Expression Of Mesenchymal Cell Markers Research Articles

Plasma-derived exosomes contributes to endothelial-to-mesenchymal transition in Moyamoya disease

BackgroundMoyamoya disease (MMD) is a cerebrovascular disease with a high disability rate; however, its pathogenesis remains unknown. Endothelial-mesenchymal transition (EndMT) is the pathological basis of many vascular diseases; however, the key role of EndMT in MMD has not yet been reported. MethodWe collected vascular tissues from three control samples and six patients with MMD to detect the expression of EndMT-related genes. To elucidate the mechanism of EndMT in MMD, we performed in vitro cell experiments. Plasma-derived exosomes (PDEs) can transmit information between cells and tissues and are of considerable importance in several disease studies. PDEs were used to stimulate EndMT phenotype in cerebrovascular endothelial cells. ResultsMultiplex fluorescent immunohistochemistry staining confirmed that CD31, VE-cadherin and E-cadherin down-regulated, whereas α-SMA and vimentin were significantly up-regulated in moyamoya vascular endothelial cells than in control samples. PDEs from MMD patients significantly promoted cell proliferation and migration, resulting in slender cells. PDEs induce EndMT-related phenotype changes in cerebral vascular endothelial cells, including decreased endothelial cell marker expression and increased mesenchymal cell marker expression. We demonstrated that EndMT phenotypic alterations are mediated, in part, by microRNA(miRNAs). ConclusionThis study was the first to propose that EndMT may exist in the vessels of patients with MMD. PDEs induce the EndMT phenotype to promote the development of MMD. This study aimed to provide a new theoretical basis for elucidating the pathogenesis of MMD.

Treatment with Amniotic Fluid Stem Cell Extracellular Vesicles Promotes Fetal Lung Branching and Cell Differentiation at Canalicular and Saccular Stages in Experimental Pulmonary Hypoplasia Secondary to Congenital Diaphragmatic Hernia

Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired branching morphogenesis and differentiation. We have previously demonstrated that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues development of hypoplastic lungs at the pseudoglandular and alveolar stages in rodent models of CDH. Herein, we tested whether AFSC-EVs exert their regenerative effects at the canalicular and saccular stages, as these are translationally relevant for clinical intervention. To induce fetal pulmonary hypoplasia, we gavaged rat dams with nitrofen at embryonic day 9.5 and demonstrated that nitrofen-exposed lungs had impaired branching morphogenesis, dysregulated signaling pathways relevant to lung development (FGF10/FGFR2, ROBO/SLIT, Ephrin, Neuropilin 1, β-catenin) and impaired epithelial and mesenchymal cell marker expression at both stages. AFSC-EVs administered to nitrofen-exposed lung explants rescued airspace density and increased the expression levels of key factors responsible for branching morphogenesis. Moreover, AFSC-EVs rescued the expression of alveolar type 1 and 2 cell markers at both canalicular and saccular stages and restored markers of club, ciliated epithelial, and pulmonary neuroendocrine cells at the saccular stage. AFSC-EV-treated lungs also had restored markers of lipofibroblasts and PDGFRA+ cells to control levels at both stages. EV tracking showed uptake of AFSC-EV RNA cargo throughout the fetal lung and an mRNA-miRNA network analysis identified that several miRNAs responsible for regulating lung development processes were contained in the AFSC-EV cargo. These findings suggest that AFSC-EV-based therapies hold potential for restoring fetal lung growth and maturation in babies with pulmonary hypoplasia secondary to CDH.

Engineering naturally-derived human connective tissues for clinical applications using a serum-free production system

