Exosomal miR-205-5p Improves Endometrial Receptivity by Upregulating E-Cadherin Expression through ZEB1 Inhibition.

Endometrial receptivity is a complex process that prepares the uterine endometrium for embryo implantation; insufficient endometrial receptivity is one of the causes of implantation failure. Here, we analyzed the microRNA expression profiles of exosomes derived from both receptive (RL95-2) and non-receptive (AN3-CA) endometrial epithelial cell (EEC) lines to identify exosomal miRNAs closely linked to endometrial receptivity. Among the 466 differentially expressed miRNAs, miR-205-5p was the most highly expressed in exosomes secreted from receptive RL95-2 cells. miR-205-5p, enriched at the adhesive junction, was closely related to endometrial receptivity. ZEB1, a transcriptional repressor of E-cadherin associated with endometrial receptivity, was identified as a direct target of miR-205-5p. miR-205-5p expression was significantly lower in the endometrial tissues of infertile women than in that of non-infertile women. In vivo, miR-205-5p expression was upregulated in the post-ovulatory phase, and its inhibitor reduced embryo implantation. Furthermore, administration of genetically modified exosomes overexpressing miR-205-5p mimics upregulated E-cadherin expression by targeting ZEB1 and improved spheroid attachment of non-receptive AN3-CA cells. These results suggest that the miR-205-5p/ZEB1/E-cadherin axis plays an important role in regulating endometrial receptivity. Thus, the use of exosomes harboring miR-205-5p mimics can be considered a potential therapeutic approach for improving embryo implantation.

Regulating POLR3G by MicroRNA-26a-5p as a promising therapeutic target of lung cancer stemness and chemosensitivity.

Cancer stem cells (CSCs) identified in lung cancer exhibit resistance to chemotherapy, radiotherapy, and targeted therapy. Therefore, a technology for controlling CSCs is needed to overcome such resistance to cancer therapy. Various evidences about the association between epithelial-mesenchymal transition related transcriptomic alteration and acquisition of CSC phenotype have been proposed recently. Down-regulated miR-26a-5p is closely related to mesenchymal-like lung cancer cell lines. These findings suggest that miR-26a-5p might be involved in lung cancer stemness. RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. It was found that miR-26a-5p directly regulates the expression of POLR3G.Overexpression of miR-26a-5p induced a marked reduction of colony formation and sphere formation. Co-treatment of miR-26a-5p and paclitaxel decreased cell growth, suggesting that miR-26a-5p might play a role as a chemotherapy sensitizer. In the cancer genome atlas data, high miR-26a-5p and low POLR3G expression were also related to higher survival rate of patients with lung adenocarcinoma. These results suggest that miR-26a-5p can suppress lung cancer stemness and make cancer cell become sensitive to chemotherapy. This finding provides a novel insight into a potential lung cancer treatment by regulating stemness.

The miR-182-5p/NDRG1 Axis Controls Endometrial Receptivity through the NF-κB/ZEB1/E-Cadherin Pathway.

Endometrial receptivity is essential for successful pregnancy, and its impairment is a major cause of embryo-implantation failure. MicroRNAs (miRNAs) that regulate epigenetic modifications have been associated with endometrial receptivity. However, the molecular mechanisms whereby miRNAs regulate endometrial receptivity remain unclear. Therefore, we investigated whether miR-182 and its potential targets influence trophoblast cell attachment. miR-182 was expressed at lower levels in the secretory phase than in the proliferative phase of endometrium tissues from fertile donors. However, miR-182 expression was upregulated during the secretory phase in infertile women. Transfecting a synthetic miR-182-5p mimic decreased spheroid attachment of human JAr choriocarcinoma cells and E-cadherin expression (which is important for endometrial receptivity). miR-182-5p also downregulated N-Myc downstream regulated 1 (NDRG1), which was studied further. NDRG1 was upregulated in the secretory phase of the endometrium tissues and induced E-cadherin expression through the nuclear factor-κΒ (NF-κΒ)/zinc finger E-box binding homeobox 1 (ZEB1) signaling pathway. NDRG1-overexpressing or -depleted cells showed altered attachment rates of JAr spheroids. Collectively, our findings indicate that miR-182-5p-mediated NDRG1 downregulation impaired embryo implantation by upregulating the NF-κΒ/ZEB1/E-cadherin pathway. Hence, miR-182-5p is a potential biomarker for negative selection in endometrial receptivity and a therapeutic target for successful embryo implantation.

