Long non-coding RNAs (lncRNAs) have been shown to be implicated in the complex network of cancer including malignant melanoma and play important roles in tumorigenesis and progression. However, their functions and downstream mechanisms are largely unknown. This study aimed to investigate whether BRAF-activated non-coding RNA (BANCR), a novel and potential regulator of melanoma cell, participates in the proliferation of malignant melanoma and elucidate the underlying mechanism in this process. We found that BANCR was abnormally overexpressed in human malignant melanoma cell lines and tissues, and increased with tumor stages by quantitative PCR. BANCR knockdown induced by shRNA transfection significantly inhibited proliferation of tumor cells and inactivated MAPK pathway, especially by silencing the ERK1/2 and JNK component. Moreover, combination treatment of BANCR knockdown and suppression ERK1/2 or JNK (induced by specific inhibitors U0126 or SP600125 respectively) produced synergistic inhibitory effects in vitro. And the inhibitory effects induced by ERK1/2 or JNK could be rescued by BANCR overexpression. By tumorigenicity assay in BALB/c nude mice, we further found that BANCR knockdown inhibited tumor growth in vivo. In addition, patients with high expression of BANCR had a lower survival rate. Taken together, we confirmed the abnormal upregulation of a novel lncRNA, BANCR, in human malignant melanoma. BANCR was involved in melanoma cell proliferation both in vitro and in vivo. The linkage between BANCR and MAPK pathway may provide a novel interpretation for the mechanism of proliferation regulation in malignant melanoma.

Melanoma is the most aggressive type of skin cancer with a rapid progression and a limited efficiency of therapeutics. Recently, studies have identified some microRNAs playing important roles in the development of melanoma. Syndecan-1 (Syn-1), dysregulated in many cancers, plays important roles in tumor progression by controlling cell proliferation. In this study, we investigated whether microRNA-143 (miR-143) is involved in the regulation of Syn-1 and thus plays a functional role in melanoma. We found that miR-143 expression was significantly lower in melanoma tissues than in normal tissues and its low expression was closely related to clinical stages of melanoma. Further experiments showed that consistent with the inhibitory effects induced by knockdown of Syn-1, overexpression of miR-143 suppressed cell proliferation, promoted G1 phase arrest and induced apoptosis in melanoma. Downregulation of miR-143 apparently produced opposite effects. Combined treatment of miR-143 overexpression and Syn-1 knockdown induced remarkable synergistic effects, while reconstitution of miR-143-resistant Syn-1 reversed the inhibitory activity of miR-143. Moreover, miR-143 level was inversely correlated with Syn-1 expression in melanoma cells. miR-143 directly targeted the 3′-untranslated regions of Syn-1 mRNA and they were in the same Argonaute2 complex. Taken together, this study revealed a link between miRNA-143 and Syn-1 in the pathogenesis of melanoma. MiR-143 plays an important role in the regulation of cell growth in melanoma. Restoration of miR-143 expression may represent a promising and efficient therapeutic approach for targeting malignant melanoma.

The present study aimed to examine the effects of isopsoralen against postmenopausal osteoporosis in an ovariectomized rat model. The ovariectomized rats were treated with three days 10 mg/kg isopsoralen or with three days 20 mg/kg isopsoralen. Alkaline phosphatase, the oxidative stress indicators and caspase‑3/9 were measured using ELISA assay kits. Reverse transcription‑quantitative polymerase chain reaction was used to measure collagen type I (Col I), osteocalcin and osteoprotegerin mRNA levels. Wnt, β‑catenin and peroxisome proliferators‑activated receptor γ (PPAR‑γ) were analyzed using western blot analysis. Isopsoralen suppressed mature adipocyte differentiation of C2C12 cells, inhibited serum calcium and urinary calcium levels, and reduced the structural scores of articular cartilage and cancellous bone in the proximal tibia metaphysis of mice with postmenopausal osteoporosis. Isopsoralen also promoted the activity of alkaline phosphatase and the mRNA expression levels of Col 1, osterix and osteopontin in mice with postmenopausal osteoporosis. Oxidative stress and activities of caspase‑3/9 in the mice with postmenopausal osteoporosis were effectively suppressed by isopsoralen treatment, which upregulated the protein expression of Wnt/β‑catenin and downregulated the protein expression of PPAR‑γ. These findings demonstrated that isopsoralen prevented osteoporosis through the regulation of PPAR‑γ/WNT, inhibiting oxidative stress by targeting the PPAR‑γ/WNT pathway. These results provide evidence of the potential targeted therapy for isopsoralen in the clinical treatment of postmenopausal osteoporosis.

BackgroundPyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox active secondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatory response triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to be clarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction and the signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined.ResultsIt was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA) differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after 10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also found that within 10 min of PCN incubation, the level of p-I-κBα in the cytosol was increased, which returned to baseline level after 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high in total cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-κB inhibitors inhibited PCN-induced NF-κB activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of their concentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine, an antioxidant, was able to inhibit PCN-induced IL-8 protein expression.ConclusionsIt is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937 cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF-κΒ signaling pathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.

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Porokeratosis is a chronic skin disorder characterized by the presence of patches with elevated, thick, keratotic borders, with histological cornoid lamella. Classic porokeratosis of Mibelli (PM) frequently appears in childhood with a risk of malignant transformation. Disseminated superficial actinic porokeratosis (DSAP) is the most common subtype of porokeratosis with genetic heterogeneities, and mevalonate kinase gene (MVK) mutations have been identified in minor portion of DSAP families of Chinese origin. To confirm the previous findings about MVK mutations in DSAP patients and test MVK's role(s) in PM development, we performed genomic sequence analysis for 3 DSAP families and 1 PM family of Chinese origin. We identified a splicing mutation of MVK gene, designated as c.1039+1G>A, in the PM family. No MVK mutations were found in three DSAP families. Sequence analysis for complementary DNA templates from PM lesions of all patients revealed a mutation at splice donor site of intron 10, designated as c.1039+1G>A, leading to the splicing defect and termination codon 52 amino acids after exon 10. Although no MVK mutations in DSAP patients were found as reported previously, we identified MVK simultaneously responsible for PM development.

BackgroundAtherosclerosis is considered to be a chronic inflammatory disease. Astragali Radix extract (ARE) is one of the major active ingredients extracted from the root of Astragalus membranaceus Bge. Although ARE has an anti-inflammatory function, its anti-atherosclerotic effects and mechanisms have not yet been elucidated.MethodsMurine endothelial SVEC4-10 cells were pretreated with different doses of ARE at different times prior to induction with tumor necrosis factor (TNF)-α. Cell adhesion assays were performed using THP-1 cells and assessed by enzyme-linked immunosorbent assay, western blotting and immunofluorescence analyses to detect the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), phosphorylated inhibitor of κB (p-iκB) and nuclear factor (NF)-κB. We also examined the effect of ARE on atherosclerosis in the aortic endothelium of apolipoprotein E-deficient (apoE−/−) mice.ResultsTNF-α strongly increased the expression of VCAM-1 and ICAM-1 accompanied by increased expression of p-iκB and NF-κB proteins. However, the expression levels of VCAM-1 and ICAM-1 were reduced by ARE in dose- and time-dependent manners, with the strongest effect at a dose of 120 μg/ml incubated for 4 h. This was accompanied by significantly decreased expression of p-iκB and inhibited activation of NF-κB. Immunofluorescence analysis also revealed that oral administration of ARE resulted in downregulation of adhesion molecules and decreased expression of macrophages in the aortic endothelium of apoE−/− mice. ARE could suppress the inflammatory reaction and inhibit the progression of atherosclerotic lesions in apoE−/− mice.ConclusionThis study demonstrated that ARE might be an effective anti-inflammatory agent for the treatment of atherosclerosis, possibly acting via the decreased expression of adhesion molecules.

Cellular repressor of E1A stimulated genes (CREG) is a novel modulator that maintains the homeostasis of vascular cells. The present study aimed to investigate the effects of CREG on tumor necrosis factor (TNF)-α-mediated inflammatory injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured and CREG overexpressing (VC), knockdown (VS) and mock-transfected (VE) HUVECs were challenged with TNF-α. We demonstrated that TNF-α prompted robust intercellular filamentous actin (F-actin) stress fiber formation as examined by rhodamin-phalloidin staining. Transwell assay and rhodamine B isothiocyanate-dextran staining indicated that TNF-α induced intercellular hyperpermeability of the HUVEC monolayers. These effects were attenuated in VC cells with forced CREG overexpression but significantly potentiated in VS cells with CREG silencing. After TNF-α stimulation, interleukin (IL)-6 and IL-8 secretions in VE cells were markedly increased and inducible nitric oxidase (iNOS) expression substantially elevated, whereas these effects were pronouncedly damped in VC cells. Conversely, in VS cells, the increase in inflammatory markers was substantially potentiated. Immunofluorescence staining demonstrated that nuclear factor κB (NF-κB) slowly and transiently translocated into the nuclei of VC cells upon TNF-α stimulation. However, a more swift and sustained nuclear translocation was observed in VS as compared to VE cells. Corresponding changes in the pattern of its protein expression was also observed. These data suggested that CREG can inhibit NF-κB activation, TNF-α-induced inflammatory responses and the hyperpermeability of endothelial cells, and may therefore represent a potential therapeutic target for pathological vascular injury.