The increasing need for tissue substitutes in reconstructive surgery spurs the development of engineering methods suited for clinical applications. Cell culture and tissue production traditionally require the use of fetal bovine serum (FBS) which is associated with various complications especially from a translational perspective. Using the self-assembly approach of tissue engineering, we hypothesized that all important parameters of tissue reconstruction can be maintained in a production system devoid of FBS from cell extraction to tissue reconstruction. We studied two commercially available serum-free medium (SFM) and xenogen-free serum-free medium (XSFM) for their impact on tissue reconstruction using human adipose-derived stem/stromal cells (ASCs) in comparison to serum-containing medium. Both media allowed higher ASC proliferation rates in primary cultures over five passages compared with 10% FBS supplemented medium while maintaining high expression of mesenchymal cell markers. For both media, we evaluated extracellular matrix production and deposition necessary to engineer manipulatable tissues using the self-assembly approach. Tissues produced in SFM exhibited a significantly increased thickness (up to 6.8-fold) compared with XSFM and FBS-containing medium. A detailed characterization of tissues produced under SFM conditions showed a substantial 50% reduction of production time without compromising key tissue features such as thickness, mechanical resistance and pro-angiogenic secretory capacities (plasminogen activator inhibitor 1, hepatocyte growth factor, vascular endothelial growth factor, angiopoietin-1) when compared to tissues produced in the control FBS-containing medium. Furthermore, we compared ASCs to the frequently used human dermal fibroblasts (DFs) in the SFM culture system. ASC-derived tissues displayed a 2.4-fold increased thickness compared to their DFs counterparts. In summary, we developed all-natural human substitutes using a production system compatible with clinical requirements. Under culture conditions devoid of bovine serum, the resulting engineered tissues displayed similar and even superior structural and functional properties over the classic FBS-containing culture conditions with a considerable 50% shortening of production time.

Elabela Attenuates the TGF-β1-Induced Epithelial-Mesenchymal Transition of Peritoneal Mesothelial Cells in Patients Receiving Peritoneal Dialysis.

Peritoneal fibrosis (PF), a common complication in patients receiving peritoneal dialysis (PD), is primarily caused by the epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). PF is the main reason for patients on PD to withdraw from PD. Effective treatment is unavailable for this complication at present. Elabela (ELA) is a polypeptide hormone secreted by the vascular endothelium and kidney. Peptide hormones ELA and apelin (APLN) have various protective effects on the cardiovascular and urinary systems and have potential therapeutic effects on organ fibrosis. ELA and APLN are less studied in PD population. Here, we aimed to investigate the clinical significance of ELA in patients on PD and to evaluate the therapeutic effect of ELA on EMT of HPMCs. Compared with those in patients with stage 5 chronic kidney disease who are not on dialysis, serum ELA levels in patients on PD increased with the improvement of residual renal function at PD duration <36 months and decreased to pre-dialysis levels at PD duration ≥36 months, suggesting that dialysis duration is the main risk factor affecting serum ELA levels in patients on PD. In addition, serum APLN levels decreased in the early stage of PD and recovered to the pre-dialysis level with the prolongation of dialysis time. Notably, serum APLN levels were positively correlated with dialysis duration in patients undergoing PD. To establish the EMT model, we stimulated HPMCs using transforming growth factor-beta 1 (TGF-β1) in cell experiments performed in vitro. ELA-32 treatment reversed the TGF-β1-induced reduction in the expression of the epithelial cell marker and suppressed the expression of mesenchymal cell markers by inhibiting the phosphorylation of SMAD2/3, ERK1/2, and AKT. Therefore, our findings imply that ELA-32 can interfere with the EMT of HPMCs by inhibiting the activation of the TGF-β/SMAD2/3, ERK1/2, and AKT pathways, providing novel insights on the potential therapeutic use of ELA for treating PD-related PF.

Abstract 954: Combined treatment of γ-tocotrienol, and vitamin D3 synergistically inhibits breast cancer cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT)

