[Effect of the focal adhesion kinase inhibitor TAE226 on the epithelial-mesenchymal transition in human oral squamous cell carcinoma cell line].

OBJECTIVE: To study the effect of the focal adhesion kinase inhibitor TAE226 on epithelial-mesenchymal transition (EMT) in human oral squamous cell carcinoma (OSCC) cell line. METHODS: HSC-3 and HSC-4 cells were cultured with TAE226 under different concentrations (0, 1, 5, and 10 µmol·L⁻¹) for 24, 48, and 72 h. Real-time quantitative polymerase chain reaction was performed to detect the mRNA expressions of E-cadherin and Vimentin. The protein expressions of E-cadherin and Vimentin were determined by Western blot assay after 48 h of TAE226 treatment. RESULTS: Real-time quantitative polymerase chain reaction showed that increasing the TAE226 dose and reaction time resulted in increased and decreased E-cadherin and Vimentin mRNA expressions, respectively (P<0.05). Western blot assays showed that increasing the TAE226 dose resulted in increased and decreased E-cadherin and Vimentin protein expressions, respectively (P<0.05). CONCLUSIONS: TAE226, which is expected to be an effective drug for OSCC treatment, can effectively inhibit the EMT of the OSCC cell line.

Oct-4 Enhanced the Therapeutic Effects of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Acute Kidney Injury.

BACKGROUND/AIMS: Acute kidney injury (AKI) is a common clinical condition that can lead to chronic kidney failure. Although mesenchymal stem cell-derived extracellular vesicles (MSC EVs) are regarded as a potent AKI treatment, the mechanisms underlying their beneficial effects remain unclear. Oct-4 may play an important role in tissue injury repair. We thus hypothesized that oct-4 overexpression might enhance the therapeutic effects of MSC EVs in AKI treatment. METHODS: Renal tubular epithelial cells were cultured in a low oxygen environment, then cocultured with MSC EVs or control medium for 48 h. BrdU and transferase-mediated dUTP nick-end labeling (TUNEL) staining were used to assess cell proliferation and apoptosis. Mice subjected to ischemia reperfusion were randomly divided into 4 groups, then injected with either phosphate-buffered saline (vehicle), EVs, EVs overexpressing oct-4 (EVs+Oct-4), and EVs not expressing Oct-4 (EVs-Oct-4). Blood creatinine (CREA) and urine nitrone levels were assessed 48 h and 2 weeks after injection. After ischemia reperfusion, renal tissues from each group were stained with TUNEL and proliferating cell nuclear antigen (PCNA) to determine the degree of apoptosis and proliferation. Masson trichrome staining was used to evaluate renal fibrosis progression. Snail gene expression was assessed using polymerase chain reaction (PCR). RESULTS: At 48 h after hypoxic treatment, TUNEL and BrdU staining indicated that the EVs+Oct-4 group had the least apoptosis and the most proliferation, respectively. Treatment with EVs overexpressing Oct-4 significantly decreased serum Crea and blood urea nitrogen levels and rescued kidney fibrosis, as indicated by the low proportion of Masson staining, high number of PCNA-positive cells, and low number of TUNEL-positive cells. PCR analysis indicated that Snail was most upregulated in the vehicle group and least upregulated in the EVs+Oct-4 group. CONCLUSIONS: MSC EVs had a pronounced therapeutic effect on ischemic reperfusion injury-related AKI, and Oct-4 overexpression enhanced these therapeutic effects. Our results may inspire a new direction for AKI treatment with MSC EVs.

Comprehensive miRNA Analysis of Human Umbilical Cord-Derived Mesenchymal Stromal Cells and Extracellular Vesicles.

