tenascin-C mRNA Research Articles

Tenascin-C induces transdifferentiation of retinal pigment epithelial cells in proliferative vitreoretinopathy

Proliferation and transdifferentiation of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy (PVR); however, the critical regulators of this process remain to be elucidated. Here, we investigated the role of tenascin-C in PVR development. In vitro, exposure of human ARPE-19 (hRPE) cells to TGF-β2 increased tenascin-C expression. Tenascin-C was shown to be involved in TGF-β2-induced transdifferentiation of hRPE cells, which was inhibited by pretreatment with tenascin-C siRNA. In PVR mouse models, a marked increase in the expression of tenascin-C mRNA and protein was observed. Additionally, immunofluorescence analysis demonstrated a dramatic increase in the colocalization of tenascin-C with RPE65 or α-smooth muscle actin(α-SMA) in the epiretinal membranes of patients with PVR. There was also abundant expression of integrin αV and β-catenin in the PVR membranes. ICG-001, a β-catenin inhibitor, efficiently attenuated PVR progression in a PVR animal model. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of PVR via the integrin αV and β-catenin pathways. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of epiretinal membrane development associated with PVR.

In vitro anticancer activity of melanin-like nanoparticles for multimodal therapy of glioblastoma

Abstract Glioblastoma (GBM) is one of the most aggressive and hard to treat cancers. Traditional anti-cancer treatment methods have low efficiency and the lifespan after diagnosis is only 12–18 months. Brain tumor cells overexpress many proteins that play an important role in tumor progression and can be used as therapeutic targets. One of the promising approaches in cancer treatment is down-regulation of an extracellular matrix glycoprotein – Tenascin-C (TN-C) through RNA interference therapy. However, the effective delivery of double stranded RNA with one strand complementary to TN-C mRNA sequence is difficult due to rapid degradation by nucleases and low intracellular uptake. Polydopamine (PDA), a biomimetic polymer characterized by high biocompatibility and simple modification ability, is commonly used in nanobiomedicine to create a drug/gene delivery vehicle. Furthermore, photothermal characteristics of this polymer enable its application in photothermal therapy (PTT), which is a great option for cancer treatment. Here we synthesize PDA nanoparticles (NPs) coated with polyamidoamine dendrimers generation 3.0 (DD3.0) for therapeutic anti-TN-C RNA and doxorubicin delivery. As prepared PDA@DD3.0 NPs are then used in combined drug delivery, gene silencing, and PTT of GBM. The obtained materials are analyzed in terms of physicochemical and photothermal properties as well as their cytotoxicity, using human GBM cells. The results demonstrate that the obtained nanocarriers are effective non-viral vehicle for combined therapeutic approach for killing glioma cells via anti-TN-C RNA delivery and combined chemo-PTT therapy (CT-PTT). The application of PDA@DD3.0 NPs contributed to the 3-fold reduction in the proliferation rate of GBM cells, a decrease in the level of TN-C expression (by 30%) and a reduction in the number of viable cells by up to 20%.

Abstract 1585: Tenascin C in pancreatic cancer-associated fibroblasts enhances epithelial mesenchymal transition and is associated with resistance to immune checkpoint inhibitor