SIRT1 suppresses in vitro decidualization of human endometrial stromal cells through the downregulation of forkhead box O1 expression.

SIRT1 regulates survival, DNA repair, and metabolism in human cells and has pleiotropic effects on age-related diseases through either deacetylating target proteins or inhibiting gene transcription. Forkhead box O1 (FOXO1) is one of the most important transcription factors during decidualization. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) are well-known FOXO1-dependent genes in decidualizing cells. To determine whether SIRT1 plays a role in decidualization, we investigated morphological changes in cells following artificially stimulated decidualization and expression levels of PRL, IGFBP1, and FOXO1 in the immortalized non-neoplastic human endometrial stromal cell line T HESCs. SIRT1 expression decreased in the decidualization condition and SIRT1 inhibited morphological changes caused by decidualization of T HESCs. SIRT1 suppressed PRL, IGFBP1, and FOXO1 expression; inhibited FOXO1, PRL, and IGFBP1 promoter activity; and decreased histone protein acetylation of the FOXO1 promoter. We found that FOXO1 expression increased in the secretory phase compared with the proliferative phase, whereas SIRT1 expression decreased in the secretory phase in the human endometrium. We also revealed that SIRT1 may inhibit embryo implantation according to the blastocyst-like spheroid implantation assay. Collectively, these results indicate that SIRT1 suppresses decidualization of human endometrial stromal cells by inhibiting FOXO1 expression.

Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosome storage disease (LSD) characterized by significantly reduced intracellular autophagy function. This contributes to the progression of intracellular pathologic signaling and can lead to organ injury. Phospholipid-polyethyleneglycol-capped Ceria-Zirconia antioxidant nanoparticles (PEG-CZNPs) have been reported to enhance autophagy flux. We analyzed whether they suppress globotriaosylceramide (Gb3) accumulation by enhancing autophagy flux and thereby attenuate kidney injury in both cellular and animal models of FD. RESULTS: Gb3 was significantly increased in cultured human renal proximal tubular epithelial cells (HK-2) and human podocytes following the siRNA silencing of α galactosidase A (α-GLA). PEG-CZNPs effectively reduced the intracellular accumulation of Gb3 in both cell models of FD and improved both intracellular inflammation and apoptosis in the HK-2 cell model of FD. Moreover these particles attenuated pro fibrotic cytokines in the human podocyte model of FD. This effect was revealed through an improvement of the intracellular autophagy flux function and a reduction in reactive oxygen species (ROS). An FD animal model was generated in which 4-week-old male B6;129-Glatm1Kul/J mice were treated for 8 weeks with 10 mg/kg of PEG-CZNPs (twice weekly via intraperitoneal injection). Gb3 levels were reduced in the kidney tissues of these animals, and their podocyte characteristics and autophagy flux functions were preserved. CONCLUSIONS: PEG-CZNPs alleviate FD associated kidney injury by enhancing autophagy function and thus provide a foundation for the development of new drugs to treat of storage disease.

Impairment of Decidualization of Endometrial Stromal Cells by hsa-miR-375 Through NOX4 Targeting.