Abstract Metastatic breast cancer is a leading cause of death of women in the United States. The metastatic process involves epithelial cells losing their cellular polarity, adhesion to the basement membrane, and acquiring migratory and invasive properties. Metastatic epithelial cells take on a mesenchymal phenotype, hence this process is termed epithelial-to-mesenchymal transition (EMT). EMT is characterized by a reduction in epithelial cell marker expression (E-cadherin, cytokeratin 8, and cytokeratin 18), and an increased expression of mesenchymal cell markers (N-cadherin, vimentin, and fibronectin). Previous studies have demonstrated that dietary intake of γ-tocotrienol and/or vitamin D3 may provide benefit in the preventing the growth and development of metastatic breast cancer. γ-Tocotrienol is a natural isoform of vitamin E that shows potent anticancer activity against a variety of cancer cell types at treatment doses that have little or no effect on normal cell growth or viability. Vitamin D3 has also been shown to display potent antiproliferative and antiangiogenic activity against human breast cancer cells, particularly triple negative breast cancers. Studies were conducted here to examine the effects of γ-tocotrienol and vitamin D3 given alone or in combination on MDA-MB-231 triple negative breast cancer (TNBC) cellular proliferation, migration, and epithelial to mesenchymal transition. Treatment with 10-16 µM γ-tocotrienol or 20-40 nM vitamin D3 induced a significant dose-responsive decrease in MDA-MB-231 growth after a 4-day culture period. Combined treatment with sub-effective doses of γ-tocotrienol (8 μM) and vitamin D3 (10 nM) induced a synergistically inhibition of MDA-MB-231 cell growth, as determined isobologram analysis. Studies using the matrigel invasion assay showed that similar combination treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly inhibited MDA-MB-231 motility. These effects appear to result from a reversal in EMT. Western blot analysis shows that combined treatment resulted in an increased expression of the epithelial cell markers E-cadherin, cytokeratins 8 and cytokeratins 18 and a corresponding reduction in mesenchymal cell markers N-cadherin and vimentin. Taken together, these findings indicate that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3 synergistically inhibit TNBC MDA-MB-231 breast cancer cell growth, viability, motility, and induced a reversal of EMT. These findings strongly suggest that combined γ-tocotrienol and vitamin D3 treatment may provide significant benefits in the treatment of metastatic TNBC in women. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Md. Rafi Anwar, Taylor A. Hurst, Paul W. Sylvester. Combined treatment of γ-tocotrienol, and vitamin D3 synergistically inhibits breast cancer cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 954.

Ras sumoylation in cell signaling and transformation.

Ras proteins are small GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. Mutations or deregulated activities of Ras are frequently the driving force for oncogenic transformation and tumorigenesis. Posttranslational modifications play a crucial role in mediating the stability, activity, or subcellular localization/trafficking of numerous cellular regulators including Ras proteins. A series of recent studies reveal that Ras proteins are also regulated by sumoylation. All three Ras protein isoforms (HRas, KRas, and NRas) are modified by SUMO3. The conserved lysine42 appears to be the primary site for mediating sumoylation. Expression of KRasV12/R42 mutants compromised the activation of the Raf/MEK/ERK signaling axis, leading to a reduced rate of cell migration and invasion in vitro in multiple cell lines. Moreover, treatment of transformed pancreatic cells with a SUMO E2 inhibitor blocks cell migration in a concentration-dependent manner, which is associated with a reduced level of both KRas sumoylation and expression of mesenchymal cell markers. Furthermore, mouse xenograft experiments reveal that expression of a SUMO-resistant mutant appears to suppress tumor development in vivo. Combined, these studies indicate that sumoylation functions as an important mechanism in mediating the roles of Ras in cell proliferation, differentiation, and malignant transformation and that the SUMO-modification system of Ras oncoproteins can be explored as a new druggable target for various human malignancies.

A Non-canonical Wnt Signature Correlates With Lower Survival in Gastric Cancer.

Genetic evidence suggests a role for the Wnt/β-catenin pathway in gastric cancer. However, Wnt5a, regarded as a prototypical non-canonical Wnt ligand, has also been extensively associated with this disease. Therefore, the roles of the Wnt signaling pathway in gastric cancer initiation and progression, and particularly the precise mechanisms by which the non-canonical Wnt pathway might promote the development and progression of gastric cancer, are not entirely well understood. This article analyzes publicly available gene and protein expression data and reveals the existence of a WNT5A/FZD2/FZD7/ROR2 signature, which correlates with tumor-infiltrating and mesenchymal cell marker expression. High expression of FZD7 and ROR2 correlates with a shared gene and protein expression profile, which in turn correlates with poor prognosis. In summary, the findings presented in this article provide an updated view of the relative contributions of the Wnt/β-catenin and non-canonical Wnt pathways in gastric cancer.

Histochemical localization of putative stem cells in irreversible pulpitis.

