Extracellular Vesicles Derived from Bone Mesenchymal Stem Cells Carrying circ_0000075 Relieves Cerebral Ischemic Injury by Competitively Inhibiting miR-218-5p and Up-regulating E3 Ubiquitin Ligase SMURF2.

Extracellular vesicle (EV)-encapsulated circRNAs have the potential role in affecting brain disorders. However, the role of circ_0000075 in cerebral ischemic injury remains unclear. Here, we tried to investigate the mechanism of bone marrow mesenchymal stem cell (BMSC)-derived EVs carrying circ_0000075 in the control of cerebral ischemic injury. Initially, a mouse model with cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO), followed by the determination of circ_0000075 expression. Then, neurons were isolated and subjected to oxygen-glucose deprivation/reperfusion. BMSCs were isolated for extraction of EVs. The correlation among circ_0000075, microRNA (miR)-218-5p, and Smad ubiquitination regulatory factor 2 (SMURF2) was detected with their roles in cerebral ischemic injury analyzed in vivo and in vitro. circ_0000075 was down-regulated in MCAO mice and engineered RVG-EVs were internalized by neurons to up-regulate circ_0000075 expression. Treatment of RVG-circ_0000075-EVs reduced brain tissue damage, increased neuronal count, and significantly curtailed apoptosis rate, suppressing cerebral ischemic injury in vitro and in vivo. miR-218-5p was targeted by circ_0000075 in neurons, which promoted SMURF2 expression. A negative correlation between SMURF2 and transcriptional regulator Yin Yang 1 (YY1) was identified. In vitro experiments further proved that circ_ 00,000 75 could down-regulate the expression of YY1 through SMURF2, and finally relieving cerebral ischemic injury. Collectively, engineered EVs delivered circ_0000075 into brain tissues and increased circ_0000075 expression, which down-regulated miR-218-5p and up-regulated SMURF2, thus alleviating cerebral ischemic injury.

Induction of cancer cell stemness in glioma through glycolysis and the long noncoding RNA HULC-activated FOXM1/AGR2/HIF-1α axis.

Gliomas are the most common primary intracranial tumor, accounting for more than 70% of brain malignancies. Studies indicate that highly upregulated in liver cancer (HULC), a long noncoding RNA (lncRNA), functions as an oncogene in gliomas. However, the underlying mechanism of HULC in gliomas remains under-studied and was subsequently investigated in the current study. Brain tissues were clinically collected from 50 patients with glioblastoma (GBM) and 35 patients with acute craniocerebral injury, followed by immunohistochemical detection of the expression patterns of Forkhead box M1 (FOXM1), anterior gradient 2 (AGR2), and hypoxia-inducible factor-1α (HIF-1α). After flow cytometry-based sorting of the CD133+ glioma stem cells (GSCs) from the U251 cell line, the obtained cells were subjected to lentivirus infection. Afterwards, the proliferation, stemness, and apoptosis of GSCs were evaluated using sphere formation, immunofluorescence, and flow cytometry assays, respectively. In addition, the interactions among HULC, FOXM1, AGR2, and HIF-1α were identified using RNA immunoprecipitation (RIP), RNA pull-down, Chromatin immunoprecipitation (ChIP), IP, and dual luciferase reporter assays. Last, the specific effects were validated in vivo. HULC was upregulated in GBM tissues and GSCs, which may promote the progression of glioma. On the other hand, silencing of HULC reduced the stemness, inhibited the proliferation, and promoted the apoptosis and differentiation of GSCs. In addition, HULC further stabilized FOXM1 expression in GSCs through ubiquitination, while FOXM1 activated AGR2 transcription to promote HIF-1α expression. Moreover, HULC promoted the glycolysis and stemness of GSCs through its regulation of the FOXM1/AGR2/HIF-1α axis, consequently exacerbating the occurrence and development of glioma. The findings obtained in our study indicate that HULC stabilizes the FOXM1 protein by ubiquitination to upregulate the expression of AGR2 and HIF-1α, which further promote the glycolysis of and maintain the stemness of GSCs, to enhance the tumorigenicity of GSCs, highlighting a novel therapeutic target for glioma.

Erratum: MicroRNA-181 inhibits the proliferation, drug sensitivity and invasion of human glioma cells by targeting Selenoprotein K (SELK).

[This corrects the article on p. 6632 in vol. 11, PMID: 31737213.].

Extracellular vesicles derived from M2 microglia reduce ischemic brain injury through microRNA-135a-5p/TXNIP/NLRP3 axis.