Decidualization of the endometrial stromal cells (ESCs) is essential for successful embryo implantation. It involves the transformation of fibroblastic cells into epithelial-like cells that secrete cytokines, growth factors, and proteins necessary for implantation. Previous studies have revealed altered expression of miR-375 in the endometrium of patients with recurrent implantation failure and the ectopic stromal cells of patients with endometriosis. However, the exact molecular mechanisms, particularly the role of microRNAs (miRNAs) in the regulation of decidualization, remain elusive. In this study, we investigated whether decidualization is affected by miR-375 and its potential target(s). The findings demonstrated the downregulation of the expression of miR-375 in the secretory phase compared to its expression in the proliferative phase of the endometrium in normal donors. In contrast, it was upregulated in the secretory phase of the endometrium in infertility patients. Furthermore, during decidualization of ESCs in vitro, overexpression of miR-375 significantly reduced the transcript-level expression of forkhead box protein O1 (FOXO1), prolactin (PRL), and insulin-like growth factor binding protein-1 (IGFBP1), the well-known decidual cell markers. Overexpression of miR-375 also resulted in reduced decidualization-derived intracellular and mitochondrial reactive oxygen species (ROS) levels. Using the luciferase assay, we confirmed that NADPH oxidase 4 (NOX4) is a direct target of miR-375. Collectively, the study showed that the miR-375-mediated NOX4 downregulation reduced ROS production and attenuated the decidualization of ESCs. It provides evidence that miR-375 is a negative regulator of decidualization and could serve as a potential target for combating infertility.

Transcriptomic analysis and competing endogenous RNA network in the human endometrium between proliferative and mid-secretory phases.

Successful embryo implantation is the first step for establishing natural pregnancy and is dependent on the crosstalk between the embryo and a receptive endometrium. However, the molecular signaling events for successful embryo implantation are not entirely understood. To identify differentially expressed transcripts [long-noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs] and competing endogenous RNA (ceRNA) networks associated with endometrial receptivity, the current study analyzed gene expression profiles between proliferative and mid-secretory endometria in fertile women. A total of 247 lncRNAs, 67 miRNAs and 2,154 mRNAs were identified as differentially expressed between proliferative and mid-secretory endometria. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these differentially expressed genes were significantly enriched for 'cell adhesion molecules.' Additionally, 98 common mRNAs were significantly involved in tryptophan metabolism, metabolic pathways and FoxO signaling. From the differentially expressed lncRNA/miRNA/mRNA ceRNA network, hub RNAs that formed three axes were identified: The DLX6-AS1/miR-141 or miR-200a/OLFM1 axis, the WDFY3-AS2/miR-135a or miR-183/STC1 axis, and the LINC00240/miR-182/NDRG1 axis. These may serve important roles in the regulation of endometrial receptivity. The hub network of the current study may be developed as a candidate marker for endometrial receptivity.

LncRNA LINC00240 suppresses invasion and migration in non-small cell lung cancer by sponging miR-7-5p.

BACKGROUND: lncRNAs have important roles in regulating cancer biology. Accumulating evidence has established a link between the dysregulation of lncRNAs and microRNA in cancer progression. In previous studies, miR-7-5p has been found to be significantly down-regulated in mesenchymal-like lung cancer cell lines and directly regulated EGFR. In this work, we investigated the lncRNA partner of miR-7-5p in the progression of lung cancer. METHODS: We investigated the expression of miR-7-5p and the lncRNA after transfection with an miR-7-5p mimics using a microarray. The microarray results were validated using quantitative real time-polymerase Chain Reaction (qRT-PCR). The regulatory effects of lncRNA on miR-7-5p and its target were evaluated by changes in the expression of miR-7-5p after transfection with siRNAs for lncRNA and the synthesis of full-length lncRNA. The effect of miR-7-5p on lncRNA and the miRNA target was evaluated after transfection with miRNA mimic and inhibitor. The role of lncRNA in cancer progression was determined using invasion and migration assays. The level of lncRNA and EGFR in lung cancer and normal lung tissue was analyzed using TCGA data. RESULTS: We found that LINC00240 was downregulated in lung cancer cell line after miR-7-5p transfection with an miR-7-5p mimic. Further investigations revealed that the knockdown of LINC00240 induced the overexpression of miR-7-5p. The overexpression of miR-7-5p diminished cancer invasion and migration. The EGFR expression was down regulated after siRNA treatment for LINC00240. Silencing LINC00240 suppressed the invasion and migration of lung cancer cells, whereas LINC00240 overexpression exerted the opposite effect. The lower expression of LINC00240 in squamous lung cancer was analyzed using TCGA data. CONCLUSIONS: Taken together, LINC00240 acted as a sponge for miR-7-5p and induced the overexpression of EGFR. LINC00240 may represent a potential target for the treatment of lung cancer.