Our study aimed to observe the distribution of putative stem cells in irreversible pulpitis and to investigate the expression of specific molecules. Extracted third molar teeth were collected and divided into two groups: the normal pulp group and inflamed pulp group. Real-time PCR was applied to detect the expression of several embryonic and dentinogenic genes. The expression of mesenchymal cell markers (STRO-1, CD90, and CD146) and stromal cell-derived factor 1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) proteins was examined by immunohistochemical analysis. The expression levels of most embryonic and dentinogenic genes were not statistically different between the two groups. Immunohistochemical analysis revealed that in inflamed pulp, cells with positive expression for STRO-1, CD90, and CD146 mainly resided in two specific niches, both adjacent to inflammatory sites: one in the pulp core and another in the odontoblast layer. SDF-1α- and CXCR4-positive cells were significantly correlated with STRO-1-positive cells. Double immunofluorescence analysis indicated that STRO-1-positive cells overlapped with SDF-1α- and CXCR4-positive cells near the inflammatory site. This study gave a direct observation of putative stem cells distributed in irreversible pulpitis and implied a role of SDF-1α/CXCR4 signaling in stem cell-based therapies for reparative dentinogenesis.

Identification of Radil as a Ras binding partner and putative activator

Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anticancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediate its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our present studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial–mesenchymal transition and may provide new directions for Ras-related anticancer drug development.

Evaluation of epithelial and mesenchymal cell markers in canine urinary bladder transitional cell carcinoma

Canine transitional cell carcinoma (cTCC) is the most common malignant tumour in the urinary bladder: it is highly invasive and exhibits metastatic characteristics. Inflammation is also strongly related to cTCC. Epithelial tumours often exhibit a mesenchymal cell phenotype during tumour invasion and metastasis owing to epithelial-mesenchymal transition (EMT), which is often induced in chronic inflammation. The aim of this retrospective study was to investigate the expression of epithelial and mesenchymal cell markers in tumour cells and to evaluate its relationship with prognosis of cTCC. In this study, 29 dogs with cTCC who underwent surgical treatment were enrolled. Clinical parameters were reviewed using medical records. Tissue expression of epithelial and mesenchymal markers was evaluated by immunohistochemical analysis. The association between the expression of mesenchymal cell markers and clinical parameters, including prognosis, was statistically examined.In five normal bladder tissues used as controls, no expression of mesenchymal markers was observed, except for one tissue that expressed fibronectin. Conversely, epithelial tumour cells expressed vimentin and fibronectin in 23/29 and 19/28 cTCC tissues, respectively. Regarding clinical parameters, vimentin score in Miniature Dachshunds was significantly higher than those in other dog breeds (P < 0.001). Multivariate survival analyses revealed that age>12 years was related to shorter progression-free survival (P = 0.02). Higher vimentin score, lower fibronectin score, and advanced clinical T stage were significantly correlated with shorter median survival time (P < 0.05). The results of this study indicate that vimentin expression was associated with cTCC progression. Further studies are needed to examine the incidence and relevance of EMT in cTCC.

Role of TGF-β1-mediated epithelial-mesenchymal transition in the pathogenesis of tympanosclerosis.

The present study aimed to explore the role of TGF-β1-mediated epithelial-mesenchymal transition (EMT) in the pathogenesis of tympanosclerosis. Sprague Dawley rats were injected with inactivated Streptococcus pneumoniae suspension to establish a rat model of tympanosclerosis. The rats were sacrificed 8 weeks after the model was established. H&E and von Kossa staining was used to observe the morphological changes of middle ear mucosa. Western blotting was used to detect the expression of TGF-β1 and EMT-associated proteins in the mucosa samples. Middle ear mucosal epithelial cells of rats were collected to establish a primary culture. The cultured cells were stimulated with TGF-β1 and the expression of EMT-associated proteins was detected by western blotting and immunofluorescence. In addition, the cells were treated with TGF-β receptor type I/II inhibitor and the expression level of EMT-associated proteins was detected by western blotting. Sclerotic lesions appeared on 72.4% of tympanic membranes, and marked inflammation, inflammatory cell infiltration and fibrosis were found in the middle ear mucosa of rat models of tympanosclerosis. In middle ear mucosa of rats with tympanosclerosis, the expression of mesenchymal cell markers increased and that of epithelial cell markers decreased compared with the control group. TGF-β1 stimulated the activation of the EMT pathway in middle ear mucosal epithelial cells, resulting in an increased expression of fibronectin and N-cadherin. In addition, a decreased expression level of EMT-associated proteins was observed when TGF-β1 was inhibited. In conclusion, the present study indicated that TGF-β1-mediated EMT may play an important role in the pathogenesis of tympanosclerosis.

