Wnt/β-catenin signaling pathway is frequently dysregulated in human tumors and plays a critical role in tumorigenesis; however, the roles of microRNAs in mediating Wnt/β-catenin pathway are not well understood. Herein, we show that miR-30a-5p is activated by Wnt/β-catenin pathway through direct binding of β-catenin/TCF4 to two sites in the promoter region of miR-30a-5p. We also found that miR-30a-5p represses neural cell adhesion molecule (NCAM) expression by directly targeting two sites in the 3' untranslated region (3'-UTR) of NCAM mRNA. Moreover, Wnt/β-catenin pathway represses NCAM expression in glioma cells, which depends on miR-30a-5p. Finally, we found that miR-30a-5p promotes glioma cell growth invasion by repressing NCAM. Our findings demonstrate a novel Wnt/β-catenin-miR-30a-5p-NCAM regulatory axis which plays important roles in controlling glioma cell invasion and tumorigenesis.

Membranous nephropathy (MN) is the major cause of adult nephrotic syndrome, which severely affects patients' quality of life. Currently, percutaneous renal biopsy is required to definitively diagnose MN. However, this technique is invasive and may cause severe complications. Therefore, an urgent clinical need exists for dynamic noninvasive monitoring of the renal state. In-depth molecular imaging studies could assist in finding a solution. Membrane attack complex C5b-9 is the key factor in the development of MN, and this protein primarily deposits in the glomerulus. The present study bound polyclonal antibodies to C5b-9 with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to obtain C5b-9-targeted magnetic resonance molecular imaging probes. The probes were injected intravenously into rats with Heymann nephritis, a classic disease model of MN. The signal intensity in the T2*-weighted imaging of kidneys in vivo using 7.0 Tesla magnetic resonance imaging decreased significantly 24 hours after injection compared to the untargeted and control groups. This signal change was consistent with the finding of nanoparticle deposits in pathological glomeruli. This study demonstrated a novel molecular imaging technique for the assessment of MN.

Excessive reactive oxygen species (ROS) play an important role in the development of osteoporosis. Ophiopogonin D (OP-D), isolated from the traditional Chinese herbal agent Radix Ophiopogon japonicus, is a potent anti-oxidative agent. We hypothesized that OP-D demonstrates anti-osteoporosis effects via decreasing ROS generation in mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells and a macrophage cell line RAW264.7 cells. We investigated OP-D on osteogenic and osteoclastic differentiation under oxidative status. Hydrogen peroxide (H2O2) was used to establish an oxidative damage model. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Then, we searched the molecular mechanism of OP-D against osteoporosis. Our results revealed that OP-D significantly promoted the proliferation of MC3T3-E1 cells and improved some osteogenic markers. Moreover, OP-D reduced TRAP activity and the mRNA expressions of osteoclastic genes in RAW264.7 cells. OP-D suppressed ROS generation in both MC3T3-E1 and RAW264.7 cells. OP-D treatment reduced the activity of serum bone degradation markers, including CTX-1 and TRAP. Further research showed that OP-D displayed anti-osteoporosis effects via reducing ROS through the FoxO3a-β-catenin signaling pathway. In summary, our results indicated that the protective effects of OP-D against osteoporosis are linked to a reduction in oxidative stress via the FoxO3a-β-catenin signaling pathway, suggesting that OP-D may be a beneficial herbal agent in bone-related disorders, such as osteoporosis.

As human phosphodiesterase (PDE) proteins are attractive drug targets, a large number of selective PDE inhibitors have been developed. However, since the catalytic sites of PDE isoforms are conserved in sequence and structure, it remains unclear how these inhibitors discriminate PDE isoforms in a selective manner. Here we perform long-time scale molecular dynamics (MD) simulations to investigate the spontaneous association processes of a highly selective PDE2A inhibitor (BAY60-7550) with the catalytic pockets of six PDE isoforms. We found that the free-energy landscapes of PDE:BAY60-7550 interactions on the PDE surfaces are very different between various PDE isoforms; and the free-energy landscape of PDE2A forms a favorable low-energy pathway that not only drives BAY60-7550 toward the target binding site, but also guides BAY60-7750 to adopt its native binding conformation known from crystal structure. Thus, this study reveals that the inhibitor interactions with the PDE surface residues play an important role in its high selectivity for PDE2A, and thereby provides new fundamental insights into the PDE isoform-specific inhibitor selectivity.

