Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines, anti-PD-1 and poly I:C.

Glioblastoma (GBM) is a lethal cancer with limited therapeutic options. Dendritic cell (DC)-based cancer vaccines provide a promising approach for GBM treatment. Clinical studies suggest that other immunotherapeutic agents may be combined with DC vaccines to further enhance antitumor activity. Here, we report a GBM case with combination immunotherapy consisting of DC vaccines, anti-programmed death-1 (anti-PD-1) and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy, and the patient remained disease-free for 69 months. The patient received DC vaccines loaded with multiple forms of tumor antigens, including mRNA-tumor associated antigens (TAA), mRNA-neoantigens, and hypochlorous acid (HOCl)-oxidized tumor lysates. Furthermore, mRNA-TAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histocompatibility complex (MHC) class I and II antigen presentation. The treatment consisted of 42 DC cancer vaccine infusions, 26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions. The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells. No immunotherapy-related adverse events were observed during the treatment. Robust antitumor CD4+ and CD8+ T-cell responses were detected. The patient remains free of disease progression. This is the first case report on the combination of the above three agents to treat glioblastoma patients. Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient. A large-scale trial to validate these findings is warranted.

Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines,anti-PD-1 and poly I:C / 药物分析学报

Glioblastoma(GBM)is a lethal cancer with limited therapeutic options.Dendritic cell(DC)-based cancer vaccines provide a promising approach for GBM treatment.Clinical studies suggest that other immu-notherapeutic agents may be combined with DC vaccines to further enhance antitumor activity.Here,we report a GBM case with combination immunotherapy consisting of DC vaccines,anti-programmed death-1(anti-PD-1)and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy,and the patient remained disease-free for 69 months.The patient received DC vaccines loaded with multiple forms of tumor antigens,including mRNA-tumor associated antigens(TAA),mRNA-neoantigens,and hypochlorous acid(HOCl)-oxidized tumor lysates.Furthermore,mRNA-TAAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histo-compatibility complex(MHC)class Ⅰ and Ⅱ antigen presentation.The treatment consisted of 42 DC cancer vaccine infusions,26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions.The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells.No immunotherapy-related adverse events were observed during the treatment.Robust antitumor CD4+and CD8+T-cell responses were detected.The patient remains free of disease progression.This is the first case report on the combination of the above three agents to treat glioblastoma patients.Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient.A large-scale trial to validate these findings is warranted.

Monitored anesthesia care and asleep-awake-asleep techniques combined with multiple monitoring for resection of gliomas in eloquent brain areas: a retrospective analysis of 225 patients.

BACKGROUND: Awake craniotomy (AC) has become gold standard in surgical resection of gliomas located in eloquent areas. The conscious sedation techniques in AC include both monitored anesthesia care (MAC) and asleep-awake-asleep (AAA). The choice of optimal anesthetic method depends on the preferences of the surgical team (mainly anesthesiologist and neurosurgeon). The aim of this study was to compare the difference in physiological and blood gas data, dosage of different drugs, the probability of switching to endotracheal intubation, and extent of tumor resection and dysfunction after operation between AAA and MAC anesthetic management for resection of gliomas in eloquent brain areas. METHODS: Two-hundred and twenty-five patients with super-tentorial tumor located in eloquent areas underwent AC from 2009 to 2021 in Xijing Hospital. Forty-one patients underwent AAA technique, and the rest one-hundred eighty-four patients underwent MAC technique. Anesthetic management, dosage of different drugs, intraoperative complications, postoperative outcomes, adverse events, extent of resection and motor, and sensory and language dysfunction after operation were compared between MAC and AAA. RESULT: There was no significant difference in gender, KPS score, MMSE score, glioma grade, type, and growth site between the patients in the two groups, except the older age of patients in MAC group than that in AAA group. During the whole process of operation, there were greater pulse pressure difference (P = 0.046), shorter operation time (P = 0.039), less dosage of remifentanil (P = 0.000), more dosage of dexmedetomidine (P = 0.013), more use of antiemetics (81%, P = 0.0067), lower use of vasoactive agent (45.1%, P = 0.010), and lower probability of conversion to general anesthesia (GA, P = 0.027) in MAC group than that in AAA group. Blood gas analysis showed that PetCO2 (P = 0.000), Glu concentration (P = 0.000), and PaCO2 (P = 0.000) were higher, but SPO2 (P = 0.002) and PaO2 (P = 0.000) were lower in MAC group than that in AAA group. In the postoperative recovery stage, compared with that of AAA group, the probability of dysfunction in MAC group at 1, 3, 5, and 7 days after operation was lower, which were 27.8% vs 53.6% (P = 0.003), 31% vs 68.3% (P = 0.000), 28.8% vs 63.4% (P = 0.000), and 25.6% vs 58.5% (P = 0.000), respectively. CONCLUSION: Compared with AAA, it seems that MAC has more advantages in the management for resection of gliomas in eloquent brain areas, and MAC combined with multiple monitoring such as cerebral cortical mapping, neuronavigation, and ultrasonic detection is worthy of popularization for the resection of gliomas in eloquent brain areas.