MiR-494 induces EndMT and promotes the development of HCC (Hepatocellular Carcinoma) by targeting SIRT3/TGF-\u03b2/SMAD signaling pathway

EndMT has an important effect on metastasis and progression of tumor. This work will elucidate the effect of miR-494 on EndMT and development of HCC. Therefore, the differential miRNA expression among non-tumorous, para-tumorous and tumorous tissues was analyzed. Moreover, luciferase activities of SIRT3 3′UTR treated with miR-494 were determined. Then human hepatoma cell lines were dealt with mimics or inhibitors of miR-494, migration and proliferation ability were assessed. The expression of SIRT3 and markers of mesenchymal cell were analyzed. The influences of miR-494 on development of HCC through inducing EndMT by targeting SIRT3 and TGF-β/SMAD signaling pathways in hepatoma cell lines were investigated. Xenograft mice were used to explore the potential roles of miR-494 on EndMT and development of HCC in vivo. Our results showed that, compared with non-tumorous tissues, 17 miRNAs were upregulated and 3 miRNAs were down-regulated in tumor tissues. In tumor tissues, the miR-494 expression level was much more than the expression of para-tumorous and non-tumorous tissues. MiR-494 suppressed SIRT3 expression, additionally enhanced expression of mesenchymal cell markers, while exerted effects on cell proliferation and migration of hepatoma cell lines. Moreover, the antagomir of miR-494 could protect against development process in xenogarft murine model. In conclusions, our work demonstrated that miR-494 targeted to SIRT3, and was a crucial mediator of EndMT and development of HCC through regulating SIRT3/TGF-β/SMAD signaling pathway. It suggested that aim at SIRT3/TGF-β/SMAD signaling pathway through suppressing the miR-494 expression level, was a feasible therapy strategy for HCC.

MiR-27a promotes EMT in ovarian cancer through active Wnt/𝜷-catenin signalling by targeting FOXO1.

Ovarian cancer (OC) is the fifth most common type of cancer in women worldwide. MiR-27a plays an important role in the development of ovarian cancer. However, the exact function and molecular mechanism of miR-27a in epithelial-mesenchymal transition (EMT) has not been thoroughly elucidated to date. Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-27a and FOXO1 mRNA in ovarian tissues and cells. The function of miR-27a in ovarian cancer was investigated through overexpression and knockdown of miR-27a in vitro. Wound healing and Transwell assays were performed to evaluate the migration and invasive capacity of the cells. A luciferase reporter assay was conducted to confirm the interaction between miR-27a and FOXO1. Western blotting was used to evaluate FOXO1, EMT and Wnt/β-catenin relative protein expression. In our study, we found that the mRNA expression level of miR-27a was significantly higher in ovarian cancer tissues and in HO8910 and OV90 cells. Functional experiments showed that miR-27a overexpression potentiated the migration and invasion of HO8910 and OV90 cells, while miR-27a inhibition reduced the cells' migration and invasion. Moreover, miR-27a upregulated the expression of mesenchymal cell markers and downregulated the expression of epithelial cell markers, which were restored via silencing of miR-27a expression. Subsequently, miR-27a was found to directly target and suppress the expression of FOXO1. Finally, we demonstrated that miR-27a promoted the progression of ovarian cancer cells and induced the process of EMT via the Wnt/β-catenin signalling pathway through inhibition of FOXO1. Taken together, these results indicate that targeting miR-27a and FOXO1 could represent a strategy for anticancer therapy in ovarian cancer.

Role of microRNA-150-5p/SRCIN1 axis in the progression of breast cancer.