Accumulating evidences have suggested that extracellular vesicles (EVs) are crucial players in the pathogenesis of ischemic brain injury. This study was designed to explore the specific functions of M2 phenotype microglia-derived EVs in ischemic brain injury progression. The expression of microRNA-135a-5p (miR-135a-5p) in M2 microglia-derived EVs was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), followed by the identification of expression relationship among miR-135a-5p, thioredoxin-interacting protein (TXNIP), and nod-like receptor protein 3 (NLRP3) by dual luciferase reporter gene assay. After construction of an oxygen-glucose deprivation/reperfusion (OGD/R) cell model, the effects of miR-135a-5p on the biological characteristics of HT-22 cells were assessed by cell counting kit 8 (CCK-8) assay and flow cytometry. Finally, a mouse model of transient middle cerebral artery occlusion (tMCAO) was established and cerebral infarction volume was determined by triphenyltetrazolium chloride (TTC) staining and the expression of IL-18 and IL-1ß in the brain tissue was determined by enzyme-linked immunosorbent assay (ELISA). We found that M2 microglia-derived EVs had high expression of miR-135a-5p, and that miR-135a-5p in M2 microglia-derived EVs negatively regulated the expression of NLRP3 via TXNIP. Overexpression of miR-135a-5p promoted the proliferation but inhibited the apoptosis of neuronal cells, and inhibited the expression of autophagy-related proteins. M2 microglia-derived EVs delivered miR-135a-5p into neuronal cells to inhibit TXNIP expression, which further inhibited the activation of NLRP3 inflammasome, thereby reducing neuronal autophagy and ischemic brain injury. Hence, M2 microglia-derived EVs are novel therapeutic targets for ischemic brain injury treatment.

MicroRNA-181 inhibits the proliferation, drug sensitivity and invasion of human glioma cells by targeting Selenoprotein K (SELK).

Gliomas are aggressive type of brain tumors and cause significant human mortality world over. The frequent relapses, development of drug resistance, the adverse effects of the chemotherapy and dearth of the therapeutic targets form the major hurdles in glioma treatment. Several studies suggest that microRNAs (miRs) are involved in the development and progression of different cancers. Herein, the therapeutic potential of miR-181 was explored in human glioma cells. The results showed that miR-181 is significantly downregulated in human glioma cells. Overexpression of miR-181 caused significant inhibition in the proliferation of U87 and U118 glioma cells. The miR-181 triggered growth inhibition was found to be mainly due to the induction of apoptosis which was concomitant with increase in the Bax/Bcl-2 ratio. Additionally, miR-181 enhanced the chemosensitivity of the glioma cells to temozolomide and suppressed their invasion. Bioinformatic analysis showed that miR-181 exerts its effects by inhibiting the expression of Selenoprotein K (SELK). The expression of SELK was found to be significantly upregulated in glioma cells and silencing of SELK suppressed the proliferation of glioma cells. Nonetheless, overexpression of SELK could nullify the effects of miR-181 on the proliferation of the glioma cells. Taken together, miR-181 may exhibit therapeutic implications in the treatment of glioma.

Silencing microRNA-221/222 cluster suppresses glioblastoma angiogenesis by suppressor of cytokine signaling-3-dependent JAK/STAT pathway.

Angiogenesis is a major pathologic characteristic of glioblastoma, which is one aggressive primary brain tumor. MicroRNA-221/222 (miR-221/222) cluster has been previously reported to function importantly in malignant glioma biological process. The current study aims at evaluating the effects of miR-221/222 cluster on angiogenesis of glioblastoma cells. Microarray data were analyzed to select glioblastoma-associated differentially expressed genes, and dual-luciferase reporter assay was performed to assess targeting correlation between miR-221/222 cluster and suppressor of cytokine signaling-3 (SOCS3). Subsequently, the expression patterns of miR-221 and miR-222 in glioblastoma cells were identified. miR-221 and miR-222 were overexpressed or silenced in glioblastoma cells to identify the effect of miR-221/222 cluster in cell invasion, migration, proliferation, and angiogenesis. To define downstream pathway of miR-221/222 cluster or SOCS3 in glioblastoma, levels of Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway-related proteins were assessed. Additionally, the functions of miR-221/222 on glioblastoma cell angiogenesis were measured in vivo with microvessel density assayed. miR-221 and miR-222 were expressed at a high level and SOCS3 was at a low level in glioblastoma. Downregulation of the miR-221/222 cluster diminished the invasion, migration, proliferation, and angiogenesis with reduced protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9, and vascular endothelial growth factor in glioblastoma cells. Also, silencing miR-221/222 cluster reduced p-JAK2/JAK2 and p-STAT3/STAT3. Consistently, the inhibitory role of silencing miR-221/222 cluster on tumorigenesis of glioblastoma cells was confirmed in vivo. Collectively, the inhibition of miR-221/222 cluster could attenuate the glioblastoma angiogenesis through inactivation of the JAK/STAT pathway by upregulating SOCS3.

Combined Toxicity of an Environmental Remediation Residue, Magnetite Fe3O4 Nanoparticles/Cr(VI) Adduct.