Ceria-Zirconia Antioxidant Nanoparticles Attenuate Hypoxia-Induced Acute Kidney Injury by Restoring Autophagy Flux and Alleviating Mitochondrial Damage.

Oxidative stress is one of the principal causes of hypoxia-induced kidney injury. The ceria nanoparticle (CNP) is known to exhibit free radical scavenger and catalytic activities. When zirconia is attached to CNPs (CZNPs), the ceria atom tends to remain in a Ce3+ form and its efficacy as a free radical scavenger thus increases. We determined the effectiveness of CNP and CZNP antioxidant activities against hypoxia-induced acute kidney injury (AKI) and observed that these nanoparticles suppress the apoptosis of hypoxic HK-2 cells by restoring autophagy flux and alleviating mitochondrial damage. In vivo experiments revealed that CZNPs effectively attenuate hypoxia-induced AKI by preserving renal structures and glomerulus function. These nanoparticles can successfully diffuse into HK-2 cells and effectively counteract reactive oxygen species (ROS) to block hypoxia-induced AKI. This suggests that these particles represent a novel approach to controlling this condition.

MicroRNA-7-5p's role in the O-GlcNAcylation and cancer metabolism.

O-GlcNAc Transferase (OGT) is a complementary enzyme that regulates O-linked N-acetylglucosaminylation(O-GlcNAcylation) and plays a critical role in various cancer phenotypes, including invasion, migration, and metabolic reprogramming. In our previous study we found that miR-7-5p was downregulated at lung cancer cells with highly metastatic capacity. In the in-silico approach, OGT is the predicted target of miR-7-5p. To identify miR-7-5p's role in cell growth and metabolism, we transfected various lung cancer cell lines with miR-7-5p. The expression level of miR-7-5p was confirmed by qRT-PCR in lung cancer cell lines. Western blot assays and qRT-PCR were performed to demonstrate miR-7-5p's effect. Bioinformatic analysis indicated that OGT is a direct target of miR-7-5p. The binding sites of miR-7-5p in the OGT 3' UTR were verified by luciferase reporter assay. To investigate the role of miR-7-5p in the cancer metabolism of non-small cell lung cancer (NSCLC) cells, mimic of miR-7-5p was transfected into NSCLC cells, and the effect of miR-7-5p on cancer metabolism was analyzed by LDH assays, glucose uptake, and mitochondrial ATP synthase inhibitor assay. O-GlcNAcylated protein level was determined by Western blot. The role of miR-7-5p in lung cancer growth was measured by MTS assays. To identify the delivery of miR-7-5p via PLGA, an in vitro release assay of PLGA-miR-7-5p was done. miR-7-5p was highly expressed whereas OGT showed low expression in H358, H827. However, miR-7-5p exhibited low expression while OGT had high expression in H522, H460, and H1299 cell lines. OGT were repressed by binding of miR-7a-5p to the 3'-UTR. Overexpression of miR-7-5p also diminished anaerobic glycolysis. miR-181a-5p transfection induced expression levels of OGT were diminished compared to those in the control group. O-GlcNAcylation was suppressed by miR-7-5p. Moreover, the overexpression of miR-7-5a suppressed lung cancer cell growth. miR-7-5p was released via PLGA for up to 10 days. In the present study, inhibition of OGT by miR-7-5p decreased the growth and cancer metabolism of lung cancer.