In China, breast cancer is the most commonly occurring cancer in women. MicroRNAs (miRs) are a group of endogenous small non-coding RNAs, which serve a role in many biological processes through the regulation of target genes. In the current study, miR-150-5p expression was significantly up-regulated in breast cancer tissues and cell lines. To investigate the cellular function and underlying molecular mechanism of miR-150-5p in breast cancer, TargetScan7.2 was used to identify miR-150-5p target genes. SRC kinase signaling inhibitor 1 (SRCIN1) was identified as a direct target gene of miR-150-5p and the current study demonstrated that SRCIN1 was negatively regulated by miR-150-5p in breast cancer cells. Furthermore, SRCIN1 expression was significantly down-regulated in breast cancer tissues and cell lines. Taken together, these results demonstrated that there was a negative association between miR-150-5p and SRCIN1 in breast cancer. The CCK-8 and Transwell assays were used to examine breast cancer cell viability, invasion and migration ability. The current study demonstrated that over-expression of miR-150-5p enhanced breast cancer cell proliferation, invasion and migration. In addition, miR-150-5p over-expression increased the expression of mesenchymal cell markers (vimentin, N-cadherin and β-catenin) and decreased the expression of epithelial cell markers (E-cadherin and zonula occludens-1). By contrast, miR-150-5p knockdown inhibited breast cancer cell viability, invasion and migration. Additionally, miR-150-5p knockdown decreased the expression of mesenchymal cell markers and increased the expression of epithelial cell markers. Taken together, these results suggest that the miR-150-5p/SRCIN1 axis may be a potential target in the treatment of breast cancer.

MiR-449a induces EndMT, promotes the development of atherosclerosis by targeting the interaction between AdipoR2 and E-cadherin in Lipid Rafts

EndMT plays an important role in the relationship between endothelial dysfunction and atherosclerosis. This work will elucidate the biofunction induced by miR-449a and lipid rafts in EndMT and development of atherosclerosis. The differential miRNA expression between atherosclerotic plaques and normal arteries were analyzed. The luciferase activities of AdipoR2 3’ UTR treated with miR-449a were determined. ECs were dealt with miR-449a mimics or inhibitors, then cell proliferation and migration were assessed. Moreover, the expression of AdipoR2 and mesenchymal cell markers were analyzed. The influences of lipid rafts related to reciprocity between E-cadherin and AdipoR2 on TNF-α-induced damage in ECs were investigated. ApoE KO diabetic mice were used to explore the potential roles of miR-449a on atherosclerosis. Our results indicated that compared with normal arteries, 17 miRNAs were upregulated and 3 miRNAs were down-regulated in atherosclerotic plaques. The relative expression of miR-449a in plaques was significantly higher than that in normal arteries. MiR-449a suppressed AdipoR2 expression, additionally its interaction protein E-cadherin in ECs. MiR-449a enhanced expression of mesenchymal cell markers, induced cell proliferation and migration of ECs, regulated the interaction between E-cadherin and AdipoR2 interceded by lipid rafts. The miR-449a antagomir could protect against the development process of atherosclerosis in ApoE KO diabetic mice. In conclusion, miR-449a targeted to AdipoR2, and was a crucial mediator of EndMT and atherosclerosis in ECs through regulating E-cadherin bindability with AdipoR2 in lipid rafts. These results suggested that aim to lipid rafts and miR-449a in chronic EC inflammation response, was a feasible therapy strategy for atherosclerosis.

Inhibition of protein kinase C activity inhibits osteosarcoma metastasis.

IntroductionFor some cancers bone is the preferred site for metastasis and involves a cascade involving transition of epithelial cells to mesenchymal cells and subsequent intravasation to the blood and lymph vessels, and finally hematogenous dissemination to perivascular niches of the bone marrow sinusoids. It has been shown that protein kinase C can aid metastasis to bone. Hence, pharmacological inhibition of protein kinase C (PKC) activity is thought of as a potential therapeutic option in bone metastatic lesions. The objective of the current study was to investigate how PKCs exert their effect on bone cancer metastasis and to test the efficacy of pharmacological inhibition of PKC on bone metastasis.Material and methodsThe effect of the PKC inhibitor Go6983 on epithelial and mesenchymal cell marker expression in the osteosarcoma cell line DAN was determined by immunoblot and immunofluorescence analysis. The in vivo effect of Go6983 was evaluated with a xenograft model using DAN cells.ResultsTreatment with transforming growth factor β (TGF-β) led to loss of the epithelial cell marker and gain of mesenchymal cell markers in the osteosarcoma cell line, DAN. This transition occurred concomitantly with PKC activation. TGF-β-mediated PKC activation resulted in activation of ribosomal protein 6 (S6), but not S6K1. Pharmacological inhibition of PKC activation attenuated these effects. In a xenograft model of experimental metastasis, pharmacological inhibition of PKC activation over a period of 4 weeks reduced both tumor burden and metastasis to lungs.ConclusionsOur results indicate that PKC potentiates tumor metastasis to the bone by potentiating translation increase and can be putatively inhibited by pharmacological inhibition.

