Genome-wide DNA methylation analysis identifies potent CpG signature for temzolomide response in non-G-CIMP glioblastomas with unmethylated MGMT promoter: MGMT-dependent roles of GPR81.

PURPOSES: To identify potent DNA methylation candidates that could predict response to temozolomide (TMZ) in glioblastomas (GBMs) that do not have glioma-CpGs island methylator phenotype (G-CIMP) but have an unmethylated promoter of O-6-methylguanine-DNA methyltransferase (unMGMT). METHODS: The discovery-validation approach was planned incorporating a series of G-CIMP-/unMGMT GBM cohorts with DNA methylation microarray data and clinical information, to construct multi-CpG prediction models. Different bioinformatic and experimental analyses were performed for biological exploration. RESULTS: By analyzing discovery sets with radiotherapy (RT) plus TMZ versus RT alone, we identified a panel of 64 TMZ efficacy-related CpGs, from which a 10-CpG risk signature was further constructed. Both the 64-CpG panel and the 10-CpG risk signature were validated showing significant correlations with overall survival of G-CIMP-/unMGMT GBMs when treated with RT/TMZ, rather than RT alone. The 10-CpG risk signature was further observed for aiding TMZ choice by distinguishing differential outcomes to RT/TMZ versus RT within each risk subgroup. Functional studies on GPR81, the gene harboring one of the 10 CpGs, indicated its distinct impacts on TMZ resistance in GBM cells, which may be dependent on the status of MGMT expression. CONCLUSIONS: The 64 TMZ efficacy-related CpGs and in particular the 10-CpG risk signature may serve as promising predictive biomarker candidates for guiding optimal usage of TMZ in G-CIMP-/unMGMT GBMs.

High expression of RRM2 mediated by non-coding RNAs correlates with poor prognosis and tumor immune infiltration of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is known to have a poor prognosis. Accumulating evidence indicates that RRM2 plays a critical role in the occurrence and progression of multiple human cancers. However, the knowledge about RRM2 in HCC is still insufficient, and further research is needed. Here, we first analyzed the expression and prognosis of RRM2 using TCGA and GTEx data, and found that RRM2 may play a potential carcinogenic role in HCC. Then, through a series of comprehensive analysis, including expression analysis, correlation analysis or survival analysis, non-coding RNAs (ncRNAs) that regulate RRM2 overexpression were identified. Finally, MIR4435-2HG/CYTOR were observed to be the most promising upstream lncRNAs for the miR-125b-5p/RRM2 axis in HCC. In addition, RRM2 expression was significantly positively related to immune cell infiltration, immune cell biomarker or immune checkpoint expression in HCC. Altogether, the upregulation of RRM2 mediated by ncRNAs correlates with poor prognosis and tumor immune infiltration of HCC.

High Expression of RRM1 Mediated by ncRNAs Correlates with Poor Prognosis and Tumor Immune Infiltration of Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinoma (HCC) is one of several tumors with poor prognosis and causes a significant social burden. A growing number of studies have shown that RRM1 plays a crucial role in the development and progression of multiple human cancers. However, the specific role and mechanism of RRM1 have not been fully defined in HCC. Methods: TCGA and GTEx data were used for the first time to conduct a pan-cancer analysis of RRM1 expression and prognosis, and identified RRM1 as a possible potential oncogene in HCC. At the same time, a combination of analyses (including expression analysis, correlation analysis or survival analysis) identified non-coding RNAs (ncRNAs) that contribute to RRM1 overexpression. Results: MIR4435-2HG/miR-22-3p and SNHG6/miR-101-3p were identified as the most promising RRM1 upstream ncRNA-related pathways in HCC. In addition, RRM1 levels were significantly and positively correlated with tumor immune cell infiltration, immune cell biomarker or immune checkpoint expression. Conclusion: These results suggest that high expression of RRM1 mediated by ncRNAs is associated with poor prognosis and tumor immune infiltration in HCC.

A Novel Glycolysis and Hypoxia Combined Gene Signature Predicts the Prognosis and Affects Immune Infiltration of Patients with Colon Cancer.