miR­497/MIR497HG inhibits glioma cell proliferation by targeting CCNE1 and the miR­588/TUSC1 axis.

Emerging evidence has shown that microRNA (miR)­497 serves pivotal roles in tumorigenesis, cancer progression, metastasis and chemotherapy resistance in several types of cancer. In the present study, the expression and biological functions of miR­497 host gene (MIR497HG) were investigated in glioma tissue. The expression levels of miR­497 and MIR497HG were measured in glioma, adjacent non­cancerous and normal brain tissue and their association with the prognosis of patients with glioma were analyzed. The biological roles of miR­497 and MIR497HG were investigated in glioma cell lines. In addition, bioinformatics analysis, luciferase reporter assay and functional experiments were performed to identify and validate the downstream targets of miR­497 or MIR497HG. The expression levels of miR­497 and MIR497HG were downregulated in glioma tissue and cell lines compared with those in adjacent non­cancerous and normal brain tissue and normal human cortical neuron cell line. Patients with low miR­497 or MIR497HG expression levels exhibited a poor prognostic outcome. In addition, forced overexpression of miR­497 or MIR497HG significantly inhibited the proliferation and cell cycle progression of glioma cell lines. Furthermore, the results indicated that miR­497 and MIR497HG exerted their biological functions by direct targeting of cyclin E1 and miR­588/tumor suppressor candidate 1. In summary, the data indicated that miR­497 and MIR497HG served as tumor suppressors and may be used as potential therapeutic targets and prognostic biomarkers in glioma.

miR-139/PDE2A-Notch1 feedback circuit represses stemness of gliomas by inhibiting Wnt/ß-catenin signaling.

Rationale: The malignant phenotypes of glioblastomas (GBMs) are primarily attributed to glioma stem cells (GSCs). Our previous study and other reports have suggested that both miR-139 and its host gene PDE2A are putative antitumor genes in various cancers. The aim of this study was to investigate the roles and mechanisms of miR-139/PDE2A in GSC modulation. Methods: Clinical samples were used to determine miR-139/PDE2A expression. Patient-derived glioma stem-like cells (PD-GSCs) were stimulated for immunofluorescent staining, sphere formation assays and orthotopic GBM xenograft models. Bioinformatic analysis and further in vitro experiments demonstrated the downstream molecular mechanisms of miR-139 and PDE2A. OX26/CTX-conjugated PEGylated liposome (OCP) was constructed to deliver miR-139 or PDE2A into glioma tissue specifically. Results: We demonstrated that miR-139 was concomitantly transcribed with its host gene PDE2A. Both PDE2A and miR-139 indicated better prognosis of gliomas and were inversely correlated with GSC stemness. PDE2A or miR-139 overexpression suppressed the stemness of PD-GSCs. FZD3 and ß-catenin, which induced Wnt/ß-catenin signaling activation, were identified as targets of miR-139 and mediated the effects of miR-139 on GSCs. Meanwhile, PDE2A suppressed Wnt/ß-catenin signaling by inhibiting cAMP accumulation and GSK-3ß phosphorylation, thereby modulating the self-renewal of PD-GSCs. Notably, Notch1, which is also a target of miR-139, suppressed PDE2A/miR-139 expression directly via downstream Hes1, indicating that miR-139 promoted its own expression by the miR-139-Notch1/Hes1 feedback circuit. Expectedly, targeted overexpression miR-139 or PDE2A in glioma with OCP system significantly repressed the stemness and decelerated glioma progression. Conclusions: Our findings elaborate on the inhibitory functions of PDE2A and miR-139 on GSC stemness and tumorigenesis, which may provide new prognostic markers and therapeutic targets for GBMs.