Abstract Backgrounds: Tenascin C (TNC) is an extracellular matrix glycoprotein that is abundantly expressed in cancer stroma and its overexpression correlates with tumor progression in various types of cancer including pancreatic adenocarcinoma (PAAD). However, the role of TNC in regards to response to immune checkpoint inhibitor (ICI) still remains to be identified. In this study, we aimed to investigate the potential involvement of TNC in the response to ICI among PAAD patients. Materials and Methods: Transcriptomic profiles were downloaded by TCGA and GTEx database and TNC mRNA levels were compared between tumors and normal tissues. These transcriptomic data were deconvoluted using CIBERSORTx to identify specific enriched pathways in cancer cells and cancer-associated fibroblasts (CAFs) in PAAD patients. Bioinformatic programs were used to predict paracrine communications between cancer cells and cancer-associated fibroblasts (CAFs), and the Tumor Immune Dysfunction and Exclusion (TIDE) score was calculated to predict response to ICI treatment in PAAD patients. An independent immunotherapeutic cohort was applied to validate prediction values for immunotherapy. Results: TNC mRNA levels were significantly upregulated in tumors compared to normal tissues in eight cancer types including PAAD. TNC-high (TNC-H) PAAD patients (n = 138) had significant shorter overall survival than TNC-low (TNC-L) patients (n = 40) (p = 0.0125, Log-rank test). Using CIBERSORTx, TNC was estimated to be predominantly expressed in CAFs in PAAD. In pathway analysis, both cancer cells and CAFs had significant enrichments of the epithelial mesenchymal transition pathway in TNC-H patients. Ligand-receptor interaction analysis showed that TNC produced in CAFs had the highest interaction potentials to bind to integrin families such as ITGαV and ITGβ3 in cancer cells. In an independent immunotherapy data cohort, patients with TNC-H and ITGαV-high or ITGβ3-high expression were associated with poor response to ICI treatment and had shorter overall survival than those with both low expressions. Conclusions: In conclusion, these findings suggest that TNC-high CAFs can play a crucial role in tumor progression and resistance to ICI therapy in PAAD patients, and targeting TNC and its interactions with cancer cells may provide a potential strategy for improving the efficacy of ICI therapy in PAAD. Citation Format: Satoru Furuhashi, Yoshifumi Morita, Akio Matsumoto, Shinya Ida, Ryuta Muraki, Ryo Kitajima, Makoto Takeda, Hirotoshi Kikuchi, Yoshihiro Hiramatsu, Hiroya Takeuchi. Tenascin C in pancreatic cancer-associated fibroblasts enhances epithelial mesenchymal transition and is associated with resistance to immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1585.

Tenascin-C affects invasiveness of EGFR-mutated lung adenocarcinoma through a putative paracrine loop

Tenascin C (TNC) is an extracellular matrix (ECM) protein and a potential biomarker affecting progression of different tumor types, such as pancreatic and lung cancer. Alternative splicing variants of TNC are known to have an impact on interaction partners like other ECM proteins or cell surface receptors, including epidermal growth factor receptor (EGFR), leading to numerous and sometimes opposite roles of TNC in tumor cell dissemination and proliferation. Only little is known about the impact of TNC on biologic characteristics of lung cancer, such as invasion and metastatic potential. In the present study, we could link an increased expression of TNC in lung adenocarcinoma (LUAD) tissues with an unfavorable clinical outcome of patients. Furthermore, we investigated the functional role of TNC in LUAD. Immunohistochemical staining of TNC revealed a significant increase of TNC levels in primary tumours and metastases compared to normal lung tissue. Additionally, a significant correlation between TNC mRNA expression and EGFR copy number and protein expression levels has been determined. Moreover, inhibition of TNC in lung fibroblasts led to reduced invasiveness of LUAD cells harboring EGFR-activating mutations and to a shorter lamellipodia perimeter and a reduced lamellipodia area on the surface of LUAD cells. This study provides the evidence that TNC expression might be a biological relevant factor in LUAD progression in an EGFR-dependent manner and that it regulates tumor cell invasion by rearrangement of the actin cytoskeleton, especially affecting lamellipodia formation.

TNC Accelerates Hypoxia-Induced Cardiac Injury in a METTL3-Dependent Manner.

Cardiac fibrosis and cardiomyocyte apoptosis are reparative processes after myocardial infarction (MI), which results in cardiac remodeling and heart failure at last. Tenascin-C (TNC) consists of four distinct domains, which is a large multimodular glycoprotein of the extracellular matrix. It is also a key regulator of proliferation and apoptosis in cardiomyocytes. As a significant m6A regulator, METTL3 binds m6A sites in mRNA to control its degradation, maturation, stabilization, and translation. Whether METTL3 regulates the occurrence and development of myocardial infarction through the m6A modification of TNC mRNA deserves our study. Here, we have demonstrated that overexpression of METTL3 aggravated cardiac dysfunction and cardiac fibrosis after 4 weeks after MI. Moreover, we also demonstrated that TNC resulted in cardiac fibrosis and cardiomyocyte apoptosis after MI. Mechanistically, METTL3 led to enhanced m6A levels of TNC mRNA and promoted TNC mRNA stability. Then, we mutated one m6A site "A" to "T", and the binding ability of METTL3 was reduced. In conclusion, METTL3 is involved in cardiac fibrosis and cardiomyocyte apoptosis by increasing m6A levels of TNC mRNA and may be a promising target for the therapy of cardiac fibrosis after MI.