A Synthetic CPP33-Conjugated HOXA9 Active Domain Peptide Inhibits Invasion Ability of Non-Small Lung Cancer Cells.

Homeobox A9 (HOXA9) expression is associated with the aggressive growth of cancer cells and poor prognosis in lung cancer. Previously, we showed that HOXA9 can serve as a potential therapeutic target for the treatment of metastatic non-small cell lung cancer (NSCLC). In the present study, we have carried out additional studies toward the development of a peptide-based therapeutic agent. Vectors expressing partial DNA fragments of HOXA9 were used to identify a unique domain involved in the inhibition of NSCLC cell invasion. Next, we performed in vitro invasion assays and examined the expression of EMT-related genes in transfected NSCLC cells. The C-terminal fragment (HOXA9-C) of HOXA9 inhibited cell invasion and led to upregulation of CDH1 and downregulation of SNAI2 in A549 and NCI-H1299 cells. Reduced SNAI2 expression was consistent with the decreased binding of transcription factor NF-kB to the SNAI2 promoter region in HOXA9-C overexpressing cells. Based on the above results, we synthesized a cell-permeable peptide, CPP33-HADP (HOXA9 active domain peptide), for lung-specific delivery and tested its therapeutic efficiency. CPP33-HADP effectively reduced the invasion ability of NSCLC cells in both in vitro and in vivo mouse models. Our results suggest that CPP33-HADP has significant potential for therapeutic applications in metastatic NSCLC.

Decursinol from Angelica gigas Nakai enhances endometrial receptivity during implantation.

BACKGROUND: Embryo implantation is essential for a successful pregnancy, and an elaborate synchronization between the receptive endometrium and trophoblast is required to achieve this implantation. To increase 'endometrial receptivity', the endometrium undergoes transformation processes including responses of adhesion molecules and cellular and molecular cell to cell communication. Many natural substances from traditional herbs have been studied to aid in the achievement of successful implantation. In this study, we investigated positive effects on embryonic implantation with decursinol that is a major compound extracted from Angelica gigas Nakai known to be associated with promotion of healthy pregnancy in the traditional Korean herbal medicine. METHODS: Expression of cell adhesion molecules after treatment of endometrial epithelial cells by decursinol (40 or 80 µM) was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analysis. The alteration of endometrial receptivity by decursinol (40 or 80 µM) was identified with the in vitro implantation model between Ishikawa cells and JAr cell spheroids (diameter, 143 ± 16 µm). Exosomes secreted from Ishikawa cells after treatment of 80 µM decursinol or dimethyl sulfoxide (DMSO) as the vehicle were investigated with invasion of JAr cells and attachment of JAr spheroids to Ishikawa cells. RESULTS: Decursinol significantly (P < 0.05) increased the expression of important endometrial adhesion molecules such as integrin ß1, ß3, ß5 and L-selectin mRNAs and integrin ß5 and L-selectin in protein. The adhesion rate of JAr spheroids to decursinol-treated Ishikawa cells also increased significantly which was 2.4-fold higher than that of the control (P < 0.05). Furthermore, decursinol induced an increase in the release of exosomes from Ishikawa cells and decursinol-induced exosomes showed autocrine (to Ishikawa cells) and paracrine (to JAr cells) positive effects on our implantation model. CONCLUSION: These results propose that decursinol could serve as a new and alternative solution for patients who are infertile.

NADPH oxidase 4 mediates TGF-ß1/Smad signaling pathway induced acute kidney injury in hypoxia.