Targeted delivery of miR-99b reprograms tumor-associated macrophage phenotype leading to tumor regression.

BACKGROUND: Accumulating evidence has shown that tumor-associated macrophages (TAMs) play a critical role in tumor progression. Targeting TAMs is a potential strategy for tumor immunotherapy. However, the mechanism underlying the TAM phenotype and function needs to be resolved. Our previous studies have demonstrated that miR-125a can reverse the TAM phenotype toward antitumor. Meanwhile, we have found that miR-125a and miR-99b cluster in the first intron of the same host gene, and are transcribed simultaneously in bone marrow-derived macrophages (BMDMs) following LPS+IFNγ stimulation. However, it remains unclear whether miR-99b by itself can exert an antitumor effect by regulating macrophage phenotype. METHODS: miR-99b and/or miR-125a were delivered into TAMs of orthotopic hepatocellular carcinoma (HCC) or subcutaneous Lewis lung cancer (LLC) mice. The effect of treatment was evaluated by live imaging, TUNEL staining and survival tests. The phenotype of the immune cells was determined by qRT-PCR, ELISA, western blot and FACS. The capability of miR-99b-mediated macrophage phagocytosis and antigen presentation was detected by FACS and immunofluorescence staining. The underlying molecular mechanism was examined by qRT-PCR, reporter assay and western blot, and further verified in the tumor model. The expression of miR-99b and its target genes was determined in TAMs sorted from tumor and adjacent tissues in patients with liver cancer. RESULTS: Targeted delivery of miR-99b and/or miR-125a into TAMs significantly impeded the growth of HCC and LLC, especially after miR-99b delivery. More importantly, the delivery of miR-99b re-educated TAM toward antitumor phenotype with enhanced immune surveillance. Further investigation of mechanisms showed that macrophage-specific overexpression of miR-99b promoted M1 while suppressing M2 macrophage polarization by targeting κB-Ras2 and/or mTOR, respectively. miR-99b-overexpressed M1 macrophage was characterized by stronger capability of phagocytosis and antigen presentation. Additionally, delivery of simTOR or siκB-Ras2 into TAMs inhibited miR-99b antagomir-triggered tumor growth. Finally, miR-99b expression was lower in TAMs of patients with liver cancer than that in adjacent tissues, while the expression of κB-Ras2 and mTOR was reversed. CONCLUSIONS: Our results reveal the mechanism of miR-99b-mediated TAM phenotype, indicating that TAM-targeted delivery of miR-99b is a potential strategy for cancer immunotherapy.

Downregulation of FHL1 protein in glioma inhibits tumor growth through PI3K/AKT signaling.

Human four-and-a-half LIM domains protein 1 (FHL1) is a member of the FHL protein family, which serves an important role in multiple cellular events by interacting with transcription factors using its cysteine-rich zinc finger motifs. A previous study indicated that FHL1 was downregulated in several types of human cancer and served a role as a tumor suppressive gene. The overexpression of FHL1 inhibited tumor cell proliferation. However, to the best of our knowledge, there is no evidence to confirm whether FHL1 affected glioma growth, and the molecular mechanisms through which FHL1 represses tumor development remain unclear. In the present study, the expression level of FHL1 was determined using immunohistochemical staining in 114 tumor specimens from patients with glioma. The results indicated that FHL1 expression was negatively associated with the pathological grade of gliomas. Furthermore, Kaplan-Meier survival curves demonstrated that the patients with an increased FHL1 expression exhibited a significantly longer survival time, suggesting that FHL1 may be a prognostic marker for glioma. The protein level of FHL1 was relatively increased in the U251 glioma cell line compared with that in the U87 cell line. Therefore, FHL1 was knocked down in U251 by siRNA and overexpressed in U87, and it was identified that FHL1 significantly decreased the activation of PI3K/AKT signaling by interacting with AKT. Further experiments verified that FHL1 inhibited the growth of gliomas in vivo by modulating PI3K/AKT signaling. In conclusion, the results of the present study demonstrated that FHL1 suppressed glioma development through PI3K/AKT signaling.

MicroRNA-144 represses gliomas progression and elevates susceptibility to Temozolomide by targeting CAV2 and FGF7.

