TNC Genes Research Articles

Altered Expressions of IL-15, IFNG, and HPRT1 Genes in the Thin Endometria of Patients with Reproductive Disorders: A Prospective Comparative Study

Reproductive disorders are common events in modern reproductive medicine, occurring both in spontaneous and assisted pregnancies. Studies on the molecular mechanisms of implantation disorders in thin endometria, including the study of gene transcriptional activities, have shed light on the identification of the potential biological markers of endometrial receptivity. Background/Objectives: The goal of this study was to reveal the significantly dysregulated selected gene expressions between RIF and RPL patients with thin endometria. Methods: Endometrial samples were collected from RIF patients (n = 20) and RPL patients (n = 19) during the implantation window days (LH + 7—LH + 10) of their natural menstrual cycles. Ten genes were chosen as the target genes regarding their possible relations with the implantation process. The total RNA was purified and reverse-transcribed, and gene expressions were quantified by RT-PCR. Results: The expressions of the IL-15, INFG, and HPRT1 genes were significantly decreased in the RIF patients with thin endometria compared to the RPL patients (log2 fold change = 0.92, p = 0.023 for IL-15; log2 fold change = 1.24, p = 0.046 for INFG; and log2 fold change = 0.579, p = 0.046 for HPRT1). There were no significant differences in the expressions of the CXCL8, CXCL1, MMP10, C4BPA, TNC, VEGFB, and HAND2 genes between the groups. Conclusions: Decreased expressions of the IL-15, INFG, and HPRT1 genes were found in patients with RIF with thin endometria compared to the endometria of women with RPL. This has practical significance for clinicians for the differentiated prescription of immunomodulatory therapy in patients undergoing ART programs.

α9β1 integrin & its ligands as new potential biomarkers in FMF.

Background & objectives Familial Mediterranean Fever (FMF) manifests as a hereditary condition characterized by repeated bouts of fever, abdominal, chest, and joint discomfort, and swelling. Colchicine is the most common form of treatment, but it does not eliminate the disease. The underlying causes of the inflammatory mechanism are still not fully known. Methods A total of 20 healthy controls, 16 individuals with FMF in the attack period, and 14 in the remission period participated in the study. ITGA9, ITGB1, OPN, TNC, VEGF, VCAM-1, TGM2, TSP-1, Emilin-1, and vWF levels were measured by ELISA by obtaining serum from blood samples of individuals. In addition, gene expressions of α9β1 (ITGA9, ITGB1) and its best known ligands (TNC, SPP1) were analyzed by quantitative real-time PCR (qPCR). Results The findings of this study showed that serum levels of α9β1 and its ligands were higher in individuals with FMF in the attack period than in the healthy controls and the FMF group in the remission period (P<0.05). The marker levels of the healthy group were also higher than those in the remission period (p<0.05). In addition, when the gene expressions were compared between the healthy controls and FMF group, no significant difference was found for ITGA9, ITGB1, TNC, and SPP1 genes. Interpretation & conclusions The function of α9β1 and its ligands in FMF disease was investigated for the first time in this study as per our knowledge. Serum levels of these biomarkers may help identify potential new targets for FMF disease diagnosis and treatment approaches.

A novel splice-altering TNC variant (c.5247A > T, p.Gly1749Gly) in an Chinese family with autosomal dominant non-syndromic hearing loss

BackgroundThis study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene.MethodsA five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV_AdaBoost, dbscSNV_RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis.ResultsThe family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon–intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19.ConclusionThis novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families.

Genome-wide identification and analysis of epithelial-mesenchymal transition-related RNA-binding proteins and alternative splicing in a human breast cancer cell line

Exploring the mechanism of breast cancer metastasis and searching for new drug therapeutic targets are still the focuses of current research. RNA-binding proteins (RBPs) may affect breast cancer metastasis by regulating alternative splicing (AS) during epithelial-mesenchymal transition (EMT). We hypothesised that during EMT development in breast cancer cells, the expression level of RBPs and the gene AS pattern in the cell were significantly changed on a genome-wide scale. Using GEO database, this study identified differentially expressed RBPs and differential AS events at different stages of EMT in breast cancer cells. By establishing the correlation network of differential RBPs and differential AS events, we found that RBM47, PCBP3, FRG1, SRP72, RBMS3 and other RBPs may regulate the AS of ITGA6, ADGRE5, TNC, COL6A3 and other cell adhesion genes. By further analysing above EMT-related RBPs and AS in breast cancer tissues in TCGA, it was found that the expression levels of ADAT2, C2orf15, SRP72, PAICS, RBMS3, APOBEC3G, NOA1, ACO1 and the AS of TNC and COL6A3 were significantly correlated with the prognosis of breast cancer patients. The expression levels of all 8 RBPs were significantly different in breast cancer tissues without metastasis compared with normal breast tissues. Conclusively, eight RBPs such as RBMS3 and AS of TNC and COL6A3 could be used as predictors of breast cancer prognosis. These findings need to be further explored as possible targets for breast cancer treatment.