Mesangial cell-derived tenascin-C contributes to mesangial cell proliferation and matrix protein production in IgA nephropathy.

Tenascin-C (TNC), a non-structural extracellular matrix glycoprotein, is transiently expressed during development or after injury, playing an important role in injury and repair process. The potential role of TNC in the pathogenesis of IgA nephropathy (IgAN) remains to be clarified. Immunohistochemistry staining for TNC was conducted on paraffin-embedded slices from renal biopsies of 107 IgAN patients, and correlation analysis was made between mesangial TNC expression and clinic-pathological parameters. In situ hybridization for TNC mRNA was further performed to figure out the cells that express TNC within glomeruli. In vitro experiments were also carried out on mouse mesangial cells (SV40 MES13) to elucidate the effect of TNC on mesangial cells. TNC was expressed in the mesangial area of IgAN, as well as in fibrotic regions. Correlation analysis showed that higher mesangial TNC was associated with higher level of proteinuria, lower estimated glomerular filtration rate and more serious pathological lesions (MEST score). In situ hybridization revealed that abundant TNC mRNA expression was observed in the affected glomeruli of IgAN, but not in minimal change disease. Moreover, TNC mRNA co-localized with PDGFRβ mRNA, but not with PODXL mRNA, suggesting that TNC mRNA was expressed in the mesangial cells within glomeruli in IgAN. In vitro experiments showed that exogenous TNC promoted matrix protein production and mesangial cell proliferation, which was attenuated by an epidermal growth factor receptor inhibitor. Taken together, these results suggest that mesangial cell-derived TNC contributes to mesangial matrix expansion and mesangial cell proliferation, which is a potential therapeutic target in IgAN.

Mechanical Compression of Human Airway Epithelial Cells Induces Release of Extracellular Vesicles Containing Tenascin C.

Aberrant remodeling of the asthmatic airway is not well understood but is thought to be attributable in part to mechanical compression of airway epithelial cells. Here, we examine compression-induced expression and secretion of the extracellular matrix protein tenascin C (TNC) from well-differentiated primary human bronchial epithelial (HBE) cells grown in an air–liquid interface culture. We measured TNC mRNA expression using RT-qPCR and secreted TNC protein using Western blotting and ELISA. To determine intracellular signaling pathways, we used specific inhibitors for either ERK or TGF-β receptor, and to assess the release of extracellular vesicles (EVs) we used a commercially available kit and Western blotting. At baseline, secreted TNC protein was significantly higher in asthmatic compared to non-asthmatic cells. In response to mechanical compression, both TNC mRNA expression and secreted TNC protein was significantly increased in both non-asthmatic and asthmatic cells. TNC production depended on both the ERK and TGF-β receptor pathways. Moreover, mechanically compressed HBE cells released EVs that contain TNC. These data reveal a novel mechanism by which mechanical compression, as is caused by bronchospasm, is sufficient to induce the production of ECM protein in the airway and potentially contribute to airway remodeling.

Abstract P776: Post-Stroke Tenascin-C Induction Mediates the Ischemic Pathogenesis