An important therapeutic method of glioblastoma, the most common primary brain tumor, is radiotherapy. However, several studies reported recently that radiation could also promote the invasion and migration of malignant tumor. Herein, we have identified that a significant increase of migration and invasiveness of human glioma U251 cells undergoing X-ray was observed compared to controls, accompanied by the increase of cathepsin L (CTSL), which is a lysosomal cysteine protease overexpressed and secreted by tumor cells. To verify if there was a relationship between CTSL and the X-ray-induced glioma invasion, a CTSL specific inhibitor Z-FY-CHO or a short hairpin RNA interference was used to pretreat U251 cells. As a result, the cell invasion and migration was impaired via down-regulation of CTSL. Additionally, a marked reduction of the cell-signaling molecules Rho kinase was also detected compared with controls. We also found that CTSL is involved in EMT progress: both in vitro and in clinical specimens. Overall, our findings show that CTSL is an important protein which mediates cell invasion and migration of human glioma U251 cells induced by X-ray, and the inhibition of CTSL expression might diminish the invasion of U251 cells by reducing the activity of RhoA and CDC42 as well as EMT positive markers.

Acute aortic dissection (AAD) is a life-threatening disease. Despite the higher risk of mortality, currently there are no effective therapies that can ameliorate AAD development or progression. Identification of meaningful clusters of co-expressed genes or representative biomarkers for AAD may help to identify new pathomechanisms and foster development of new therapies. To this end, we performed a weighted gene co-expression network analysis (WGCNA) and calculated module-trait correlations based on a public microarray dataset (GSE 52093) and discovered 9 modules were found to be related to AAD. The module which has the strongest positive correlation with AAD was further analyzed and the top 10 hub genes SLC20A1, GINS2, CNN1, FAM198B, MAD2L2, UBE2T, FKBP11, SLMAP, CCDC34, and GALK1 were identified. Furthermore, we validated the data by qRT-PCR in an independent sample set originated from our study center. Overall, the qRT-PCR results were consistent with the results of the microarray analysis. Intriguingly, the highest change was found for FKBP11, a protein belongs to the FKBP family of peptidyl-prolyl cis/trans isomerases, which catalyze the folding of proline-containing polypeptides. In congruent with the gene expression analysis, FKBP11 expression was induced in cultured endothelial cells by angiotensin II treatment and endothelium of the dissected aorta. More importantly we show that FKBP11 provokes inflammation in endothelial cells by interacting with NF-kB p65 subunit, resulting in pro-inflammatory cytokines production. Accordingly, siRNA mediated knockdown of FKBP11 in cultured endothelial cells suppressed angiotensin II induced monocyte transmigration through the endothelial monolayer. Based on these data, we hypothesize that pro-inflammatory cytokines elicited by FKBP11 overexpression in the endothelium under AAD condition could facilitate transendothelial migration of the circulating monocytes into the aorta, where they differentiate into active macrophages and secrete MMPs and other extracellular matrix (ECM) degrading proteins, contributing to sustained inflammation and AAD. Taken together, our data identify important role of FKBP11 which can serve as biomarker and/or therapeutic target for AAD.

Based on the hybrid pharmacophore design concept, a novel series of dual diaryl urea and N-acylhydrazone derivatives were synthesized and evaluated for their in vitro cytotoxicity by the standard MTT assay. The pharmacological results indicated that most compounds exhibited moderate to excellent activity. Moreover, compound 2g showed the most potent cytotoxicity against HL-60, A549 and MDA-MB-231 cell lines, with IC50 values of 0.22, 0.34 and 0.41 μM, respectively, which was 3.8 to 22.5 times more active than the reference compounds sorafenib and PAC-1. The promising compound 2g thus emerges as a lead for further structural modifications.

Protein kinases have been found to possess two characteristic conformations in their activation-loops: the active DFG-in conformation and the inactive DFG-out conformation. Recently, it has been very interesting to develop type-II inhibitors which target the DFG-out conformation and are more specific than the type-I inhibitors binding to the active DFG-in conformation. However, solving crystal structures of kinases with the DFG-out conformation remains a challenge, and this seriously hampers the application of the structure-based approaches in development of novel type-II inhibitors. To overcome this limitation, here we present a computational approach for predicting the DFG-out inactive conformation using the DFG-in active structures, and develop related conformational selection protocols for the uses of the predicted DFG-out models in the binding pose prediction and virtual screening of type-II ligands. With the DFG-out models, we predicted the binding poses for known type-II inhibitors, and the results were found in good agreement with the X-ray crystal structures. We also tested the abilities of the DFG-out models to recognize their specific type-II inhibitors by screening a database of small molecules. The AUC (area under curve) results indicated that the predicted DFG-out models were selective toward their specific type-II inhibitors. Therefore, the computational approach and protocols presented in this study are very promising for the structure-based design and screening of novel type-II kinase inhibitors.