PURPOSE: We aimed to characterize the expression patterns of glycolysis and hypoxia genes in colon cancers as well as their value in prognosis and immune microenvironment. METHODS: The expression profiles were acquired from the Cancer Genome Atlas database. Enrichment of hypoxia and glycolysis gene sets in colon cancer was identified by gene set enrichment analysis. Then, a prognostic signature was built up after Cox regression analyses, and overall survival analysis validated the predictive ability. Immune status and infiltration in cancer tissues were explored using the single sample gene set enrichment analysis and CIBERSORT algorithm. A nomogram model integrating clinical variables and the gene signature was established and assessed. RESULTS: Altogether, 378 cancer and 39 control cases were enrolled. Three glycolysis gene sets and two hypoxia gene sets were enriched in colon cancer (P < 0.05). Five independent genes (ENO3, GPC1, P4HA1, SPAG4, and STC2) were significantly correlated with prognosis of colon cancer patients. Patients with higher risks had significantly better prognosis than those with lower risks (P = 0.002 and AUC = 0.750), which was also observed in the elderly, female and stage I-II subgroups (P < 0.05). In high-risk cases, proportion of NK cells resting increased (P < 0.05) while that of dendritic cells activated (P < 0.05), dendritic cells resting (P < 0.01) and monocytes (P < 0.01) decreased. Besides, expressions of 22 checkpoint genes were found abnormal in groups with different risks (P < 0.05). The predictive nomogram presented satisfactory performance with C-index of 0.771 (0.712-0.830). The area under ROC curve was 0.796 and 0.803 for 3- and 5-year survival prediction, respectively. CONCLUSION: A glycolysis and hypoxia combined gene signature was a promising method to evaluate the prognosis and immune infiltration of colon cancer patients, which may provide a new tool for cancer management.

Bioinformatic Analysis of Prognostic Value, Genetic Interaction, and Immune Infiltration of Chromobox Family Proteins in Breast Cancer.

INTRODUCTION: Breast cancer (BC) has become the malignant tumor with the highest incidence worldwide. As a critical components of epigenetic regulation complexes, chromobox (CBX) family members inhibit the transcription of target genes through chromatin modification, leading to the progression of various human diseases and cancers. So far, little is known about the role of different CBX members in BC, especially their association with immune cells. METHODS: We conducted the analysis of differential expression of CBXs using Oncomine and GEPIA, prognostic value of CBXs using GEPIA and Kaplan-Meier, genetic interaction of CBXs using cBioPortal and GeneMANIA, and immune cell infiltration of CBXs in BC patients using TIMER. RESULTS: The CBX2/3/4/8 expression levels were increased significantly, while the CBX6/7 expression levels were decreased. We found that CBX3 was significantly correlated with clinicopathological staging and short DFS in BC patients. High CBX3/5 expression was correlated with short OS in BC patients, while high expression of CBX4 was correlated with long OS in BC patients. In addition, the functions of CBXs family members mainly focus on methylated histone residue binding and chromatin organization. The CBXs expressions were closely related to the infiltration level of a variety of immune cells, including CD4/8+ T cells, B cells, neutrophils, macrophages and dendritic cells in BC cancers. The correlation between CBXs and immune cell infiltration was more common in Luminal BC than in Basal and Her-2 type. CONCLUSION: This study may provide a new understanding for selection of molecular typing, therapeutic and prognostic biomarkers of CBX family in BC.

A glycolysis-related gene signature predicts prognosis of patients with esophageal adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma (EAC) is a growing problem with a rapidly rising incidence and carries a poor prognosis. We aimed to develop a glycolysis-related gene signature to predict the prognostic outcome of patients with EAC. RESULTS: Five genes (CLDN9, GFPT1, HMMR, RARS and STMN1) were correlated with prognosis of EAC patients. Patients were classified into high-risk and low-risk groups calculated by Cox regression analysis, based on the five gene signature risk score. The five-gene signature was an independent biomarker for prognosis and patients with low risk scores showed better prognosis. Nomogram incorporating the gene signature and clinical prognostic factors was effective in predicting the overall survival. CONCLUSION: An innovative identified glycolysis-related gene signature and an effective nomogram reliably predicted the prognosis of EAC patients. METHODS: The Cancer Genome Atlas database was investigated for the gene expression profile of EAC patients. Glycolytic gene sets difference between EAC and normal tissues were identified via Gene set enrichment analysis (GSEA). Univariate and multivariate Cox analysis were utilized to construct a prognostic gene signature. The signature was evaluated by receiver operating characteristic curves and Kaplan-Meier curves. A prognosis model integrating clinical parameters with the gene signature was established with nomogram.

The Effect of Metformin on the Clinicopathological Features of Breast Cancer With Type 2 Diabetes.

BACKGROUND: The present study aimed to review the use of hypoglycemic drugs and clinicopathological data in breast cancer patients with type 2 diabetes mellitus (T2DM), and to investigate the effect of metformin on the clinicopathological features of breast cancer in patient with T2DM. METHODS: Eighty-nine patients with breast cancer hospitalized in the Second Affiliated Hospital of Xi'an Jiaotong University from January 2012 to December 2014 were included. Thirty-three patients were on metformin (metformin group) and 56 patients were on control group. Streptavidin-peroxidase (SP) method was used to quantify protein expression of molecular markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)), molecular markers of proliferation (Ki-67 and epidermal growth factor receptor (EGFR)) and epithelial-mesenchymal transition (EMT) molecular markers (matrix metalloproteinase-2 (MMP-2), E-cadherin and downstream N-cadherin). Fluorescence in situ hybridization was used to detect HER-2 (+ and ++). RESULTS: The rate of lymph node metastasis and the level of Ki-67/MMP-2 in the metformin group were significantly lower than those in the control group (P < 0.05). The ratio of luminal pattern in metformin group was higher than that in the control group (P < 0.05). However, there were no differences in the parameters of age, duration of diabetes, body mass index, tumor size, histological grade of cancer and clinical pathological features between the two groups. No significant difference was observed in the expressions of ER, PR, HER-2, EGFR, E-cadherin, N-cadherin and the recurrence rate between two groups. CONCLUSIONS: Metformin is associated with luminal breast cancer and can inhibit breast cancer invasion and metastasis in some cases. It may be associated with EMT and is beneficial to the prognosis of breast cancer.

Chikungunya-vesicular stomatitis chimeric virus targets and eliminates brain tumors.

Vesicular stomatitis virus (VSV) shows potential for targeting and killing cancer cells, but can be dangerous in the brain due to its neurotropic glycoprotein. Here we test a chimeric virus in which the VSV glycoprotein is replaced with the Chikungunya polyprotein E3-E2-6K-E1 (VSVΔG-CHIKV). Control mice with brain tumors survived a mean of 40 days after tumor implant. VSVΔG-CHIKV selectively infected and eliminated the tumor, and extended survival substantially in all tumor-bearing mice to over 100 days. VSVΔG-CHIKV also targeted intracranial primary patient derived melanoma xenografts. Virus injected into one melanoma spread to other melanomas within the same brain with little detectable infection of normal cells. Intravenous VSVΔG-CHIKV infected tumor cells but not normal tissue. In immunocompetent mice, VSVΔG-CHIKV selectively infected mouse melanoma cells within the brain. These data suggest VSVΔG-CHIKV can target and destroy brain tumors in multiple animal models without the neurotropism associated with the wild type VSV glycoprotein.

Zika Virus Targeting in the Developing Brain.

Zika virus (ZIKV), a positive-sense RNA flavivirus, has attracted considerable attention recently for its potential to cause serious neurological problems, including microcephaly, cortical thinning, and blindness during early development. Recent findings suggest that ZIKV infection of the brain can occur not only during very early stages of development, but also in later fetal/early neonatal stages of maturation. Surprisingly, after peripheral inoculation of immunocompetent mice on the day of birth, the first cells targeted throughout the brain were isolated astrocytes. At later stages, more neurons showed ZIKV immunoreactivity, in part potentially due to ZIKV release from infected astrocytes. In all developing mice studied, we detected infection of retinal neurons; in many mice, this was also associated with infection of the lateral geniculate, suprachiasmatic nuclei, and superior colliculus, suggesting a commonality for the virus to infect cells of the visual system. Interestingly, in mature mice lacking a Type 1 interferon response (IFNR-/-), after inoculation of the eye, the initial majority of infected cells in the visual system were glial cells along the optic tract. ZIKV microinjection into the somatosensory cortex on one side of the normal mouse brain resulted in mirror infection restricted to the contralateral somatosensory cortex without any infection of midline brain regions, indicating the virus can move by axonal transport to synaptically coupled brain loci. These data support the view that ZIKV shows considerable complexity in targeting the CNS and may target different cells at different stages of brain development.SIGNIFICANCE STATEMENT Zika virus (ZIKV) can cause substantial damage to the developing human brain. Here we examine a developmental mouse model of ZIKV infection in the newborn mouse in which the brain is developmentally similar to a second-trimester human fetus. After peripheral inoculation, the virus entered the CNS in all mice tested and initially targeted astrocytes throughout the brain. Infections of the retina were detected in all mice, and infection of CNS visual system nuclei in the brain was common. We find that ZIKV can be transported axonally, thereby enhancing virus spread within the brain. These data suggest that ZIKV infects multiple cell types within the brain and that astrocyte infection may play a more important role in initial infection than previously appreciated.

Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain.

Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain.IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism.

MicroRNA-128-3p Protects Mouse Against Cerebral Ischemia Through Reducing p38α Mitogen-Activated Protein Kinase Activity.

The p38α, also named Mapk14, is a pro-apoptotic protein, which is reported to be downregulated 2 h after cerebral ischemia. However, little is known what causes the downregulation of p38α protein level. Here, we studied the effect of cerebral ischemia on p38α mRNA expression and p38α protein level in brain of mice. We found that p38α protein level is reduced after middle cerebral artery occlusion. However, at the meantime, p38α mRNA expression has no detectable changes, suggesting that the possible posttranscription is regulated by ischemia. To reveal the mechanism for posttranscription of p38α protein, we tested the effect of miR-128-3p. Using luciferase reporter assay, we found that miR-128-3p could directly target p38α 3'UTR. We further tested the effect of miR-128-3p on the p38α protein level. We found that miR-128-3p strongly decreased the p38α protein level in SH-SY5Y cells after the cells were transfected with miR-128-3p using lentivirus vector containing precursor its RNA sequences. We further found that inhibition of miR-128-3p enhanced the infarct volume of brain in mice. Our study thus confirms that miR-128-3p can downregulate p38α protein level through posttranscription and increase of miR-128-3p level may contribute to neuronal survival in ischemia-induced brain injury.

Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Into Neuron-Like Cells In Vitro and Co-Cultured with Biological Scaffold as Transplantation Carrier.

BACKGROUND Autograft and allograft transplantation are used to prompt the regeneration of axons after nerve injury. However, the poor self-regeneration caused by the glial scar and growth inhibitory factors after neuronal necrosis limit the efficacy of these methods. The purpose of this study was to develop a new chitosan porous scaffold for cell seeding. MATERIAL AND METHODS The bone marrow mesenchymal stem cells (BMSCs) and tissue-engineered biomaterial scaffold compound were constructed and co-cultured in vitro with the differentiated BMSCs of Wistar rats and chitosan scaffold in a 3D environment. The purity of the third-generation BMSCs culture was identified using flow cytometry and assessment of induced neuronal differentiation. The scaffolds were prepared by the freeze-drying method. The internal structure of scaffolds and the change of cells' growth and morphology were observed under a scanning electron microscope. The proliferation of cells was detected with the MTT method. RESULTS On day 5 there was a significant difference in the absorbance value of the experimental group (0.549±0.0256) and the control group (0.487±0.0357) (P>0.05); but on day 7 there was no significant difference in the proliferation of the experimental group (0.751±0.011) and the control group and (0.78±0.017) (P>0.05). CONCLUSIONS Tissue engineering technology can provide a carrier for cells seeding and is expected to become an effective method for the regeneration and repair of nerve cells. Our study showed that chitosan porous scaffolds can be used for such purposes.

Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke.

In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold in vitro, transplanted either the composite or bone marrow mesenchymal stem cells alone into the ischemic area in animal models, and compared their effects. At 14 days after co-transplantation of bone marrow mesenchymal stem cells and the hitosan-collagen scaffold, neurological function recovered noticeably. Vascular endothelial growth factor expression and nestin-labeled neural precursor cells were detected in the ischemic area, surrounding tissue, hippocampal dentate gyrus and subventricular zone. Simultaneously, a high level of expression of glial fibrillary acidic protein and a low level of expression of neuron-specific enolase were visible in BrdU-labeled bone marrow mesenchymal stem cells. These findings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold has a neuroprotective effect following ischemic stroke.