The mechanistic role of Tenascin-C (TNC) in the pathogenesis of acute ischemic stroke is not known despite its prognostic association with cerebrovascular diseases. We currently observed that transient middle cerebral artery occlusion (MCAO) upregulated cerebral TNC mRNA and protein expression between 3h and 24h reperfusion in adult mice of both sexes. We then evaluated the effect of TNC knockdown on ischemic outcome in adult mice of both sexes by treating with either TNC siRNA or control siRNA (intravenous) at 5 min of reperfusion following transient MCAO. TNC siRNA treatment significantly reduced the post-ischemic TNC protein induction tested at 72h reperfusion compared with the sex-matched control siRNA treated cohorts (n=6/group/sex). TNC siRNA cohorts showed significantly improved post-stroke motor function identified by beam walk test and rotarod test between days 1 and 14 of reperfusion compared with the sex-matched control siRNA cohorts (n=7/group/sex). TNC siRNA cohorts of both sexes also showed decreased post-ischemic BBB disruption (evaluated with T1-weighted MRI with gadobenate dimeglumine as contrast agent) and reduced infarction (assessed with T2-weighted MRI) at 3 days of reperfusion compared with the sex-matched control siRNA treated cohorts (n =4/group for BBB and n =12/group/sex for infarct). At day 21 of reperfusion, the survival rate was observed to be higher in the TNC siRNA treated mice compared with the control siRNA treated mice (n =7/group/sex). These studies thus show that induction of TNC during the acute period after stroke might be a mediator of ischemic brain damage and its knockdown is neuroprotective. Importantly this effect is independent of sex. The study was funded by the Department of Neurological Surgery, Univ. of Wisconsin-Madison.

Down-regulation of tenascin-C inhibits breast cancer cells development by cell growth, migration, and adhesion impairment.

Tenascin-C (TNC) is an extracellular matrix (ECM) glycoprotein that plays an important role in cell proliferation, migration, and tumour invasion in various cancers. TNC is one of the main protein overexpressed in breast cancer, indicating a role for this ECM molecule in cancer pathology. In this study we have evaluated the TNC loss-off-function in breast cancer cells. In our approach, we used dsRNA sharing sequence homology with TNC mRNA, called ATN-RNA. We present the data showing the effects of ATN-RNA in MDA-MB-231 cells both in monolayer and three-dimensional culture. Cells treated with ATN-RNA were analyzed for phenotypic alterations in proliferation, migration, adhesion, cell cycle, multi-caspase activation and the involvement in epithelial to mesenchymal transition (EMT) processes. As complementary analysis the oncogenomic portals were used to assess the clinical implication of TNC expression on breast cancer patient’s survival, showing the TNC overexpression associated with a poor survival outcome. Our approach applied first in brain tumors and then in breast cancer cell lines reveals that ATN-RNA significantly diminishes the cell proliferation, migration and additionally, reverses the mesenchymal cells phenotype to the epithelial one. Thus, TNC could be considered as the universal target in different types of tumors, where TNC overexpression is associated with poor prognosis.

Periostin and tenascin-C interaction promotes angiogenesis in ischemic proliferative retinopathy

Ischemic proliferative retinopathy (IPR), such as proliferative diabetic retinopathy (PDR), retinal vein occlusion and retinopathy of prematurity is a major cause of vision loss. Our previous studies demonstrated that periostin (PN) and tenascin-C (TNC) are involved in the pathogenesis of IPR. However, the interactive role of PN and TNC in angiogenesis associated with IPR remain unknown. We found significant correlation between concentrations of PN and TNC in PDR vitreous humor. mRNA and protein expression of PN and TNC were found in pre-retinal fibrovascular membranes excised from PDR patients. Interleukin-13 (IL-13) promoted mRNA and protein expression of PN and TNC, and co-immunoprecipitation assay revealed binding between PN and TNC in human microvascular endothelial cells (HRECs). IL-13 promoted angiogenic functions of HRECs. Single inhibition of PN or TNC and their dual inhibition by siRNA suppressed the up-regulated angiogenic functions. Pathological pre-retinal neovessels of oxygen-induced retinopathy (OIR) mice were attenuated in PN knock-out, TNC knock-out and dual knock-out mice compared to wild-type mice. Both in vitro and in vivo, PN inhibition had a stronger inhibitory effect on angiogenesis compared to TNC inhibition, and had a similar effect to dual inhibition of PN and TNC. Furthermore, PN knock-out mice showed scant TNC expression in pre-retinal neovessels of OIR retinas. Our findings suggest that interaction of PN and TNC facilitates pre-retinal angiogenesis, and PN is an effective therapeutic target for IPR such as PDR.

Transforming Growth Factor-Beta and Sonic Hedgehog Signaling in Palatal Epithelium Regulate Tenascin-C Expression in Palatal Mesenchyme During Soft Palate Development.