P53 Silencing and Mammosphere Formation in Breast Cancer Cells Harbouring P53 Gain-of-Function Mutations Trigger Epithelial-Mesenchymal Transition

Background: Mutations in p53 gene are observed in ∼50% of all human cancers. In breast cancer alone, 12%-32% of luminal, 84% of basal-like and 75% of HER-2 expressing tumors have apparent p53 mutations. Tumor cells undergo epithelial-mesenchymal transition (EMT) to metastasise from primary sites to form secondary tumors at distant regions of the body. EMT is a complex biologic phenomenon which governs the transition of cancer cells with epithelial characteristics to mesenchymal traits, gaining new properties such as aggressiveness and invasiveness. Recent studies revealed that mutations in the p53 gene can give rise to alternate functional phenotypes leading to tumor initiation and progression. Aim: The aim of this study is to develop a robust human breast cancer cellular model to investigate p53 gain-of-function (GOF) mutations and EMT as well as evaluating the EMT phenotype associated with these mutations. Methods: Two breast cancer cell lines, namely MDA-MB-468 and HCC38 carrying the R273H and R273L missense mutations, respectively, were subjected to p53 knockdown using shRNA directed against p53 gene through lentiviral vector transduction. The transduced cells were then harvested for Western blotting to evaluate the protein expression of EMT markers which includes E-cadherin, SNAIL, ZEB1 and vimentin compared with the nontransduced control cells. Subsequently, both cell lines were subjected to mammosphere generation and redifferentiation to determine the basal expression of the EMT markers. Results: Silencing of p53 using siRNA in MDA-MB-468 and HCC38 downregulated E-cadherin expressions but upregulated vimentin levels. Furthermore, E-cadherin levels reduced significantly after conversion from adherent parental cells to mammospheres, but rebound upon redifferentiation. Conversely, vimentin was upregulated in mammospheres as compared with the parental and redifferentiated groups. Conclusion: p53 knockdown in breast cancer cells harboring R273H and R273L mutations favor vimentin expression but not E-cadherin, suggesting that p53-GOF mutants are involved in EMT and the development of metastatic tumors. The mammosphere model accurately recapitulates cell plasticity between epithelial and mesenchymal states, as evidenced by the expression of mesenchymal cell markers in the mammospheres, and epithelial cell markers in adherent and redifferentiated cells.

Programmed cell death 4 overexpression enhances sensitivity to cisplatin via the JNK/c-Jun signaling pathway in bladder cancer.

The aim of the present study was to evaluate the effects of programmed cell death4 (PDCD4) on cell proliferation and apoptosis, and to elucidate the potential role of the Jun N-terminal kinase(JNK)/c-Jun pathway in human bladder cancer (BCa) cells. Mixed BCa cells were transfected with plasmids containing PDCD4 (PDCD4-pcDNA3). The sensitivity to cisplatin was analyzed using cell viability, invasion/migration, apoptosis, flow cytometry, wound healing and Transwell assays at different transfection times. Furthermore, epithelial-to-mesenchymal transition (EMT) markers were detected by immunofluorescence staining, and the protein expression of c-Jun, and phosphorylated Jun N-terminal kinase(p-JNK) and c-Jun (p-c-Jun, Ser-73) were also tested using western blotting. It was observed that BCa cell proliferation and invasion and tumor growth were significantly inhibited, whereas apoptosis was enhanced in PDCD4-transfected cells treated with cisplatin compared with controls. Moreover, the western blotting and immunofluorescence results demonstrated that PDCD4 upregulated the expression of epithelial cell markers, but downregulated the expression of mesenchymal cell markers. Furthermore, overexpression of PDCD4 reduced the protein levels of p-JNK and p-c-Jun. Taken together, the findings of the present study indicate that PDCD4 enhances the sensitivity of BCa cells to cisplatin, partially via regulation of the JNK/c-Jun pathway, and reverses EMT. In conclusion, the results of the present study suggested that PDCD4, a nuclear/cytoplasmic shuttling protein with multiple functions, plays an important role in the development and progression of human BCa.