Malignant gliomas are the most common tumor in central nervous system with poor prognosis. Due to the limitation of histological classification in earlier diagnosis and individualized medicine, it is necessary to combine the molecular signatures and the pathological characteristics of gliomas. Lots of microRNAs presented abnormal expression in gliomas and modulated gliomas development. Exploration the miRNAs profile is helpful for the diagnosis, therapy and prognosis of gliomas. It has been demonstrated that miR-144 plays important roles in solid tumors. However, the detail mechanisms remained unrevealed. In this study, we have demonstrated the level of miR-144 decreased in glioma tissues from patients, especially in gliomas with higher grades. MiR-144 was also validated have lower expression in glioma cell lines compared with cortical neuron cell by using qRT-PCR. The in vitro functional experiment indicated miR-144 improved gliomas progression through repressing proliferation, sensitizing to chemotherapeutics and inhibiting metastasis. We further identified fibroblast growth factor 7 (FGF7) and Caveolin 2 (CAV2) were target genes of miR-144 by luciferase reporter assay and western blotting. The mechanisms study suggested forced FGF7 expression elevated Akt activation and decreased reactive oxygen species (ROS) generation. The MTT and cell cycle assay indicated miR-144 suppressed glioma cells proliferation through modulating FGF mediated Akt signaling pathway. Meanwhile, miR-144 promoted Temozolomide (TMZ) induced apoptosis in glioma cells via increasing ROS production by using FACS. On the other hand, CAV2, as another target of miR-144, accelerated glioma cells migration and invasion via promoting glioma cells EMT progress. Retrieved expression of FGF7 or CAV2 rescued the proliferation and migration function mediated by miR-144. Furthermore, the in vivo experiments in PDX models displayed the anti-tumor function of miR-144, which could be retrieved by overexpression of FGF7 and CAV2. Taken together, these findings indicated miR-144 acted as a potential target against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new therapeutic strategy and prognostic indicator for gliomas.

A ketogenic diet attenuates proliferation and stemness of glioma stem­like cells by altering metabolism resulting in increased ROS production.

Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem­like cells (GSCs), by culturing patient­derived primary GSCs as well as a GSC cell line in glucose­restricted, ß­hydroxybutyrate­containing medium (BHB­Glow) which was used to mimic clinical KD treatment. GSCs cultured in BHB­Glow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self­renewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB­Glow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB­Glow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia­inducible factor 1 and B­cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB­Glow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.

Is Platelet-rich Plasma Injection Effective for Chronic Achilles Tendinopathy? A Meta-analysis.

BACKGROUND: Chronic Achilles tendinopathy is common in the general population, and platelet-rich plasma (PRP) is seeing increased use to treat this problem. However, studies disagree as to whether PRP confers a beneficial effect for chronic Achilles tendinopathy, and no one to our knowledge has pooled the available randomized trials in a formal meta-analysis to try to reconcile those differences. QUESTIONS/PURPOSES: In the setting of a systematic review and meta-analysis of randomized controlled trials (RCTs), we asked: Does PRP plus eccentric strength training result in (1) greater improvements in Victorian Institute of Sports Assessment-Achilles (VISA-A) scores; (2) differences in tendon thickness; or (3) differences in color Doppler activity compared with placebo (saline) injections plus eccentric strength training in patients with chronic Achilles tendinopathy? METHODS: A search of peer-reviewed articles was conducted to identify all RCTs using PRP injection with eccentric training for chronic Achilles tendinopathy in the electronic databases of PubMed, Web of Science (SCI-E/SSCI/A&HCI), and EMBASE from January 1981 to August 2017. Results were limited to human RCTs and published in all languages. Two reviewers assessed study quality using the Cochrane Collaboration risk-of-bias tool. All the included studies had low risk of bias. The primary endpoint was improvement in the VISA-A score, which ranges from 0 to 100 points, with higher scores representing increased activity and less pain; we considered the minimum clinically important difference on the VISA-A to be 12 points. Secondary outcomes were tendon thickness change (with a thicker tendon representing more severe disease), color Doppler activity (with more activity representing a poorer result), and other functional measures (such as pain and return to sports activity). Four RCTs involving 170 participants were eligible and included 85 participants treated with PRP injection and eccentric training and 85 treated with saline injection and eccentric training. The patients in both PRP and placebo (saline) groups seemed comparable at baseline. We assessed for publication bias using a funnel plot and saw no evidence of publication bias. Based on previous studies, we had 80% power to detect a 12-point difference on the VISA-A score with the available sample size in each group. RESULTS: With the numbers available, there was no difference between the PRP and saline groups regarding the primary outcome (VISA-A score: mean difference [MD], 5.3; 95% confidence interval [CI], -0.7 to 11.3; p = 0.085). Likewise, we found no difference between the PRP and saline groups in terms of our secondary outcomes of tendon thickness change (MD, 0.2 mm; 95% CI, 0.6-1.0 mm; p = 0.663) and color Doppler activity (MD, 0.1; 95% CI, -0.7 to 0.4; p = 0.695). CONCLUSIONS: PRP injection with eccentric training did not improve VISA-A scores, reduce tendon thickness, or reduce color Doppler activity in patients with chronic Achilles tendinopathy compared with saline injection. Larger randomized trials are needed to confirm these results, but until or unless a clear benefit has been demonstrated in favor of the new treatment, we cannot recommend it for general use. LEVEL OF EVIDENCE: Level I, therapeutic study.

miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1.

OBJECTIVE: To investigate the role of FOXO1 and miR-183-96-182 clusters in ox-LDL induced endothelial cell apoptosis. METHODS: FOXO1 overexpression (OE) and knockdown (KD) as well as AKT1 OE in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were achieved by lentiviral transduction. Upregulation of miR-183-5p, miR-182-5p or miR-96-5p was mimicked by agomir treatment. FOXO1 gene transcription was monitored by FOXO1 promotor reporter assay. Cell apoptosis in culture was monitored by TiterTACS in situ detection. Regulation of FOXO1 gene expression by an miRNA targeting mechanism was monitored by AGO2-RNA immunoprecipitation assay. RESULTS: FOXO1 mRNA and protein expression levels in ox-LDL treated HUVECs or HAECs were significantly upregulated due to transcriptional and miRNA targeting mechanisms. MiR-183-5p, miR-182-5p and miR-96-5p expression levels in HUVECs or HAECs were significantly reduced by ox-LDL treatment, the overexpression of which by agomir treatment partially reduced the FOXO1 mRNA/protein expression levels and cell apoptosis which was upregulated by ox-LDL treatment. FOXO1 overexpression antagonized the effect of the agomir treatment indicated above. MiR-183-5p, miR-182-5p and miR-96-5p agomir treatment partially rescued the FOXO1 pSer256/total FOXO1 protein ratio and the AKT1 pSer473 level that were reduced by ox-LDL treatment in the HUVECs or HAECs. AKT1 overexpression significantly reduced FOXO1 protein expression, increased miR-182-5p and miR-183-5p expression, and partially alleviated ox-LDL induced HUVEC or HAEC apoptosis in an miR-183-5p and miR-182-5p-dependent manner. CONCLUSION: miR-183-96-182 clusters could partially alleviate ox-LDL-induced apoptosis in HUVECs or HAECs by targeting FOXO1.

High expression of MMP9 in glioma affects cell proliferation and is associated with patient survival rates.

Human gliomas are a heterogeneous group of primary malignant brain tumors, which most commonly occur in the central nervous system of children and adults. Previous studies have suggested a prognostic role of matrix metalloproteinase 9 (MMP9) in glioma, however, the frequency and significance of the protein expression of MMP9 in glioma remain to be fully elucidated. In the present study, the expression of MMP9 was detected by reverse transcription-quantitative polymerase chain reaction (qPCR), western blotting and immunohistochemical staining. MTT and colony-forming assays were used to detect the role of MMP9 in the proliferation of glioma cells. MMP9 copy numbers in glioma were examined using qPCR. The results indicated that the expression level of MMP9 was significantly increased in glioma and was associated with World Health Organization (WHO) glioma grades. The high expression of MMP9 in tissues was an independent predictor of survival rates in patients with WHO grade III tumors. The overexpression of MMP9 promoted cell growth and induced a significant increase in clonogenic potential in U87 glioblastoma cell lines. These experimental data suggested that the overexpression of MMP9 in glioblastoma cells may occur primarily through an increase in gene copy number. The results of the present study suggested that the overexpression of MMP9 may be necessary for the transition to the more aggressive phenotype typical of WHO grade III gliomas, suggesting the likely involvement of the MMP9 gene in gliomagenesis and disease progression.

Systematic comparison of biologically active foreign ions-codoped calcium phosphate microparticles on osteogenic differentiation in rat osteoporotic and normal mesenchymal stem cells.