HSV-1 infection in the cornea could lead to blindness. The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. It has been previously demonstrated that miR-H6 encoded from HSV-1 genome targets ICP4 to help maintain latency. In this study, synthesized miR-H6 mimics were transfected into HSV-1-infected human cornea epithelial (HCE) cells. The inhibition of HSV-1 replication and viral ICP4 expression in miR-H6-transfected HCE was confirmed by plaque assay, immunofluorescence, and Western blot. Compared to nontransfection or mock, miR-H6 produced a low-titer HSV-1 and weak ICP4 expression. In addition, miR-H6 can decrease the interleukin 6 released into the medium, which was determined by ELISA. Taken together, the data suggests that miR-H6 targeting of ICP4 inhibits HSV-1 productive infection and decreases interleukin 6 production in HCE, and this may provide an approach to prevent HSV-1 lytic infection and inhibit corneal inflammation.

Gliomas are the most common and aggressive primary brain tumors for which unfortunately no effective treatment modalities exist despite advances in molecular biology as the knowledge base to unravel the extremely complex molecular mechanisms of tumorigenesis is limited. In this study an attempt has been made to understand the molecular pathological basis of tumorigenesis which led to an identification of an oncogene, CDC2, and an epigenetic strategy has been evaluated to control the tumorigensis by downregulating this oncogene.

Tumor metastasis is the leading cause of death in patients with advanced gastric cancer (GC). Limited therapeutic regimens are available for this condition, which is associated with a poor prognosis, and the mechanisms underlying tumor metastasis remain unclear. In the present study, increased histone methyltransferase G9A expression in GC tissues correlated with advanced stage and shorter overall survival, and in vitro and in vivo experiments revealed that G9A promoted tumor invasion and metastasis. Moreover, we observed that Reg IV induced G9A via the p-ERK/p-SP1 pathway. SP1 directly binds the G9A promoter and enhances G9A expression, and upregulated G9A then forms a transcriptional activator complex with P300 and GR, thereby promoting ITGB3 expression induced by dexamethasone (DEX) and contributing to GC metastasis. However, the G9A-mediated increase in ITGB3 expression was not dependent on the SET domain and methyltransferase activity of G9A. This study demonstrates that G9A is an independent prognostic marker and promotes metastasis in GC, thus suggesting that it may be a tumor biomarker and potential therapeutic target in GC.

Introduction: The emerging field of "radiomics" has considerable potential in disease diagnosis, pathologic grading, prognosis evaluation, and prediction of treatment response. We aimed to develop a novel radiomics nomogram based on radiomics features and clinical characteristics that could preoperatively predict early recurrence (ER) of intrahepatic cholangiocarcinoma (ICC) after partial hepatectomy. Methods: A predictive model was developed from a training cohort comprising 139 ICC patients diagnosed between January 2010 and June 2014. Radiomics features were extracted from arterial-phase image of contrast-enhanced magnetic resonance imaging. Feature selection and construction of a "radiomics signature" were through Spearman's rank correlation and least absolute shrinkage and selection operator (LASSO) logistic regression. Combined with clinical characteristics, a radiomics nomogram was developed with multivariable logistic regression. Performance of the nomogram was evaluated with regard to discrimination, calibration, and clinical utility. An independent validation cohort involving 70 patients recruited from July 2014 to March 2016 was used to evaluate the utility of the nomogram developed. Results: The radiomics signature, consisting of nine features, differed significantly between ER patients and non-ER patients in training and validation cohorts. The area under the curve (AUC) of the radiomics signature in training and validation cohorts was 0.82 (confidence interval [CI], 0.74-0.88) and 0.77 (95% CI, 0.65-0.86), respectively. The AUC of the radiomics nomogram combining the radiomics signature and clinical stage in the two cohorts was 0.90 (95%CI, 0.83-0.94) and 0.86 (95% CI, 0.76-0.93), respectively. Decision curve analysis confirmed the clinical usefulness of the radiomics nomogram. Conclusion: The non-invasive radiomics nomogram developed using the radiomics signature and clinical stage could be used to predict ER of ICC after partial hepatectomy.