OBJECTIVE: This paper aims to elucidate the combined toxicity of magnetite nanoparticles/Chromium [MNPs/Cr(VI)] adducts. METHODS: The HEK293 cell was exposed to either Cr(VI) or MNPs, or their adducts MNPs/Cr(VI). The cytotoxicity was evaluated by assessing the cell viability, apoptosis, oxidative stress induction, and cellular uptake. RESULTS: The toxicity of formed adducts is significantly reduced when compared to Cr(VI) anions. We found that the cellular uptake of MNPs/Cr(VI) adduct was rare, only few particles were endocytosed from the extracellular fluid and not accumulated in the cell nucleus. On the other hand, the Cr(VI) anions entered cells, generated oxidative stress, induced cell apoptosis, and caused cytotoxicity. CONCLUSION: The results showed minor effects of the nanoadducts on the tested cells and supported that magnetite nanoparticles could be implemented in the wastewater treatment process in which advantageous properties outweigh the risks.

Toxicity effects of di-(2-ethylhexyl) phthalate to Eisenia fetida at enzyme, cellular and genetic levels.

Di-(2-ethylhexyl) phthalate (DEHP) is a dominant phthalic acid ester (PAE) that has aroused public concern due to its resistance to degradation and its toxicity as an endocrine-disrupting compound. Effects of different concentrations of DEHP on Eisenia fetida in spiked natural soil have been studied in the body of the earthworm by means of soil cultivation tests 7, 14, 21 and 28 days after exposure. The results indicated that, in general, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, metallothionein (MT) content, the expression of heat shock protein 70 (HSP 70) and all the tested geno-toxicity parameters are promoted as time elapses and with increasing concentration of DEHP. However, peroxidase (POD) activity, neutral red retention time (NRRT) and mitochondrial membrane potential difference values were found to decrease even at a low concentration of DEHP of 1 mg kg-1 soil (p<0.05). Clear toxic effects of DEHP on E. fetida have been generally recognized by means of the disturbance of antioxidant enzyme activity/content and critical proteins, cell membrane and organelle disorder and DNA damage estimated by length of tail, tail DNA ratio, and tail moment parameters. A concentration of DEHP of 3 mg kg-1 may be recommended as a precaution against the potential risk of PAEs in soils and for indicating suitable threshold values for other soil animals and soil micro-organisms.

Oxytetracycline Toxicity and Its Effect on Phytoremediation by Sedum plumbizincicola and Medicago sativa in Metal-Contaminated Soil.

Excessive use of antibiotics potentially threatens human health, agricultural production, and soil phytoremediation. This arouses concern over the potential adverse effects of a commonly used antibiotic, oxytetracycline (OTC), on plants used for soil remediation and possible stimulation of antibiotic resistance genes in soils. A greenhouse experiment was conducted to investigate different rates (0, 1, 5, and 25 mg kg-1) and frequencies (one single high and daily low application) of OTC addition to soil on phytoremediation of a heavy metal contaminated soil by Sedum plumbizincicola and/or Medicago sativa (alfalfa). After 90 days both Cd and Zn were substantially removed by phytoextraction into S. plumbizincicola shoots especially at the high OTC (25 mg kg-1) treatment which also led to inhibition of antioxidative enzyme activities in both plant species. Soil microbial activity decreased significantly with the addition of OTC, and this was ameliorated by planting alfalfa and S. plumbizincicola together. OTC at <5 mg kg-1 increased the biomass of both plant species, but the frequency of OTC addition had no effect on the rate of metal removal. Alfalfa exhibited greater detoxification ability and effectiveness in soil microbial activity promotion than S. plumbizincicola with intercropping. Phytoremediation by alfalfa and S. plumbizincicola in association can both promote the removal of heavy metals and also alleviate the toxic effects of pollutants on plants and soil microbes even at relatively high soil OTC concentrations.

Toxicity of OTC to Ipomoea aquatica Forsk. and to microorganisms in a long-term sewage-irrigated farmland soil.