During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-β3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-β3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-β3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5–14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2fl/fl mice that lack the TGF-β type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2fl/fl mice that lack the TGF-β type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-β3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-β inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh–/+;MFCS4+/– mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-β and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-β and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior–posterior axis of the palatal shelves.

Castration-induced stromal remodeling disrupts the reconstituted prostate epithelial structure

AbstractThe normal prostate epithelial structure is maintained by homeostatic interactions with smooth muscle cells. However, structural alterations of the stroma are commonly observed in prostatic proliferative diseases, leading to the abnormalities of prostate epithelial structure. A decrease in the androgen level experimentally induces stromal remodeling, i.e., replacement of smooth muscle cells with fibroblasts or myofibroblasts. In this study, we investigated the effects of castration-induced stromal remodeling and subsequent aberrant activation of epithelial–stromal interactions on the reconstituted human prostate-like epithelial structure. We performed in vivo experiments using the human prostate epithelial cell line BPH-1 and fetal rat urogenital sinus mesenchyme to generate heterotypic tissue recombinants that form human prostate-like epithelial structure (i.e., solid- and canalized-epithelial cords). Host mice were castrated at 12 weeks post transplantation (castration) and implanted with a dihydrotestosterone pellet at 14 days post castration (androgen replacement treatment; ART). In the castration group, the percentages of fibrotic area and disrupted prostate epithelial structure without the basement membrane (BM) increased proportionally in a time-dependent manner, but were suppressed by ART. In the castration group, tenascin-C (TNC)-positive fibroblasts were abundant in the stroma surrounding disrupted prostate epithelial structure without the BM. TGF-β1 secretion from BPH-1 cells was increased by co-culturing with human primary cultured prostate fibroblasts. TNC mRNA expression was increased in fibroblasts co-culturing with BPH-1 cells and was suppressed by treatment with a TGF-β RI kinase inhibitor. Moreover, in the castration group, the percentage of p-Smad2-positive cells was significantly higher in the stroma surrounding disrupted prostate epithelial structure without the BM. Our results demonstrate that castration-induced stromal remodeling disrupted the reconstituted human prostate-like epithelial structure and induced the appearance of TNC-positive fibroblasts accompanied by activation of TGF-β signaling. The alteration of prostate stromal structure may be responsible for loss of the BM and epithelial cell polarity.

Tenascin-C Is Increased in Inflammatory Bowel Disease and Is Associated with response to Infliximab Therapy.

Tenascin-C (TNC) is an extracellular matrix glycoprotein expressed in response to inflammation and tissue damage. The role of TNC in patients with inflammatory bowel disease (IBD) is not well understood. In this study, we analyzed the expression of TNC in the inflamed mucosa of patients with ulcerative colitis (UC) and Crohn's disease (CD). Serum TNC levels were determined by the enzyme-linked immunosorbent assay (ELISA), and the levels of TNC in patients with different disease activities were compared. The expression of TNC was derived from a GEO dataset. THP-1 cells were stimulated with TNC to evaluate the proinflammatory role of TNC. We found higher TNC expression in the inflamed mucosa of patients with UC and CD compared with normal controls (NCs). TNC was mainly expressed in the stromal area of the intestinal mucosa. The median serum levels of TNC were significantly higher in UC (median 74.1 ng/ml, range 42.6–102.1 ng/ml) and CD (median 59.2 ng/ml, range 44.0–80.9 ng/ml). We also found that serum TNC levels were correlated with Mayo scores in UC and Crohn's disease activity index (CDAI) in CD. Through GSE14580, we demonstrated that patients who were nonresponsive to infliximab treatment had higher mucosal TNC mRNA expression. High TNC mRNA expression in the inflamed intestinal mucosa was associated with poor response to infliximab therapy in patients with UC. Furthermore, THP-1 cells stimulated with TNC showed increased expression of IL-6, but not TNF-α, IL-8, MCP-1, or IL-1β. Thus, increased TNC levels may participate in the pathogenesis of IBD and may serve as a biomarker for disease activity and response to treatment with infliximab.