Hypoxia is an important cause of acute kidney injury (AKI) in various conditions because kidneys are one of the most susceptible organs to hypoxia. In this study, we investigated whether nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase 4 (Nox4) plays a role in hypoxia induced AKI in a cellular and animal model. Expression of Nox4 in cultured human renal proximal tubular epithelial cells (HK-2) was significantly increased by hypoxic stimulation. TGF-ß1 was endogenously secreted by hypoxic HK-2 cells. SB4315432 (a TGF-ß1 receptor I inhibitor) significantly inhibited Nox4 expression in HK-2 cells through the Smad-dependent cell signaling pathway. Silencing of Nox4 using Nox4 siRNA and pharmacologic inhibition with GKT137831 (a specific Nox1/4 inhibitor) reduced the production of ROS and attenuated the apoptotic pathway. In addition, knockdown of Nox4 increased cell survival in hypoxic HK-2 cells and pretreatment with GKT137831 reproduce these results. This study demonstrates that hypoxia induces HK-2 cell apoptosis through a signaling pathway involving TGF-ß1 via Smad pathway induction of Nox4-dependent ROS generation. In an ischemia/reperfusion rat model, pretreatment of GKT137831 attenuated ischemia/reperfusion induced acute kidney injury as indicated by preserved kidney function, attenuated renal structural damage and reduced apoptotic cells. Therapies targeting Nox4 may be effective against hypoxia-induced AKI.

Increase of Hspa1a and Hspa1b genes in the resting B cells of Sirt1 knockout mice.

Sirt1, also known as the longevity gene, is an NAD+-dependent class III histone deacetylase that has been extensively studied in multiple areas of research including cellular metabolism, longevity, cancer, autoimmunity, and immunity. However, little is known about the function of Sirt1 in B cells. This study aimed to investigate the role of Sirt1 in the expression pattern of mRNAs in the resting B cells of mice. CD19+ B cell-specific inducible Sirt1 knockout (KO) mice were divided into tamoxifen-treated Sirt1 KO group (S19T) or control group (S19). mRNAs extracted from resting B cells of both groups were analyzed for differentially expressed genes (DEG) using microarray. DEG analysis showed significant differential expression of 20 genes, of which Hspa1a and Hspa1b showed the highest fold change (FC) in S19T compared with S19 (p value < 0.01 and FC > 3). Further, Kyoto Encyclopedia of Genes and Genomes analysis identified pathways associated with diseases, organismal systems, and antigen processing and presentation. Additionally, the pathways known to involve Hspa1a and Hspa1b were also activated in the S19T group. On the other hand, after in vitro stimulation with lipopolysaccharide, cell viability and IgM production were significantly decreased in Sirt1 KO B cells, while expressions of TNF-α, IL-6, and IL-10 were increased. In summary, our study reveals that Sirt1 may maintain the quiescent state in resting B cells by suppressing the increase of Hspa1a and Hspa1b. This work provides a foundation for further studies on the functional roles of Sirt1 in B cells.

Recombinant cell-permeable HOXA9 protein inhibits NSCLC cell migration and invasion.

PURPOSE: Previously, it has been reported that homeobox A9 (HOXA9) protein expression is downregulated in lung cancer cells, and that its expression is inversely correlated with the metastatic potential of lung cancer cells both in vitro and in vivo. As such, HOXA9 shows therapeutic potential. The development of therapeutic strategies based on this protein is, however, limited due to its poor membrane permeability. To overcome this problem, we developed a system to deliver HOXA9 protein into non-small cell lung cancer (NSCLC) cells. METHODS: First, we constructed a delivery vector expressing polyarginine, a cell-penetrating peptide, as well as HOXA9. The resulting recombinant R10-HOXA9 protein was effectively introduced into A549 and NCI-H1299 NSCLC cells. Next, we examined the roles and molecular mechanisms of recombinant R10-HOXA9 in processes involved in tumor progression. To investigate the therapeutic efficacy of the delivery system, we performed cell motility assays using both in vitro and in vivo experimental models. RESULTS: We found that recombinant R10-HOXA9 protein reduced the invasion and migration rate, but not the proliferation rate, of the NSCLC cells tested, both in vitro and in vivo. Treatment of NSCLC cells with recombinant R10-HOXA9 protein led to a significant increase in E-cadherin expression. Conversely, we found that the expression of snail family zinc finger 2 (SLUG), a transcriptional repressor of E-cadherin, was markedly decreased. In an experimental metastatic mouse model, recombinant R10-HOXA9 protein was found to effectively reduce the rate of lung cancer cell motility. CONCLUSIONS: Our data suggest that the developed cell-permeable R10-HOXA9 system may serve as a useful tool to prevent NSCLC cell migration and invasion.