Circular RNA DLGAP4 Ameliorates Ischemic Stroke Outcomes by Targeting miR-143 to Regulate Endothelial-Mesenchymal Transition Associated with Blood-Brain Barrier Integrity.

Circular RNAs (circRNAs) are highly expressed in the CNS and regulate physiological and pathophysiological processes. However, the potential role of circRNAs in stroke remains largely unknown. Here, we show that the circRNA DLGAP4 (circDLGAP4) functions as an endogenous microRNA-143 (miR-143) sponge to inhibit miR-143 activity, resulting in the inhibition of homologous to the E6-AP C-terminal domain E3 ubiquitin protein ligase 1 expression. circDLGAP4 levels were significantly decreased in the plasma of acute ischemic stroke patients (13 females and 13 males) and in a mouse stroke model. Upregulation of circDLGAP4 expression significantly attenuated neurological deficits and decreased infarct areas and blood-brain barrier damage in the transient middle cerebral artery occlusion mouse stroke model. Endothelial-mesenchymal transition contributes to blood-brain barrier disruption and circDLGAP4 overexpression significantly inhibited endothelial-mesenchymal transition by regulating tight junction protein and mesenchymal cell marker expression. Together, the results of our study are illustrative of the involvement of circDLGAP4 and its coupling mechanism in cerebral ischemia, providing translational evidence that circDLGAP4 serves as a novel therapeutic target for acute cerebrovascular protection.SIGNIFICANCE STATEMENT Circular RNAs (circRNAs) are involved in the regulation of physiological and pathophysiological processes. However, whether circRNAs are involved in ischemic injury, particularly cerebrovascular disorders, remains largely unknown. Here, we demonstrate a critical role for circular RNA DLGAP4 (circDLGAP4), a novel circular RNA originally identified as a sponge for microRNA-143 (miR-143), in ischemic stroke outcomes. Overexpression of circDLGAP4 significantly attenuated neurological deficits and decreased infarct areas and blood-brain barrier damage in the transient middle cerebral artery occlusion mouse stroke model. To our knowledge, this is the first report describing the efficacy of circRNA injection in an ischemic stroke model. Our investigation suggests that circDLGAP4 may serve as a novel therapeutic target for acute ischemic injury.

Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis

BackgroundLiver fibrosis is an excessive wound-healing reaction that requires the participation of inflammatory cells and hepatic stellate cells (HSCs). The pathogenesis of liver fibrosis caused by viruses and alcohol has been well characterized, but the molecular mechanisms underlying liver fibrosis induced by the liver fluke Clonorchis sinensis are poorly understood. Lysophospholipase A (LysoPLA), which deacylates lysophospholipids, plays a critical role in mediating the virulence and pathogenesis of parasites and fungi; however, the roles of C. sinensis lysophospholipase A (CsLysoPLA) in C. sinensis-induced liver fibrosis remain unknown.MethodsA mouse macrophage cell line (RAW264.7) was cultured and treated with CsLysoPLA. IL-25 and members of its associated signaling pathway were detected by performing quantitative real-time PCR, Western blotting and immunofluorescent staining. A human hepatic stellate cell line (LX-2) was cultured and exposed to IL-25. LX-2 cell activation markers were examined via quantitative real-time PCR, Western blotting and immunofluorescent staining. Migration was analyzed in transwell plates.ResultsTreating RAW264.7 cells with CsLysoPLA significantly induced IL-25 expression. Elevated PKA, B-Raf, and ERK1/2 mRNA levels and phosphorylated B-Raf and ERK1/2 were detected in CsLysoPLA-stimulated RAW264.7 cells. The PKA inhibitor H-89 weakened B-Raf and ERK1/2 phosphorylation whereas the AKT activator SC79 attenuated ERK1/2 phosphorylation in RAW264.7 cells. Both H-89 and SC79 inhibited CsLysoPLA-induced IL-25 upregulation. In addition, stimulation of LX-2 cells with IL-25 upregulated the expression of mesenchymal cell markers, including α-smooth muscle actin (α-SMA) and collagen type I (Collagen-I), and promoted cell migration.ConclusionsCsLysoPLA activates HSCs by upregulating IL-25 in macrophages through the PKA-dependent B-Raf/ERK1/2 pathway and potentially promotes hepatic fibrosis during C. sinensis infection.