Establishment of a Model of Microencapsulated SGC7901 Human Gastric Carcinoma Cells Cocultured with Tumor-Associated Macrophages.

The important factors of poor survival of gastric cancer (GC) are relapse and metastasis. For further elucidation of the mechanism, a culture system mimicking the microenvironment of the tumor in humans was needed. We established a model of microencapsulated SGC7901 human GC cells and evaluated the effects of coculturing spheres with tumor-associated macrophages (TAMs). SGC7901 cells were encapsulated in alginate-polylysine-sodium alginate (APA) microcapsules using an electrostatic droplet generator. MTT assays showed that the numbers of microencapsulated cells were the highest after culturing for 14 days. Metabolic curves showed consumption of glucose and production of lactic acid by day 20. Immunocytochemistry confirmed that Proliferating Cell Nuclear Antigen (PCNA) and Vascular Endothelial Growth Factor (VEGF) were expressed in microencapsulated SGC7901 cells on days 7 and 14. The expression of PCNA was observed outside spheroids; however, VEGF was found in the entire spheroids. PCNA and VEGF were increased after being cocultured with TAMs. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions were detected in the supernatant of microencapsulated cells cocultured with TAMs but not in microencapsulated cells. Our study confirms the successful establishment of the microencapsulated GC cells. TAMs can promote PCNA, VEGF, MMP-2, and MMP-9 expressions of the GC cells.

Acute downregulation of miR-199a attenuates sepsis-induced acute lung injury by targeting SIRT1.

MicroRNA-199a (miR-199a) is a novel gene regulator with an important role in inflammation and lung injury. However, its role in the pathogenesis of sepsis-induced acute respiratory distress syndrome (ARDS) is currently unknown. Our study explored the role of miR-199a in sepsis-induced ARDS and its mechanism of action. First, we found that LPS could upregulate miR-199a in alveolar macrophages. Downregulation of miR-199a inhibited the upregulation of inflammatory cytokines in alveolar macrophages and induced the remission of histopathologic changes, the reduction of proinflammatory cytokines, and the upregulation of apoptosis protein expression in an ARDS lung, showing a protective role for miR-199a. We further identified sirtuin 1 (SIRT1) as a direct target of miR-199a in alveolar macrophages, and the expression of SIRT1 was negatively correlated with the level of miR-199a. The protective role of miR-199a downregulation in LPS-stimulated alveolar macrophages and sepsis-induced ARDS could be attenuated by SIRT1 inhibitor. Taken together, these results indicate that downregulation of miR-199a might protect lung tissue against sepsis-induced ARDS by upregulation of SIRT1 through the suppression of excessive inflammatory responses and the inhibition of cellular apoptosis in lung tissue, suggesting its potential therapeutic effects on sepsis-induced ARDS.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1.

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-ß2 by targeting ZNF217 via miR-200c in keloid fibroblasts.

Abnormally high activation of transforming growth factor-ß (TGF-ß) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-ß (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-ß in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-ß, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-ß2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-ß2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-ß2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-ß2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways.

Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing.

MiR-10a and miR-181c regulate collagen type I generation in hypertrophic scars by targeting PAI-1 and uPA.

Urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) have been proposed to play key roles in extracellular matrix (ECM) deposition in hypertrophic scars (HS). Here, we found that in HS fibroblasts (HFs) miR-181c and miR-10a were differentially-expressed and targeted uPA and PAI-1, respectively. The production of Type 1 collagen (Col1) was inhibited by miR-181c knockdown or miR-10a overexpression in HFs, and this resulted in increased levels of metalloproteinase 1 (MMP1). These results suggest that the miR-181c-uPA and miR-10a-PAI-1 regulatory pathways have an integral role in HS pathogenesis.