Identification of mTOR inhibitor-resistant genes in cutaneous squamous cell carcinoma.

PURPOSE: The PI3K/AKT/mTOR pathway is frequently activated in various squamous cell carcinomas (SCCs). Although mTOR inhibitors are suggested as effective treatments in immunosuppressed patients with metastatic SCC, they are still not proven to be favorable in treating skin SCC patients not undergoing immunosuppressive therapy. Moreover, the exact mechanism of the mTOR signaling pathway in SCC has not yet been identified. In this study, we aimed to determine the genes associated with mTOR inhibitors in skin SCC. MATERIALS AND METHODS: The identification of cell viability according to concentration of everolimus and Western blot was done. To analyze the global gene expression profiles, A431 and HSC-1 cells were treated with dimethyl sulfoxide (DMSO) or 100 nM of everolimus for 72 hours. Furthermore, differentially expressed genes (DEGs) were identified using Affymetrix analysis. To identify the gene network associated with everolimus resistance in SCC cells, pathway analysis was performed using the Ingenuity Pathway Analysis (IPA) tool. RESULTS: The effects of cell death with respect to the mTOR inhibitor concentration were observed in the HSC-1 cell line; however, the mTOR inhibitor did not show effective cytotoxic activity in the A431 cell line. p-mTOR concentration also diminished with respect to everolimus concentrations in the HSC-1 cell line. Moreover, the microarray results showed that the MYC/CCND1/TP73/NUPR1/SBD/ERBB2/CDKN2B genes were related to mTOR inhibitor resistance. However, CCND1 gene overexpression was most closely related to mTOR inhibitor resistance. CONCLUSION: We identified mTOR inhibitor resistance genes, and our findings may help select therapeutic targets in skin SCC.

Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury.

Contrast-induced acute kidney injury (CIAKI) is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidases (Noxs) are important sources of reactive oxygen species (ROS). Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831). Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38) implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2'-deoxyguanosine (8OHdG) and kidney injury molecule-1 (KIM-1), and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.

TGF-ß-mediated NADPH oxidase 4-dependent oxidative stress promotes colistin-induced acute kidney injury.

Background: Colistin (polymyxin E) is an important constituent of the polymyxin class of cationic polypeptide antibiotics. Intrarenal oxidative stress can contribute to colistin-induced nephrotoxicity. Nicotinamide adenine dinucleotide 3-phosphate oxidases (Noxs) are important sources of reactive oxygen species. Among the various types of Noxs, Nox4 is predominantly expressed in the kidney. Objectives: We investigated the role of Nox4 and benefit of Nox4 inhibition in colistin-induced acute kidney injury using in vivo and in vitro models. Methods: Human proximal tubular epithelial (HK-2) cells were treated with colistin with or without NOX4 knockdown, or GKT137831 (most specific Nox1/4 inhibitor). Effects of Nox4 inhibition on colistin-induced acute kidney injury model in Sprague-Dawley rats were examined. Results: Nox4 expression in HK-2 cells significantly increased following colistin exposure. SB4315432 (transforming growth factor-ß1 receptor I inhibitor) significantly inhibited Nox4 expression in HK-2 cells. Knockdown of NOX4 transcription reduced reactive oxygen species production, lowered the levels of pro-inflammatory markers (notably mitogen-activated protein kinases) implicated in colistin-induced nephrotoxicity and attenuated apoptosis by altering Bax and caspase 3/7 activity. Pretreatment with GKT137831 replicated these effects mediated by downregulation of mitogen-activated protein kinase activities. In a rat colistin-induced acute kidney injury model, administration of GKT137831 resulted in attenuated colistin-induced acute kidney injury as indicated by attenuated impairment of glomerulus function, preserved renal structures, reduced expression of 8-hydroxyguanosine and fewer apoptotic cells. Conclusions: Collectively, these findings identify Nox4 as a key source of reactive oxygen species responsible for kidney injury in colistin-induced nephrotoxicity and highlight a novel potential way to treat drug-related nephrotoxicity.