Osteoporosis is a disease characterized by structural deterioration of bone tissue, leading to skeletal fragility with increased fracture risk. Calcium phosphates (CaPs) are widely used in bone tissue engineering strategies as they have similarities to bone apatite except for the absence of trace elements (TEs) in the CaPs. Bioactive glasses (BGs) have also been used successfully in clinic for craniomaxillofacial and dental applications during the last two decades due to their excellent potential for bonding with bone and inducing osteoblastic differentiation. In this study, we evaluated the osteogenic effects of the ionic dissolution products of the quaternary Si-Sr-Zn-Mg-codoped CaP (TEs-CaP) or 45S5 Bioglass® (45S5 BG), both as mixtures and separately, on rat bone marrow-derived mesenchymal stem cells (rOMSCs & rMSCs) from osteoporotic and normal animals, using an MTT test and Alizarin Red S staining. The materials enhanced cell proliferation and osteogenic differentiation, especially the combination of the BG and TEs-CaP. Analysis by quantitative PCR and ELISA indicated that the expression of osteogenic-specific genes and proteins were elevated. These investigations suggest that the TEs-CaP and 45S5 BG operate synergistically to create an extracellular environment that promotes proliferation and terminal osteogenic differentiation of both osteoporotic and normal rMSCs.

miR-139 Functions as An Antioncomir to Repress Glioma Progression Through Targeting IGF-1 R, AMY-1, and PGC-1ß.

Gliomas are the most common primary malignant brain tumor with poor prognosis, characterized by a highly heterogeneous cell population, extensive proliferation, and migration. A lot of molecular mechanisms regulate gliomas development and invasion, including abnormal expression of oncogenes and variation of epigenetic modification. MicroRNAs could affect cell growth and functions. Several reports have demonstrated that miR-139 plays multifunctions in kinds of solid tumors through different pathways. However, the antitumor mechanisms of this miR-139 are not unveiled in detail. In this study, we not only validated the low expression level of miR-139 in glioma tissues and cell lines but also detected the effect of miR-139 on modulating gliomas proliferation and invasion both in vitro and in vivo. We identified insulin-like growth factor 1 receptor, associate of Myc 1, and peroxisome proliferator-activated receptor γ coactivator 1ß as direct targets of miR-139 and the levels of them were all inversely correlated with miR-139 in gliomas. Insulin like growth factor 1 receptor promoted gliomas invasion through Akt signaling and increased proliferation in the peroxisome proliferator-activated receptor γ coactivator 1ß-dependent way. Associate of Myc 1 also facilitated gliomas progression by activating c-Myc pathway. Overexpression of the target genes could retrieve the antitumor function of miR-139, respectively, in different degrees. The nude mice transplantation tumor experiment displayed that glioma cells stably expressed miR-139 growth much slower in vivo than the negative control cells. Taken together, these findings suggested miR-139 acted as a favorable factor against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new evidenced prognostic marker and therapeutic target for gliomas.

In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.

Islet transplantation provides curative treatments to patients with type 1 diabetes, but donor shortage restricts the broad use of this therapy. Thus, generation of alternative transplantable cell sources is intensively investigated worldwide. We previously showed that bone marrow-derived mesenchymal stem cells (bmMSCs) can be reprogrammed to pancreatic-like cells through simultaneously forced suppression of Rest/Nrsf (repressor element-1 silencing transcription factor/neuronal restrictive silencing factor) and Shh (sonic hedgehog) and activation of Pdx1 (pancreas and duodenal transcription factor 1). We here aimed to reprogram bmMSCs further along the developmental pathway towards the islet lineages by improving our previous strategy and by overexpression of Ngn3 (neurogenin 3) and NeuroD1 (neurogenic differentiation 1), critical regulators of the development of endocrine pancreas. We showed that compared to the previous protocol, the overexpression of only Pdx1 and Ngn3 reprogrammed bmMSCs into cells with more characteristics of islet endocrine lineages verified with bioinformatic analyses of our RNA-Seq datasets. These analyses indicated 2325 differentially expressed genes including those involved in the pancreas and islet development. We validated with qRT-PCR analysis selective genes identified from the RNA-Seq datasets. Thus, we reprogrammed bmMSCs into islet endocrine-like cells and advanced the endeavor to generate surrogate functional insulin-secreting cells.