BACKGROUND/AIMS: Mesenchymal stromal cells (MSCs) participate in the tissue-specific repair of many different organs, especially the kidney. Their effects are primarily mediated by the paracrine release of factors including extracellular vesicles (EVs), which are composed of micro-vesicles and exosomes. The corresponding microRNAs (miRNAs) of EVs are considered important for their biological functions. METHODS: MSCs were cultured from the human umbilical cord, and EVs were isolated from the medium. The expression levels of miRNAs in MSCs and EVs were determined by microarray analysis, and gene ontology (GO) was used to analyze the functions of their target genes. RESULTS: MSCs and EVs had similar miRNA expression profiles, with the exception of a small number of selectively enriched miRNAs. GO analysis indicated that, unlike MSCs, the target genes of EV-enriched miRNAs were associated with calcium channel regulation and cell junction activities, which may indicate that MSC and EVs have different regulatory properties. Angiogenesis, oxidative stress, and inflammatory signaling pathways related to the repair of renal injury were also analyzed, and EV-enriched miRNAs targeted genes associated with oxidative stress, T cell activation, and Toll-like receptor signaling. The miRNAs enriched in both MSCs and EVs targeted different genes in signaling pathways regulating angiogenesis and chemokine release. CONCLUSION: MSCs and their EVs shared similar miRNA component, and some selectively enriched miRNAs observed in MSCs and EVs may affect different target genes through some specific signaling pathways.

Microvesicles derived from human umbilical cord mesenchymal stem cells facilitate tubular epithelial cell dedifferentiation and growth via hepatocyte growth factor induction.

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.

Determination of polychlorinated biphenyls and organochlorine pesticides in human serum by gas chromatography with micro-electron capture detector.

A method for determination of concentrations of polychlorinated biphenyl congeners (PCB-28, 52, 101, 118, 138, 153, 156, and 187) and organochlorine pesticides (hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, gamma-hexachlorocyclohexane, delta-hexachlorocyclohexane, p,p'-dichlorodiphenyl dichloroethylene, o,p'-dichlorodiphenyl trichloroethane, p,p'-dichlorodiphenyl dichloroethane, p,p'-dichlorodiphenyl trichloroethane, alpha-chlordane, gamma-chlordane, heptachlor, heptachlor epoxide, and aldrin) in human serum is developed. Recovery is assessed with artificial serum, in which PCBs and OCPs could not be detected. The method is then confirmed with pooled human serum. Experiments are performed by adding two concentrations of analytes (0.5 µg/L and 1.0 µg/L) to both matrices. The sample pretreatment process involves denaturing with a mixture of water-1-propanol (v:v, 85:15), extraction with a C-18 cartridge, and cleanup with an Alumina B cartridge. This process required about 2 mL of serum. The limit of detection ranged from 0.05-0.35 µg/L for all the analytes. Recovery of analytes at low and high spiking concentrations varied from 63-122% and 61-124% for artificial serum and pooled human serum, respectively. Relative standard deviation was lower than 16% and 18% for artificial serum and pooled human serum, respectively. Stability of the method, expressed as relative standard deviation, was lower than 14%. The method has been applied in epidemiological research.

The role of metabotropic glutamate receptor 5 in developmental lead neurotoxicity.

A complete explanation of the mechanisms of lead-induced developmental neurotoxicity remains unknown. The glutamate receptor is one of the most important targets of lead. More recently, metabotropic glutamate receptor 5 (mGluR5) has been shown to have a functional relationship with learning and memory. We investigated the impact of developmental lead exposure on hippocampal mGluR5 expression and its potential role in lead neurotoxicity. Both in vitro model of lead exposure with Pb(2+) concentrations of 0, 10 nM, 1 microM, and 100 microM in cultured rat embryonic hippocampal neurons, and the in vivo model of rat maternal lead exposure involving both gestational and lactational exposure with 0, 0.05%, 0.2%, and 0.5% lead acetate were utilized. Immunoperoxidase and immunofluorescent analyses, quantitative PCR and western blotting were used. In vitro studies revealed that expression of mGluR5 mRNA and protein was decreased dose-dependently after lead exposure, which was further confirmed by the results of in vivo studies. These data suggest that mGluR5 might be involved in lead-induced neurotoxicity by disturbing mGluR5-induced long-term depression and decreasing N-methyl-D-aspartic acid receptor (NMDAR)-dependent or protein synthesis-dependent long-term potentiation. These results might improve the understanding of the mechanism and potential treatments for moderate to severe lead poisoning in children.