Transgenic GFP gene mice are widely used. Given the unique advantages of immunodeficient animals in the field of oncology research, we aim to establish a nude mouse inbred strain that stably expresses enhanced GFP (EGFP) for use in transplanted tumor microenvironment (TME) research. Female C57BL/6-Tg(CAG-EGFP) mice were backcrossed with male BALB/c nude mice for 11 generations. The genotype and phenotype of novel inbred strain Foxn1nu .B6-Tg(CAG-EGFP) were identified by biochemical loci detection, skin transplantation and flow cytometry. PCR and fluorescence spectrophotometry were performed to evaluate the relative expression of EGFP in different parts of the brain. Red fluorescence protein (RFP) gene was stably transfected into human glioma stem cells (GSC), SU3, which were then transplanted intracerebrally or ectopically into Foxn1nu .B6-Tg(CAG-EGFP) mice. Cell co-expression of EGFP and RFP in transplanted tissues was further analyzed with the Live Cell Imaging System (Cell'R, Olympus) and FISH. The inbred strain Foxn1nu .B6-Tg(CAG-EGFP) shows different levels of EGFP expression in brain tissue. The hematological and immune cells of the inbred strain mice were close to those of nude mice. EGFP was stably expressed in multiple sites of Foxn1nu .B6-Tg(CAG-EGFP) mice, including brain tissue. With the dual-fluorescence tracing transplanted tumor model, we found that SU3 induced host cell malignant transformation in TME, and tumor/host cell fusion. In conclusion, EGFP is differentially and widely expressed in brain tissue of Foxn1nu .B6-Tg(CAG-EGFP), which is an ideal model for TME investigation. With Foxn1nu .B6-Tg(CAG-EGFP) mice, our research demonstrated that host cell malignant transformation and tumor/host cell fusion play an important role in tumor progression.

Olfaction is key to many insects. Odorant receptors (ORs) stand among the key chemosensory receptors mediating the detection of pheromones and kairomones. Small hive beetles (SHBs), Aethina tumida, are parasites of social bee colonies and olfactory cues are especially important for host finding. However, how interactions with their hosts may have shaped the evolution of ORs in the SHB remains poorly understood. Here, for the first time, we analyzed the evolution of SHB ORs through phylogenetic and positive selection analyses. We then tested the expression of selected OR genes in antennae, heads, and abdomens in four groups of adult SHBs: colony odor-experienced/-naive males and females. The results show that SHBs experienced both OR gene losses and duplications, thereby providing a first understanding of the evolution of SHB ORs. Additionally, three candidate ORs potentially involved in host finding and/or chemical communication were identified. Significantly different downregulations of ORs between the abdomens of male and female SHBs exposed to colony odors may reflect that these expression patterns might also reflect other internal events, e.g., oviposition. Altogether, these results provide novel insights into the evolution of SHB ORs and provide a valuable resource for analyzing the function of key genes, e.g., for developing biological control. These results will also help in understanding the chemosensory system in SHBs and other beetles.

Dysregulated circular RNAs (circRNAs) have been shown to play important roles in various cancers, and could serve as diagnostic biomarkers. However, research focusing on the roles of the circRNAs in laryngeal squamous cell carcinoma (LSCC) is limited. This research aimed to explore the expressions of hsa_circ_0036722 in LSCCs and its diagnostic significance.

Excessive consumption of saturated fat leads to non-alcoholic fatty liver disease (NAFLD), which is attenuated by supplementation of n-3 polyunsaturated fatty acids (PUFAs). Endoplasmic reticulum (ER) stress is crucial in the development of NAFLD, but how high-saturated fat diet (HFD) causes ER stress and NAFLD remains unclear. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in hepatic ER stress. We aimed to explore the roles of LOX-1 in HFD-induced ER stress. Male Sprague-Dawley rats were fed an HFD without or with supplementation of fish oil for 16 weeks. The effects of n-3 PUFAs on hepatic ER stress degrees and the expression levels of LOX-1 were examined. Then human L02 hepatoma cells were treated with palmitate or palmitate and DHA to determine the ER stress and LOX-1 expression levels in vitro. After that the expression of LOX-1 in L02 cells was either knocked-down or overexpressed to analyze the roles of LOX-1 in palmitate-induced ER stress. The feeding of HFD induced NAFLD development and ER stress in the liver, and LOX-1 expressing level, which were all reversed by fish oil supplementation. In vitro, DHA treatment reduced the expression of LOX-1, and palmitate-induced ER stress. SiRNA-mediated knock-down of LOX-1 inhibited palmitate-induced ER stress, whereas overexpression of LOX-1 dramatically induced ER stress in L02 cells.LOX-1 is critical for HFD-induced ER stress, and inhibition of its expression under the treatment of n-3 PUFAs could ameliorate HFD-induced NAFLD.

Three-dimensional (3D) tissue models replicating liver architectures and functions are increasingly being needed for regenerative medicine. However, traditional studies are focused on establishing 2D environments for hepatocytes culture since it is challenging to recreate biodegradable 3D tissue-like architecture at a micro scale by using hydrogels. In this paper, we utilized a gelatin methacryloyl (GelMA) hydrogel as a matrix to construct 3D lobule-like microtissues for co-culture of hepatocytes and fibroblasts. GelMA hydrogel with high cytocompatibility and high structural fidelity was determined to fabricate hepatocytes encapsulated micromodules with central radial-type hole by photo-crosslinking through a digital micromirror device (DMD)-based microfluidic channel. The cellular micromodules were assembled through non-contact pick-up strategy relying on local fluid-based micromanipulation. Then the assembled micromodules were coated with fibroblast-laden GelMA, subsequently irradiated by ultraviolet for integration of the 3D lobule-like microtissues encapsulating multiple cell types. With long-term co-culture, the 3D lobule-like microtissues encapsulating hepatocytes and fibroblasts maintained over 90% cell viability. The liver function of albumin secretion was enhanced for the co-cultured 3D microtissues compared to the 3D microtissues encapsulating only hepatocytes. Experimental results demonstrated that 3D lobule-like microtissues fabricated by GelMA hydrogels capable of multicellular co-culture with high cell viability and liver function, which have huge potential for liver tissue engineering and regenerative medicine applications.

Accumulating investigations have demonstrated that microRNAs (miRNAs) are promising efficient targets for the next generation of molecular therapeutics. The development of miRNA-based therapies requires the identification and validation of cancer-associated miRNAs. Herein, we identified that miR-197-3p regulates the carcinogenesis and development of prostate cancer (PCa) via bioinformatics analysis. Next, we investigated the function and regulatory mechanisms of miR-197-3p in PCa. Overexpression of miR-197-3p suppressed PCa cell proliferation and colony formation. In contrast, inhibition of miR-197-3p activity enhanced PCa cell proliferation and colony formation. Mechanistic investigations identified that voltage dependent anion channel 1 (VDAC1) is a direct target of miR-197-3p. miR-197-3p targeting of VDAC1 resulted in downregulation of p-Akt and β-catenin. Subsequently, we found that restoration of VDAC1 abolished the effects of miR-197-3p on PCa cell proliferation and AKT signaling pathway. Furthermore, we confirmed that miR-197-3p suppressed tumor xenograft growth in vivo. In conclusion, our study offers an empirical investigation of miR-197-3p, a tumor suppressor that may be a potential therapeutic target in PCa.

Osteoarthritis (OA) is a major cause of joint pain and disability, and chondrocyte senescence is a key pathological process in OA and may be a target of new therapeutics. MicroRNA-140 (miR-140) plays a protective role in OA, but little is known about its epigenetic effect on chondrocyte senescence. In this study, we first validated the features of chondrocyte senescence characterized by increased cell cycle arrest in the G0/G1 phase and the expression of senescence-associated β-galactosidase (SA-βGal), p16INK4a, p21, p53, and γH2AX in human knee OA. Then, we revealed in interleukin 1β (IL-1β)-induced OA chondrocytes in vitro that pretransfection with miR-140 effectively inhibited the expression of SA-βGal, p16INK4a, p21, p53, and γH2AX. Furthermore, in vivo results from trauma-induced early-stage OA rats showed that intra-articularly injected miR-140 could rapidly reach the chondrocyte cytoplasm and induce molecular changes similar to the in vitro results, resulting in a noticeable alleviation of OA progression. Finally, bioinformatics analysis predicted the potential targets of miR-140 and a mechanistic network by which miR-140 regulates chondrocyte senescence. Collectively, miR-140 can effectively attenuate the progression of early-stage OA by retarding chondrocyte senescence, contributing new evidence of the involvement of miR-mediated epigenetic regulation of chondrocyte senescence in OA pathogenesis.

Geriatric hip fracture patients are particularly susceptible to blood loss and venous thromboembolism (VTE) during hemiarthroplasty, yet relatively few studies have examined the safety and efficacy of tranexamic acid (TXA) in these patients.