Water spinach (Ipomoea aquatic Forsk.) was selected to investigate the effects of oxytetracycline (OTC) on the toxicity of soil contaminated by long-term sewage irrigation. After acute toxicity test in petri dish at nine different OTC-spiked levels for 48 h, the germination rate was found to be generally inhibited in all treatments treated with OTC but the root elongation and activities of several antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were either forward or backward stimulated to varying extent. During a 60-day sub-chronic toxicity test by means of a pot experiment, activities of SOD, POD and CAT in both the leaf and root tissue at 25 mg OTC per kg soil (dry weight) and in root tissue at 1 mg OTC per kg soil (dry weight) were significantly different than those in other treatments, which also indicated the higher sensitivity of the root. The foliar photosynthetic rate, stomatal conductance and transpiration rate were all gradually inhibited in spite of elevated water use efficiency under the pressure of the different OTC concentrations, which were highly significant different at 10 mg OTC per kg soil (dry weight). Indices of soil microbial diversity at 4 mg OTC kg(-1) soil were significantly different from those of the control, indicating the potential adverse effects of OTC to soil microorganisms. The results suggest that the introduction of OTC could damage both plants and soil microorganisms, and during sub-chronic incubation, the sensitivity of different indices generally followed the order of root tissue antioxidant enzyme activities, soil microbial diversity indices, leaf photosynthesis-related index and leaf tissue enzyme antioxidant activities. In addition, the application of livestock and poultry manure containing pollutants like OTC in farmland soil, especially if the soil has been contaminated before, should be taken more seriously in the context of the current pursuit of increased agricultural production.

Oxidative Stress, Cytotoxicity and Genotoxicity in Earthworm Eisenia fetida at Different Di-n-Butyl Phthalate Exposure Levels.

Recognized as ubiquitous contaminants in soil, the environmental risk of phthalic acid esters (PAEs) is of great concern recently. Effects of di-n-butyl phthalate (DnBP), an extensively used PAE compound to Eisenia fetida have been investigated in spiked natural brown yellow soil (Alfisol) for soil contact test. The toxicity of DnBP to E. fetida on the activity of superoxide dismutase (SOD) activity, peroxidase (POD), reactive oxygen species (ROS) content, and the apoptosis of coelomocytes and DNA damage at the 7th, 14th, 21st and 28th day of the incubation have been paid close attention to. In general, SOD activity and ROS content were significantly induced, opposite to total protein content and POD activity, during the toxicity test of 28 days especially under concentrations higher than 2.5 mg kg-1. The reduction in neutral red retention (NRR) time along with the increase of dead coelomocytes as the increasing of DnBP concentrations, indicating severe damage to cell viability under varying pollutant stress during cultivation, which could also be proved by comet assay results for exerting evident DNA damage in coelomocytes. DnBP in spiked natural soil could indeed cause damage to tissues, coelomocytes and the nucleus of E. fetida. The key point of the apparent change in different indices presented around 2.5 mg DnBP kg-1 soil, which could be recommended as the threshold of DnBP soil contamination, so that further investigation on threshold values to other soil animals or microorganisms could be discussed.

[Effect of various intervention factors on MMP-3 and TIMP-1 level in synovial fluid in knee joints with osteroarthritis].

OBJECTIVE: To examine the expression of MMP-3 and TIMP-1 in the synovial fluid in knee joints with osteoarthritis before and after being treated with hyaluronic acid(HA), glucosamine sulfate(GS) and arthroscopic de bridment(AD), and to explore the therapeutic mechanism. METHODS: Sixty patients (64 knees) with osteoarthritis(OA) were randomly divided into HA group AD group and GS+AD group. Some patients from the HA group and the GS+HA group were selected, and served as HA' group and GS+HA' group. The expression of MMP-3 and TIMP-1 in the synovial fluid was measured by enzyme-linked immunosorbent assay (ELISA) before and after 4 week and 6 month therapy. RESULTS: The level of MMP-3 and the ratio of MMP-3/TIMP-1 in the synovial fluid decreased after being treated for 4 weeks, and the effect on MMP-3 and MMP-3/TIMP-1 lasted for 6 months in the HA group and the GS+HA groups. The levels of TIMP-1 increased significantly after being treated for 4 weeks only in the GS+HA group. The level of MMP-3 and the ratio of MMP-3/TIMP-1 in the synovial fluid decreased, but the level of TIMP-1 increased after being treated for 4 weeks, and the effect on MMP-3 and MMP-3/TIMP-1 lasted for 6 months. The level of MMP-3 and the ratio of MMP-3/TIMP-1 in the synovial fluid increased after being treated for 6 months compared with those for 4 weeks. The level of TIMP-1 increased in the GS group more than that in the HA group after being treated for 4 weeks. The level of TIMP-1 increased in the AD group more than that in the HA' group and the GS+HA' group after being treated for 4 weeks. CONCLUSION: (1) HA, GS and AD all can decrease the level of of MMP-3 and the ratio of MMP-3/TIMP-1 in the synovial fluid in knee joints with OA. (2) The level of TIMP-1 in the synovial fluid has no difference before and after being treated with HA. The AD group and GS group can increase the level of TIMP-1, which indicates that the AD group or GS group might produce better therapeutic effect. (3) The level of TIMP-1 increased in the AD group is more than that in the HA' group and the GS+HA' group after being treated for 4 weeks, which indicates that the AD group might get better therapeutic effect than the other 2 groups. (4) One of the most important mechanisms of HA, GS and AD in treating OA might be attributed to the expression of MMPs and TIMPs in knee joints with OA.