The Predictive Role of Tenascin-C and Cellular Communication Network Factor 3 (CCN3) in Post Hepatectomy Liver Failure in a Rat Model and 50 Patients Following Partial Hepatectomy.

BackgroundMatricellular proteins of the extracellular matrix (ECM) include tenascin-C (TNC) and cellular communication network factor 3 (CCN3). This study aimed to investigate the role of TNC and CCN3 as prognostic factors for post hepatectomy liver failure (PHLF) in a rat model of partial hepatectomy and 50 patients following partial hepatectomy.Material/MethodsSprague-Dawley rats underwent 85% (n=53) or 90% hepatectomy (n=53) in the partial hepatectomy (PHx) model. TNC and CCN3 mRNA expression in residual liver tissue was evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and enzyme-linked immunoassay (ELISA) determined the serum levels of TNC and CCN3. In 50 patients who underwent partial hepatectomy, TNC and CCN3 serum levels were measured on postoperative day 1 and day 3.ResultsIn the rat partial hepatectomy model, mRNA and serum levels of TNC and CCN3 were significantly increased within the first 24 h, and were higher in the 90% PHx group compared with the 85% PHx group. Fifty patients who underwent partial hepatectomy, included patients with PHLF (n=12) and patients without PHLF (n=38). Multivariate analysis confirmed that serum levels on postoperative day 3 TNChigh+CCN3high was a significant predictor of PHLF, which was associated with more than twice the risk of severe morbidity when compared with the low-risk patients (80% vs. 30%) and a significantly longer hospital stay (17 days vs. 8 days).ConclusionsFurther studies are needed to evaluate the potential role of the matricellular proteins, TNC and CCN3 as early clinical predictors for PHLF.

Nano-mediated delivery of double-stranded RNA for gene therapy of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fueled a search for new therapeutic targets and treatment modalities. Here we report new approach towards RNA interference therapy of glioblastoma multiforme based on the magnetic nanoparticles delivery of the double-stranded RNA (dsRNA) with homological sequences to mRNA of tenascin-C (TN-C), named ATN-RNA. The obtained nanocomposite consisted of polyethyleneimine (PEI) coated magnetic nanoparticles conjugated to the dsRNA show high efficiency in ATN-RNA delivery, resulting not only in significant TN-C expression level suppressesion, but also impairing the tumor cells migration. Moreover, synthesized nanomaterials show high contrast properties in magnetic resonance imaging (MRI) and low cytotoxicity combining with lack of induction of interferon response. We believe that the present work is a successful combination of effective, functional, non-immunostimulatory dsRNA delivery system based on magnetic nanoparticles with high potential for further application in GBM therapy.

Tenascin-C expression is significantly associated with the progression and prognosis in gastric GISTs

Tenascin-C (TNC), an extracellular matrix glycoprotein, has been implicated in progression of various types of cancer. However, few reports exist on TNC expression in gastrointestinal stromal tumors (GISTs). We here attempted to investigate the expression pattern and prognostic significance of TNC in gastric GISTs. We studied TNC expression in 122 gastric GISTs tissue samples by immunohistochemistry, and examined the correlations of TNC expression with clinicopathological parameters and survival of gastric GISTs. The TNC-high expression was observed in 30 (24.6%) of 122 of gastric GISTs. The high levels of TNC expression in gastric GISTs was significantly associated with tumor size (P < .001), multivisceral resection (P = .006), metastasis at initial diagnosis (P = .006), mitotic count (P = .002) and NIH risk classification (P = .015). The TNC mRNA and protein levels were found to significantly downregulated in tumors without progression compared to those tumors which occurred tumor progression during the follow-up period (P < .05). As for the prognostic analysis, it revealed that tumor size, mitotic count, surgical margins, multivisceral resection, and TNC expression were independent predictors of PFS for gastric GISTs (P < .05). The overexpression of TNC may be as a possible marker for the metastatic potential of gastric GISTs patients.

How to detect and purify tenascin-C.