A study on the correlations study among HOMA-IR, obesity and inflammatory factors among middle aged and elderly population / 预防医学

Objective To investigate the distribution of insulin resistance (HOMA-IR) index and its influencing factorsamong middle and old aged people with normal glucose and to provide the basis for early screening and prevention of type 2diabetes. Methods A total of 229 residents were selected with health records showed normal blood glucose (fasting bloodglucose < 7.0mmol/L, postprandial 2h blood glucose<11.1 mmol/L) and more than 40 years old from July, 2012 to June,2015. Height, weight, waist and hip circumference, and the fasting plasma glucose (FPG), insulin (FINS), lowdensity lipoprotein (LDL), uric acid, tumor necrosis factor (TNF) and interleukin -6 (IL-6) were recorded to analyzethe distribution of HOMA-IR and its influencing factors. Results Totally 229 people were included, of which 113 were male(49.34%), 116 female(50.66%) . The average age was(63.58 + 8.85) years old. The average HOMA-IR index was 0.94(1.08) and there were 21 people that HOMA-IR exceed the standard (HOMA-IR≥2.68), accounting for 9.17%.TheHOMA-IR index of different gender, age, waist circumference, hip circumference, uric acid in the elderly had significantdifference (P < 0.05) .Multiple linear regression analysis showed that HOMA-IR index was positively correlated withfemale, waist circumference and IL-6 and was negatively correlated with age. Conclusion The possibility of IR was higherin women with relatively low age, female, central obesity and high IL-6 levels among the middle and old aged people withnormal blood glucose.

Combination of methylprednisolone and rosiglitazone promotes recovery of neurological function after spinal cord injury.

Methylprednisolone exhibits anti-inflammatory antioxidant properties, and rosiglitazone acts as an anti-inflammatory and antioxidant by activating peroxisome proliferator-activated receptor-γ in the spinal cord. Methylprednisolone and rosiglitazone have been clinically used during the early stages of secondary spinal cord injury. Because of the complexity and diversity of the inflammatory process after spinal cord injury, a single drug cannot completely inhibit inflammation. Therefore, we assumed that a combination of methylprednisolone and rosiglitazone might promote recovery of neurological function after secondary spinal cord injury. In this study, rats were intraperitoneally injected with methylprednisolone (30 mg/kg) and rosiglitazone (2 mg/kg) at 1 hour after injury, and methylprednisolone (15 mg/kg) at 24 and 48 hours after injury. Rosiglitazone was then administered once every 12 hours for 7 consecutive days. Our results demonstrated that a combined treatment with methylprednisolone and rosiglitazone had a more pronounced effect on attenuation of inflammation and cell apoptosis, as well as increased functional recovery, compared with either single treatment alone, indicating that a combination better promoted recovery of neurological function after injury.

Saponin 6 derived from Anemone taipaiensis induces U87 human malignant glioblastoma cell apoptosis via regulation of Fas and Bcl­2 family proteins.

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL­60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose­ and time­dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT­22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V­fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase­mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6­induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase­3, ­8 and ­9, and decreases in the levels of B­cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM.

Long non-coding RNA-DANCR in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis.

Osteoporosis is a common disease characterized by low bone mineral density (BMD) and low trauma fractures, mainly resulting from exceeding bone resorption by osteoclasts over bone formation by osteoblasts. Circulating monocytes are directly involved in osteoclastogenesis, and lncRNAs are believed to be involved in the osteoblast differentiation. However, no study has been conducted to identify the roles of lncRNA in circulating monocytes associated with human osteoporosis. In this study, we found significant upregulation of DANCR in the blood mononuclear cells (MNCs) from low-BMD patients with the qRT-PCR analyses. We further found that DANCR promoted the expression of IL6 and TNF-α at both mRNA level and protein level in MNCs. After deletion of DANCR with siRNAs, the levels of IL6 and TNF-α are decreased in the MNCs from low-BMD postmenopausal women. Moreover, DANCR level was correlated with IL6 and TNF-α in postmenopausal women with low BMD. Furthermore, we found that DANCR-induced IL6 and TNF-α in MNCs had bone-resorbing activity. These results indicate that DANCR is involved in the pathology of osteoporosis and may be as a biomarker for postmenopausal osteoporosis.

FHL1C induces apoptosis in Notch1-dependent T-ALL cells through an interaction with RBP-J.

BACKGROUND: Aberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSIs) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear. METHODS: Using RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software. RESULTS: In Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression. CONCLUSIONS: Our study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL.