Canagliflozin protects against hyperglycemia-induced cerebrovascular injury by preventing blood-brain barrier (BBB) disruption via AMPK/Sp1/adenosine A2A receptor.

Diabetes mellitus causes brain microvascular endothelial cell (MEC) damage, inducing dysfunctional angiogenic response and disruption of the blood-brain barrier (BBB). Canagliflozin is a revolutionary hypoglycemic drug that exerts neurologic and/or vascular-protective effects beyond glycemic control; however, its underlying mechanism remains unclear. In the present study, we hypothesize that canagliflozin ameliorates BBB permeability by preventing diabetes-induced brain MEC damage. Mice with high-fat diet/streptozotocin-induced diabetes received canagliflozin for 8 weeks. We assessed vascular integrity by measuring cerebrovascular neovascularization indices. The expression of specificity protein 1 (Sp1), as well as tight junction proteins (TJs), phosphorylated AMP-activated protein kinase (p-AMPK), and adenosine A2A receptors was examined. Mouse brain MECs were grown in high glucose (30 mM) to mimic diabetic conditions. They were treated with/without canagliflozin and assessed for migration and angiogenic ability. We also performed validation studies using AMPK activator (AICAR), inhibitor (Compound C), Sp1 small interfering RNA (siRNA), and adenosine A2A receptor siRNA. We observed that cerebral pathological neovascularization indices were significantly normalized in mice treated with canagliflozin. Increased Sp1 and adenosine A2A receptor expression and decreased p-AMPK and TJ expression were observed under diabetic conditions. Canagliflozin or AICAR treatment alleviated these changes. However, this alleviation effect of canagliflozin was diminished again after Compound C treatment. Either Sp1 siRNA or adenosine A2A receptor siRNA could increase the expression of TJs. Luciferase reporter assay confirmed that Sp1 could bind to the adenosine A2A receptor gene promoter. Our study identifies the AMPK/Sp1/adenosine A2A receptor pathway as a treatment target for diabetes-induced cerebrovascular injury.

Consistency of mouse models with human intracerebral hemorrhage: core targets and non-coding RNA regulatory axis.

Intracerebral hemorrhage (ICH) has a high mortality and disability rate. Numerous basic studies on pathogenesis and therapeutics have been performed in mice. However, the consistency of the experimental mouse model and the human ICH patient remains unclear. This has slowed progress in translational medicine. Furthermore, effective therapeutic targets and reliable regulatory networks for ICH are needed. Therefore, we determined the differentially expressed (DE) messenger RNAs (mRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) before and after murine ICH and analyzed their regulatory relationships. Subsequently, data on mRNAs from human peripheral blood after ICH were obtained from the Gene Expression Omnibus database. The DE mRNAs after human ICH were compared with those of the mouse. Finally, we obtained seven genes with translational medicine research value and verified them in mice. Then the regulatory network of these genes was analyzed in humans. Similarly, species homologies of these regulatory pathways were identified. In conclusion, we found that the mouse ICH model mimics the human disease mainly in terms of chemokines and inflammatory factors. This has important implications for future research into the mechanisms of ICH injury and repair.

CircTrim37 Ameliorates Intracerebral Hemorrhage Outcomes by Modulating Microglial Polarization via the miR-30c-5p/SOCS3 Axis.

Circular RNAs (circRNAs) have been progressively recognized as critical regulators in the pathology and pathophysiology of central nervous system disease. However, the potential role of circRNAs in intracerebral hemorrhage (ICH) is still largely unclear. Here, we demonstrate that circTrim37 expression was significantly upregulated at 3 days after ICH by circular RNA microarray and qPCR assays. Overexpression of circTrim37 could significantly ameliorate brain injury volume, brain edema, neurologic deficits, and inflammation in vivo after ICH. CircTrim37 promotes M2 polarization while restrains M1 polarization in vitro. Furthermore, circTrim37 acts as an endogenous sponge for miR-30c-5p, thereby inhibiting miR-30c-5p activity, leading to the upregulation of SOCS3 and making the balance of microglial response towards an M2 phenotype. Taken together, our results indicate the participation of circTrim37 and its coupling mechanism in ICH and provide a novel therapeutic target for ICH.

Intracerebral Hemorrhage-Induced Brain Injury: the Role of Lysosomal-Associated Transmembrane Protein 5.

Intracerebral hemorrhage (ICH) is a lethal stroke with high mortality or disability. However, effective therapy for ICH damage is generally lacking. Previous investigations have suggested that lysosomal protein transmembrane 5 (LAPTM5) is involved in various pathological processes, including autophagy, apoptosis, and inflammation. In this study, we aimed to identify the expression and functions of LAPTM5 in collagenase-induced ICH mouse models and hemoglobin-induced cell models. We found that LAPTM5 was highly expressed in brain tissues around the hematoma, and double immunostaining studies showed that LAPTM5 was co-expressed with microglia cells, neurons, and astrocytes. Following ICH, the mice presented increased brain edema, blood-brain barrier permeability, and neurological deficits, while pathological symptoms were alleviated after the LAPTM5 knockdown. Adeno-associated virus 9-mediated downregulation of LAPTM5 also improves ICH-induced secondary cerebral damage, including neuronal degeneration, the polarization of M1-like microglia, and inflammatory cascades. Furthermore, LAPTM5 promoted activation of the nuclear factor kappa-B (NF-κB) pathway in response to neuroinflammation. Further investigations indicated that brain injury improved by LAPTM5 knockdown was further exacerbated after the overexpression of receptor-interacting protein kinase 1 (RIP1), which is revealed to trigger the NF-κB pathway. In vitro experiments demonstrated that LAPTM5 silencing inhibited hemoglobin-induced cell function and confirmed regulation between RIP1 and LAPTM5. In conclusion, the present study indicates that LAPTM5 may act as a positive regulator in the context of ICH by modulating the RIP1/NF-κB pathway. Thus, it may be a candidate gene for further study of molecular or therapeutic targets.

MALAT1 knockdown alleviates the pyroptosis of microglias in diabetic cerebral ischemia via regulating STAT1 mediated NLRP3 transcription.

BACKGROUND: Dysregulated long non-coding RNAs participate in the development of diabetic cerebral ischemia. This study aimed to investigate the underlying mechanism of lncRNA MALAT1 in diabetic cerebral ischemia. METHOD: Middle cerebral artery occlusion (MCAO) was performed to establish diabetic cerebral I/R in vivo. TTC and neurological deficits assessment were performed to assess cerebral ischemic injury. LDH was conducted to detect cytotoxicity. RT-qPCR and western blotting assays were applied to determine mRNA and protein expression. Flow cytometry was performed to detect the pyroptosis of BV2 cells. Immunofluorescence and FISH were conducted for subcellular localization of MALAT1 and STAT1. ELISA was performed to determine cytokine release. Dual luciferase reporter, RIP, and ChIP assays were used to validate the interaction between STAT1 and MALAT1/NLRP3. Diabetes aggravated cerebral injury in vivo and in vitro. Diabetic cerebral ischemia induced inflammatory response and inflammation-induced cell pyroptosis. RESULT: MALAT1 was overexpressed in diabetic cerebral ischemia models in vivo and in vitro. However, knockdown of MALAT1 suppressed inflammatory response and the pyroptosis of BV2 cells. Moreover, MALAT1 interacted with STAT1 to transcriptionally activate NLRP3. Knockdown of STAT1 significantly reversed the effects of MALAT1. Furthermore, STAT1 promotes the MALAT1 transcription. MALAT1 interacts with STAT1 to promote the pyroptosis of microglias induced by diabetic cerebral ischemia through activating NLRP3 transcription. CONCLUSION: Thus, knockdown of MALAT1 may be a potential promising therapy target for diabetic cerebral ischemia.

Vision transformer-based weakly supervised histopathological image analysis of primary brain tumors.

Diagnosis of primary brain tumors relies heavily on histopathology. Although various computational pathology methods have been developed for automated diagnosis of primary brain tumors, they usually require neuropathologists' annotation of region of interests or selection of image patches on whole-slide images (WSI). We developed an end-to-end Vision Transformer (ViT) - based deep learning architecture for brain tumor WSI analysis, yielding a highly interpretable deep-learning model, ViT-WSI. Based on the principle of weakly supervised machine learning, ViT-WSI accomplishes the task of major primary brain tumor type and subtype classification. Using a systematic gradient-based attribution analysis procedure, ViT-WSI can discover diagnostic histopathological features for primary brain tumors. Furthermore, we demonstrated that ViT-WSI has high predictive power of inferring the status of three diagnostic glioma molecular markers, IDH1 mutation, p53 mutation, and MGMT methylation, directly from H&E-stained histopathological images, with patient level AUC scores of 0.960, 0.874, and 0.845, respectively.

Ethyl Pyruvate Alleviating Inflammatory Response after Diabetic Cerebral Hemorrhage.

OBJECTIVE: This study's purpose is to investigate the neuroprotective role of ethyl pyruvate (EP) in the pathogenesis of diabetic intracerebral hemorrhage. METHODS: The present study used a mouse model of collagenase-induced intracerebral hemorrhage (ICH) and streptozotocin-induced diabetes. The C57BL/6 mice were randomly divided into 3 groups: sham operation, diabetic cerebral hemorrhage, and diabetic cerebral hemorrhage with EP. The EP (80 mg/kg) and EP (50 mg/kg) were injected intraperitoneally one day and one hour before modeling. The protein expression levels of high mobility group box 1 (HMGB1) and NOD-like receptors 3 (NLRP3) were detected with western blot. The mRNA levels of HMGB1 and toll-like receptor 4 (TLR4) were measured by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and ELISA were performed to confirm some inflammatory factors. RESULTS: Compared to the normal diabetic intracerebral hemorrhage group, the mRNA and protein expression levels of HMGB1 and TLR4 were downregulated in the EP-affected group with diabetic cerebral hemorrhage, together with the downregulation of the expression of inflammasomes, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase 1. CONCLUSION: EP can reduce the inflammatory response after diabetic intracerebral hemorrhage and may inhibit the activation of inflammasomes by the HMGB1/TLR4 pathway.

Heme Oxygenase-1 may Mediate Early Inflammatory Response of Intracerebral Hemorrhage through Toll-like Receptor 4 Signaling Pathway.

OBJECTIVE: The aim of this study was to investigate whether heme oxygenase-1 (HO-1) promotes an early neuroinflammatory response after intracerebral hemorrhage (ICH) by regulating the toll-like receptor 4 (TLR4) signaling pathway. METHODS: We used a stereotaxic instrument to induce a mouse model of ICH through collagenase. We divided the participants into a control group, an ICH group, and an ICH and zinc protoporphyrin IX (ZnPP) group. The temporal expression pattern and cell localization of HO-1 and TLR4 after the ICH were detected by immunofluorescence and western blot; after the expression of HO-1 was inhibited, the expression levels of the TLR4 protein, the downstream molecule myeloid differentiation factor 88 (MyD88), the Toll and interleukin-1 receptor (TIR) -domain-containing adapter-inducing interferon-ß (TRIF) and the inflammatory factors were measured by western blotting. RESULTS: Immunofluorescence showed that HO-1 and TLR4 had similar temporal expression patterns and cellular localization after ICH, and we found that inhibiting HO-1 reduces the expression of TLR4 protein pathways, including TLR4, MyD88, TRIF, and related inflammatory factors, by studying the inhibitor ZnPP. CONCLUSION: These results indicate that HO-1 may promote early neuroinflammation after ICH through the TLR4/MyD88/TRIF signaling pathway.

Long Noncoding RNA MALAT1 and Colorectal Cancer: A Propensity Score Analysis of Two Prospective Cohorts.

Background: Previous researches have shown that the aberrant expression of Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) in tumour tissues may serve as a biomarker for colorectal cancer (CRC) prognosis. However, these previous studies have small sample sizes and lacked validation from independent external populations. We therefore aimed to clarify the prognostic value of MALAT1 expression status in CRC patients using a large cohort and validate the findings with another large external cohort. Methods: The prognostic association between MALAT1 expression status and CRC outcomes was evaluated initially in a prospective cohort in China (n=164) and then validated in an external TCGA population (n=596). In the initial cohort, MALAT1 expression levels were quantified by quantitative reverse transcriptase polymerase chain reaction. Propensity score (PS) adjustment method was used to control potential confounding biases. The prognostic significance was reported as PS-adjusted hazard ratio (HR) and corresponding 95% confidence interval (CI). Results: There was no statistically significant association between MALAT1 expression status and CRC patient overall survival (OS) or disease free survival (DFS) in both initial cohort and external validation cohort populations. When combining these populations together, the results did not change materially. The summarized HRPS-adjusted were 1.010 (95% CI, 0.752-1.355, P=0.950) and 1.170 (95% CI, 0.910-1.502, P=0.220) for OS and DFS, respectively. Conclusions: MALAT1 expression status is not associated with prognostic outcomes of CRC patients. However, additional larger population studies are needed to further validate these findings.

Locally secreted BiTEs complement CAR T cells by enhancing killing of antigen heterogeneous solid tumors.

Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. We hypothesized that BiTE-secreting T cells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent T cells compared with bivalent CAR T cells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

Dandelion sterol improves diabetes mellitus-induced renal injury in in vitro and in vivo study.

The purpose of our research was to evaluate Dandelion sterol's treatment effects on diabetes mellitus-induced renal injury in in vitro and in vivo study. The rats were divided into five groups as normal control (Ctrl), diabetic nephropathy model (Model), Dandelion sterol low-dose treated (Dan-Low), Dandelion sterol middle-dose treated (Dan-Middle), and Dandelion sterol high-dose treated (Dan-High). Measuring serum TNF-α, IL-1ß, and IL-6 concentrations by Elisa assay, evaluate kidney pathology by HE staining, kidney cell apoptosis of TUNEL, TLR4, and NF-κB(p65) proteins expression by IHC assay, and relative gene expressions by RT-qPCR assay. In the following step, using HK-2 treated with high glucose to model DN cell model to discuss the relative mechanisms, evaluate TNF-α, IL-1ß, and IL-6 concentrations by Elisa assay, evaluate cell apoptosis by flow cytometry, evaluate TLR4 and NF-κB(p65) proteins expression by WB assay, relative gene expression by RT-qPCR assay, and NF-κB(p65) nuclear volume by cellular immunofluorescence. Compared with Ctrl group, TNF-α, IL-1ß, and IL-6 concentrations and apoptosis cell number were significantly increased, TLR4/NF-κB(p65) pathway was significantly stimulated in Model rats and cell groups. With Dan supplement, the diabetic-induced renal injury was significantly improved (p < .05, respectively). By cell experiment, Dan improved cell apoptosis and inflammatory factors via miR-140-5p. Dan improved diabetes mellitus-induced renal injury via regulation of miR-140-5p/TLR4 axis in in vitro and in vivo study.

Berberine elevates mitochondrial membrane potential and decreases reactive oxygen species by inhibiting the Rho/ROCK pathway in rats with diabetic encephalopathy.

OBJECTIVES: Diabetic encephalopathy (DE) is a serious complication of diabetes mainly occurring in the elderly patients. Berberine (BBR) is an isoquinoline alkaloids extracted from Coptis chinensis that is applied in the treatment of diabetes clinically. This study explored the possible mechanism of BBR in relieving DE. METHODS: Wistar rats were injected with streptozotocin and fed a high fat diet to establish the model of DE. The model rats were treated with BBR. The body weight, blood glucose and insulin of rats were measured, and Morris water maze test was conducted to evaluate the learning and memory abilities. The pathological conditions of cortical tissues were detected. The cortical mitochondria membrane potential (MMP) and reactive oxygen species (ROS) were monitored. The expressions of Rho/ROCK pathway-related genes of rat cortex were detected. The changes of MMP and ROS were detected after the treatment of Rho/ROCK pathway activator. RESULTS: The body weight of model rats changed little, and levels of blood glucose and insulin were increased. The spatial learning and memory abilities were impaired, with disordered cortical neurons, and obvious neurons apoptosis and glia proliferation. BBR alleviated cognitive dysfunction and pathological damage in rats with DE. BBR enhanced cortical MMP and suppressed ROS. BBR treatment inhibited the Rho/ROCK pathway. Activation of the Rho/ROCK pathway reversed the effects of BBR on MMP and ROS. CONCLUSION: BBR elevated MMP and reduced ROS in rats with DE by inhibiting the Rho/ROCK pathway. This study may offer novel insights for the management of DE.

Androgen Receptor Splice Variant 7 Predicts Shorter Response in Patients with Metastatic Hormone-sensitive Prostate Cancer Receiving Androgen Deprivation Therapy.

BACKGROUND: Whether AR-V7 expression can predict the response in patients with metastatic hormone-sensitive prostate cancer (mHSPC) who receive androgen deprivation therapy (ADT) remains to be explored. OBJECTIVE: To evaluate the predictive value of AR-V7 expression in the prognosis of mHSPC patients receiving ADT. DESIGN, SETTING, AND PARTICIPANTS: In this multicenter prospective cohort study, 310 mHSPC patients commencing ADT were enrolled. Standard immunohistochemical staining was used to assess AR-V7 protein expression in biopsy tissues collected before initiation of ADT. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Kaplan-Meier survival estimates and Cox regression analyses were used to evaluate associations of AR-V7 status (positive vs negative) with progression-free survival (PFS) and overall survival (OS). RESULTS AND LIMITATIONS: Sixty-four (21%) patients were AR-V7-positive and 246 (79%) patients were AR-V7-negative. The median follow-up for patients not confirmed dead was 25 mo (interquartile range 10-30). Compared to AR-V7-negative patients, AR-V7-positive patients had significantly shorter PFS (hazard ratio [HR] 47.39, 95% confidence interval [CI] 25.83-86.94) and OS (HR 3.57, 95% CI 1.46-8.72). In multivariable analysis, AR-V7 was an independent predictive factor (HR 7.61, 95% CI 5.24-11.06) for shorter PFS. Limitations include the sample size and follow-up period. CONCLUSIONS: AR-V7 expression in primary cancer tissue is correlated with poor prognosis for mHSPC patients receiving ADT. PATIENT SUMMARY: In this study of men with metastatic hormone-sensitive prostate cancer, AR-V7 protein expression in primary cancer tissue was associated with poor outcomes on androgen deprivation therapy.

Protective roles of liraglutide against brain injury of streptozotocin induced diabetic rats.

Our study aimed to explore the impacts of liraglutide on brain dysfunction of type 2 diabetes mellitus. Rats in liraglutide treatment group were diabetic rats further received daily intraperitoneal administration of liraglutide for continuous 6 weeks. Body weight and blood glucose were measured weekly. Vascular structure changes in brain tissues were evaluated by Periodic acid-Schiff (PAS) staining. Angiopoietin-2 (ANG-2), high-mobility group box 1 (HMGB-1), CD105, NeuN, Oligo-2 in brain tissues were measured by immunohistochemistry staining and ANG-2, HMGB-1, and matrix metalloproteinase-9 (MMP-9) were detected by western blotting. Blood glucose levels of rats in diabetic model group were significantly elevated and blood glucose levels of rats in liraglutide treatment group were reduced to comparable levels with control group. PAS staining showed vascular basement membrane of rats in the diabetic model group was thicker than that of the control group. ANG-2, HMGB1 and MMP-9 were up-regulated in the diabetic model group comparing the control group, while down-regulated after treated with liraglutide (p<0.05). NeuN expressions were significantly higher in liraglutide treatment group. Liraglutide may have protective roles against brain injury of streptozotocin induced diabetic rats by inhibiting HMGB1, which further suppressing the MMP-9 and ANG-2.

miR-183-5p alleviates early injury after intracerebral hemorrhage by inhibiting heme oxygenase-1 expression.

Differences in microRNA (miRNA) expression after intracerebral hemorrhage (ICH) have been reported in human and animal models, and miRNAs are being investigated as a new treatment for inflammation and oxidative stress after ICH. In this study, we found that microRNA-183-5p expression was decreased in the mouse brain after ICH. To investigate the effect of miRNA-183-5p on injury and repair of brain tissue after ICH, saline, miRNA-183-5p agomir, or miRNA-183-5p antagomir were injected into the lateral ventricles of 8-week-old mice with collagenase-induced ICH. Three days after ICH, mice treated with exogenous miRNA-183-5p showed less brain edema, neurobehavioral defects, inflammation, oxidative stress, and ferrous deposition than control mice. In addition, by alternately treating mice with a heme oxygenase-1 (HO-1) inducer, a HO-1 inhibitor, a nuclear factor erythroid 2-related factor (Nrf2) activator, and Nrf2 knockout, we demonstrated an indirect, HO-1-dependent regulatory relationship between miRNA-183-5p and Nrf2. Our results indicate that miRNA-183-5p and HO-1 are promising therapeutic targets for controlling inflammation and oxidative damage after hemorrhagic stroke.

A Promising New Anti-Cancer Strategy: Iron Chelators Targeting CSCs.

Iron is a trace but vital element in the human body and is necessary for a multitude of crucial processes in life. However, iron overload is known to induce carcinogenesis via oxidative stress. Cancer cells require large amounts of iron for their rapid division and cell growth. Iron was recently found to play a role in cancer stem cells (CSCs); it maintains stemness during development. Iron also plays an important role in stemness by moderating reactive oxygen species. Thus, iron metabolism in CSCs is a promising therapeutic target. In this review, we summarize the roles of iron in cancer cells and CSCs. We also summarize anti-cancer therapeutic studies with iron chelators and describe our expectation of a new therapeutic strategy for CSCs on the basis of our findings.

The protection of New Interacting Motif E shot (NIMoEsh) in mice with collagenase-induced acute stage of intracerebral hemorrhage.

Aberrant c-Jun N terminal kinase (JNK) activation is broadly involved in the pathogenesis of several acute and chronic neurological diseases. However, the mechanism of JNK activation leading to aggravation of injury after ICH remains unclear. In this study, we confirmed that using NIMoEsh to inhibit JNK activation effectively reduced the level of brain injury following ICH. We evaluated brain outcomes by histology, immunofluorescence, Luxol fast blue/Cresyl violet staining and other experimental methods. We found that NIMoEsh could significantly inhibit the activity of JNK and thus improve inflammation, white-matter damage and neuronal cell death after ICH in mice. Our results suggest that JNK activation plays an important role of brain damage after acute stage of ICH and that NIMoEsh may be a potential target drug for the treatment of ICH.

CD4+ CD25+ regulatory T cells promote hepatocellular carcinoma invasion via TGF-ß1-induced epithelial-mesenchymal transition.

BACKGROUND: CD4+ CD25+ regulatory T cells (Tregs), a crucial component of the infiltration of immune cells in tumor microenvironment, are associated with progression and metastasis of hepatocellular carcinoma (HCC). METHODS: The mechanism of Tregs in the invasion and metastasis of HCC was investigated in vivo and in vitro using immunohistochemical analysis, western blot, and quantitative reverse transcription-PCR (qRT-PCR). RESULTS: Analysis of 78 clinical HCC samples indicated that high expression of Tregs was strongly associated with poor cancer-free survival and overall survival of patients. The reduced expression of E-cadherin and enhanced expression of Vimentin and transforming growth factor-beta 1 (TGF-ß1) were found in HCC tissue compared with normal liver tissue. The HCC Hepa1-6 cells were treated with the supernatant of Tregs-conditioned medium (Tregs-CM) to investigate the epithelial-mesenchymal transition (EMT) and TGF-ß1. Western blot and qRT-PCR also showed that down-regulated E-cadherin and up-regulated Vimentin and TGF-ß1 were found in Tregs-CM-treated Hepa1-6 cells. An experiment of tumorigenicity in C57 mice showed larger and heavier tumors in Tregs-CM-treated group than in the control group. Tregs produced higher TGF-ß1 compared with Tregs treated with FOXP3 shRNA. TGF-ß1 with neutralizing antibodies was used to deplete TGF-ß1 in Tregs-CM, which enhanced expression of E-cadherin, reduced expression of Vimentin and TGF-ß1, and decreased migratory and invasive capacity of Hepa1-6 cells. CONCLUSION: Tregs could promote the invasion and migration of Hepa1-6 cells, which are possibly maintained by TGF-ß1-induced EMT. This study showed that the development of therapeutic strategies against TGF-ß1 pathway is valuable in HCC therapy.

The IDH1 Mutation-Induced Oncometabolite, 2-Hydroxyglutarate, May Affect DNA Methylation and Expression of PD-L1 in Gliomas.

Background: Malignant gliomas are heterogeneous brain tumors with the potential for aggressive disease progression, as influenced by suppressive immunoediting. Given the success and enhanced potential of immune-checkpoint inhibitors in immunotherapy, we focused on the connections between genetic alterations affected by IDH1 mutations and immunological landscape changes and PDL-1 expression in gliomas. Methods: Paired surgically resected tumors from lower-grade gliomas (LGGs) and glioblastomas (GBM) were investigated, and a genetic analysis of patients' primary tumor samples culled from TCGA datasets was performed. Results: The results demonstrate that when compared with IDH1-mutant tumors, IDH1 wildtype tumors represent an immunosuppression landscape and elevated levels of PD-L1 expression. DNA hypo-methylation of the PD-L1 gene, as well as high gene and protein expressions, were observed in the wildtype tumors. We also found that quantitative levels of IDH1 mutant proteins were positively associated with recurrence-free survival (RFS). A key product of the IDH1 mutation (2-hydroxyglutarate) was found to transiently increase DNA methylation and suppress PD-L1 expression. Conclusions: IDH1 mutations impact the immune landscape of gliomas by affecting immune infiltrations and manipulating checkpoint ligand PD-L1 expression. Applications of immune checkpoint inhibitors may be beneficial for chemoradiation-insensitive IDH1-wildtype gliomas.