The extracellular matrix molecule tenascin-C (TNC) was discovered over 30 years ago, and its tightly regulated pattern of expression since sparked keen interest in the scientific community. In adult tissues, TNC expression is restricted to specific niches and areas of active remodeling or high mechanical strain. However, while most healthy tissues contain little TNC, its transient expression upon cellular stress or tissue injury helps to mediate repair and restore homeostasis. Persistent expression of TNC is associated with chronic inflammation, fibrosis, and cancer, where methods for its detection are emerging as a reliable means to predict disease onset, prognosis, and response to treatment. Because studying the expression of this large matrix molecule is not always straightforward, here we describe basic techniques to examine tissue levels of TNC mRNA and protein. We also describe methods for purifying recombinant TNC, knocking down its expression, and creating cell-derived matrices with or without TNC within.

The effect of CRELD1 gene on the related genes in endocardial cushion development

Objective To investigate the effect of alteration of CRELD1 gene expression on the related genes in the endocardial cushion development. Methods Over-expression and silence of CRELD1 gene were realized by the construction of lentiviral vector.Afterwards, the lentiviral vectors were used to infect the human fetal lung fibroblasts(HFL)-I.All of the cells were divided into the following 5 groups: the blank control group, the negative control group of interference, the interference group, the negative control group of over-expression, and the over-expression group.Western blot and real-time fluorescent quantitative polymerase chain reaction were applied to examine the mRNA and protein expression of CRELD1, Sox9, Aggrecan, Scleraxis and Tenascin-C. Results The DNA sequences of 2 recombinant plasmids pLV3-shRNA-CRELD1 and pLV4-CRELD1 matched very well with those which were designed according to the DNA sequence analysis.HFL-I was successfully infected with lentiviral vectors and displayed fluorescent green light under inverted fluorescence microscope.The results of real-time PCR detection and Western blot test were consistent: expressions of Sox9 and Aggrecan in the interference group were significantly higher than those in the negative control group of interference, while the expressions of the 2 genes in the over-expression group were significantly lower than those in the negative control group of over-expression.Expressions of Scleraxis increased in both the interference group and the over-expression group when compared with the negative control groups respectively.Compared to the corresponding negative control groups, Tenascin-C expression decreased markedly in the interference group, whereas it increased significantly in over-expression group. Conclusions CRELD1 gene has negative effect on the expression of the related genes Sox9 and Aggrecan in the endocardial cushion development, whereas it has positive effect on the Tenascin-C expression.It serves as a theoretical framework to illustrate the effect of CRELD1 gene on the atrioventricular septal defect. Key words: CRELD1 gene; Endocardial cushions; Interference; Over-expression

Tenascin-C secreted by transdifferentiated retinal pigment epithelial cells promotes choroidal neovascularization via integrin αV

Tenascin-C is expressed in choroidal neovascular (CNV) membranes in eyes with age-related macular degeneration (AMD). However, its role in the pathogenesis of CNV remains to be elucidated. Here we investigated the role of tenascin-C in CNV formation. In immunofluorescence analyses, tenascin-C co-stained with α-SMA, pan-cytokeratin, CD31, CD34, and integrin αV in the CNV membranes of patients with AMD and a mouse model of laser-induced CNV. A marked increase in the expression of tenascin-C mRNA and protein was observed 3 days after laser photocoagulation in the mouse CNV model. Tenascin-C was also shown to promote proliferation and inhibit adhesion of human retinal pigment epithelial (hRPE) cells in vitro. Moreover, tenascin-C promoted proliferation, adhesion, migration, and tube formation in human microvascular endothelial cells (HMVECs); these functions were, however, blocked by cilengitide, an integrin αV inhibitor. Exposure to TGF-β2 increased tenascin-C expression in hRPE cells. Conditioned media harvested from TGF-β2-treated hRPE cell cultures enhanced HMVEC proliferation and tube formation, which were inhibited by pretreatment with tenascin-C siRNA. The CNV volume was significantly reduced in tenascin-C knockout mice and tenascin-C siRNA-injected mice. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of CNV via integrin αV in a paracrine manner. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of CNV development associated with AMD.

A9.2 Tenascin-C expression during knee joint regeneration in the red-spotted NEWT notophthalmus viridescens

BackgroundIn mammals, tissue or organ damage often results in fibrotic scar formation, which in turn may impair proper organ function. Many urodele amphibians are capable of regenerating different tissues including...