Role of epigenetics in the pathogenesis of chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis (CRS) is a highly prevalent disease characterized by mucosal inflammation of the nose and paranasal sinuses. CRS can be divided into two main categories, CRS with nasal polyps (NPs; CRSwNP) and CRS without NPs (CRSsNP). Although the pathophysiology of CRS remains unclear, DNA methylation has been implicated in the etiology of CRSwNP. The aim of the present study was to elucidate whether DNA methylation of specific genes is involved in the development of NPs. In total, 18 individuals were included in the present study, and were divided into three groups: CRSwNP (n=7), CRSsNP (n=7) and healthy controls (n=4). NP tissues were obtained from the seven patients with CRSwNP and biopsies of the inferior turbinate mucosa from all three groups were used as controls. Methylated genes detected by methyl­CpG­binding domain sequencing were validated by methylation­specific polymerase chain reaction (PCR), bisulfite sequencing, and reverse transcription­quantitative PCR (RT­qPCR). Methyl­CpG­binding domain sequencing identified 43,674 CpG islands in 518 genes. The promotor regions of 10 and 30 genes were hypermethylated and hypomethylated, respectively, in NP samples compared with controls. The top four genes with altered hypomethylation in NP tissues were, Keratin 19 (KRT19), nuclear receptor subfamily 2 group F member 2 (NR2F2), A Disintegrin­like And Metallopeptidase (Reprolysin Type) with Thrombospondin type 1 motif 1 (ADAMTS1) and zinc finger protein 222 (ZNF222). RT­qPCR demonstrated that the expression levels of KRT19, NR2F2 and ADAMTS1 were significantly increased in NP tissues; however, there was no difference in the levels of ZNF222 between NP and control tissues. Further studies are required to confirm the relevance of these epigenetic modifications in the mechanisms underlying NP formation.

Aberrant expression of interleukin-10 and activation-induced cytidine deaminase in B cells from patients with Behçet's disease.

Despite extensive studies, the pathogenesis of Behçet's disease (BD) remains unclear. In particular, the roles of B cells in patients with BD have not been elucidated. Activation-induced cytidine deaminase (AID) is a critical enzyme for immunoglobulin (Ig) heavy chain class switching and somatic hypermutation in B cells and the abnormal expression of AID in various immune conditions has previously been studied. B10 cells, an interleukin (IL)-10-secreting subset of regulatory B cells, function to downregulate inflammation and autoimmunity. Thus, in the present study, the relevance of B cells in patients with BD was investigated. The plasma levels of IL-10 and IgA and the proportions of cluster of differentiation (CD)43+ B cells, excluding naïve B cells, were measured in 16 patients with BD and 16 age- and sex-matched healthy controls (HCs). Additionally, the mRNA levels of IL-10 and AID were assessed in B cells from fresh peripheral blood samples of the BD patients and HCs. The plasma level of IL-10 in patients with BD did not differ significantly from that in HCs. Similarly, there was no significant difference in the plasma level of IgA, although a slight increase was observed in patients with BD compared with that in HCs. There were no differences in CD43+CD19+ B cell numbers between patients with BD and HCs. However, IL-10 mRNA levels were significantly reduced (P<0.05), while AID mRNA levels were significantly increased (P<0.01) in the B cells of patients with BD compared with those in HCs. These results provide insight into the role of B cells in patients with BD.