM 140, are implicated in human brain gliomas. However, the roles of TMEM168 in human GBM remain poorly understood. Herein we found that mRNA levels of TMEM168 were overexpressed in GBM patients (n = 85) when compared with healthy people (n = 10), which was also supported by data from The Cancer Genome Atlas (TCGA). Kaplan-Meier analysis of Gene Expression Omnibus dataset GSE16011 suggested that enhanced TMEM168 expression was associated with shorter survival time. To investigate whether and how TMEM168 functioned in the tumorigenesis of human GBM cells, two human GBM cell lines (U87 and U373) were used for study. Lithium chloride (LiCl), an activator for Wnt/ß-catenin pathway, was used for the treatment. Our data suggested that siRNA-TMEM168 (siTMEM168) prevented viability of U87 and U373 cells, induced cell cycle arrest (G0/G1 phase) and promoted apoptosis, and the mechanisms involved in blocking Wnt/ß-catenin pathway, as evidenced by reducing expression of ß-catenin, C-myc, cyclin D1, and survivin. Furthermore, the inhibited effect of siTMEM168 on human GBM cell growth was significantly alleviated with additional LiCl treatment, substantiating the involvement of the Wnt/ß-catenin pathway in this process. In summary, our data demonstrated that TMEM168 may represent a therapeutic target for the treatment of human GBM.

MiR-223-3p overexpression inhibits cell proliferation and migration by regulating inflammation-associated cytokines in glioblastomas.

Glioblastoma(GBM) is most common brain tumor in adults. Currently standard treatments have limited effect to increase the survival, because there are still largely unclear mechanisms in glioblastoma development. miR-223 was involved in various types of cancer, however, the function of miR-223-3p in GBM was still unclear. In our study, real-time PCR was performed to exam the expression level of miR-223-3p and NLRP3 (Nucleotide-binding oligomerization domain(NOD)-like receptor family PYRIN domain containing-3) in GBM tissues. Following that, mimic or inhibitor of miR-223-3p were used to modulate miR-223-3p expression in GBM cell lines respectively. Then, we analyzed cell proliferation and migration by cell counting kit and transwell assay. Further, western blot was performed to detect several inflammation-associated cytokines level in GBM cell lines. We found that miR-223-3p was decreased but NLRP3 was increased in GBM tissues. Treatment with miR-223-3p mimic inhibits cell proliferation and migration via decreasing several inflammation-associated cytokines, including interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), IL-8 and IL-18. Importantly, these effects induced by miR-223-3p could be attenuated by NLRP3 overexpression, which was considered as one of target genes of miR-223-3p. In conclusion, these results indicated that miR-223-3p might act as a suppressor and a potential therapy target of GBM.