Dual effect of vitamin D3 on breast cancer-associated fibroblasts

BackgroundCancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Despite the well-known in vitro antitumoral effect of vitamin D3 (VD3), its impact on breast CAFs is almost unknown. In this study, we analyzed the ex vivo effects of calcitriol on CAFs isolated from breast cancer tissues.MethodsCAFs were cultured with 1 and 10 nM calcitriol and their phenotype; gene expression, protein expression, and secretion were assessed. Calcitriol-treated CAFs-conditioned media (CM) were used to analyze the effect of CAFs on the migration and protein expression of MCF-7 and MDA-MB-231 cells.ResultsTumor tissues from VD3-deficient patients exhibited lower levels of β-catenin and TGFβ1, along with higher levels of CYP24A1 compared to VD3-normal patients. In VD3-deficient patients, CAF infiltration was inversely associated with CYP24A1 levels and positively correlated with OPN levels. Calcitriol diminished CAFs’ viability, but this effect was weaker in premenopausal and VD3-normal patients. Calcitriol reduced mRNA expression of CCL2, MMP9, TNC, and increased PDPN, SPP1, and TIMP1. It also decreased the secretion of CCL2, TNC, and the activity of MMP-2, while increasing cellular levels of TIMP1 in CAFs from all patient groups. In nonmetastatic and postmenopausal patients, PDPN surface expression increased, and CAFs CM from these groups decreased MCF-7 cell migration after ex vivo calcitriol treatment. In premenopausal and VD3-deficient patients, calcitriol reduced IDO1 expression in CAFs. Calcitriol-treated CAFs CM from these patients decreased OPN expression in MCF-7 and/or MDA-MB-231 cells. However, in premenopausal patients, calcitriol-treated CAFs CM also decreased E-cadherin expression in both cell lines.ConclusionThe effects of calcitriol on breast CAFs, both at the gene and protein levels, are complex, reflecting the immunosuppressive or procancer properties of CAFs. The anticancer polarization of CAFs following ex vivo calcitriol treatment may result from decreased CCL2, TNC (gene and protein), MMP9, and MMP-2, while the opposite effect may result from increased PDPN, TIMP1 (gene and protein), and SPP1. Despite these multifaceted effects of calcitriol on molecule expression, CAFs’ CMs from nonmetastatic and postmenopausal patients treated ex vivo with calcitriol decreased the migration of MCF-7 cells.

Role of tenascin C in lesion formation in early peritoneal endometriosis

To identify cytokines or extracellular matrix components that contribute to adhesion to, and invasion of, the peritoneum, proximal to lesions in the early phase of endometriosis. Laboratory-based study. University Hospital and Laboratory of Animal Science. Five women with ovarian endometrioma, 138 wild-type (WT) C57BL/6N mice, and 48 Tenascin C (Tnc) knockout (TncKO) mice. To establish a murine endometriosis model, 20 pieces of minced uterine tissue fragments from each horn were administered intraperitoneally to syngeneic mice. Three days later, endometriotic lesions and peritoneal tissues were collected. Separately, we transfected human peritoneal mesothelial cells (HMrSV5) or human endometrial stromal cells (hESCs) with Tnc small interfering ribonucleic acid. We employed a polymerase chain reaction array to profile gene expression in the murine peritoneum, in both peritoneum distal to lesions and peritoneum surrounding lesions (PSL). The expression of upregulated genes in the PSL was verified in the peritoneal samples by real-time reverse transcription-polymerase chain reaction. TncKO mice were used to investigate the role of Tnc in the development of endometriosis. We evaluated the proliferative activity or inflammatory state of lesions by Ki67 or CD3 immunostaining. Intraperitoneal distribution of macrophages was assessed by fluorescence-activated cell sorting. Using Tnc small interfering ribonucleic acid, we examined the invasive capacity of hESCs in a coculture system with HMrSV5. Tnc gene expression was significantly higher in PSL than in peritoneum distal to lesions. The weight and number of TncKO lesions in TncKO hosts were lower than those of WT lesions in WT hosts. In contrast, the weight and number of nonattached TncKO lesions in TncKO hosts were higher than those of nonattached WT lesions in WT hosts. We observed decreased Ki67-positive cells or H-scores for CD3, a lower proportion of M1 macrophages, and a higher proportion of M2 macrophages in TncKO lesions in TncKO recipients. Silencing of Tnc expression in hESCs and HMrSV5 diminished the invasivity of hESCs. Tnc may be a crucial factor in the development of early peritoneal endometriosis.

Assessment of α9β1 ıntegrın as a new dıagnostıc and therapeutıc target ın Behcet's dısease.

This study aimed to investigate the roles of α9β1 integrin and its ligands in Behçet's disease (BD) by examining serum levels and gene expressions. 15 healthy controls and 30 BD patients (14 active and 16 inactive) were included in the study. Serum levels of ITGA9, ITGB1, TNC, OPN, VCAM-1, VEGF, TSP1, TGM2, Emilin-1, and vWF, were measured by ELISA. Gene expressions of α9β1 (ITGA9 and ITGB1) and its ligands (TNC and SPP1) were evaluated by RT-PCR. Laboratory findings (CRP, ESR, HGB, WBC, RBC, neutrophil, lymphocyte, PLT, RDW, MPV, PCT, and HLA-B51) were obtained from the electronic database. Active BD patients had higher serum levels of α9β1 integrin and its ligands than inactive patients and healthy controls. No significant difference was observed between healthy controls and inactive patients. Gene expressions of ITGB1 and SPP1 were increased in both patient groups compared to healthy controls. ITGA9 and TNC gene expression levels were lower in the active group than in the inactive group. No noticeable differences were found in ITGB1 and SPP1 gene expressions between the patient groups. BD patients exhibited elevated CRP, ESR, WBC, neutrophil, PLT, and PCT levels, while HGB, RBC, and RDW values were lower than healthy controls. Active patients had higher CRP, ESR, WBC, neutrophil, and PLT levels. Significant positive correlations were found between CRP, ESR, WBC, neutrophil, PLT, PCT and serum levels of α9β1 integrin and its ligands. Increased release of α9β1 integrin and its ligands is associated with BD, suggesting their potential as markers for disease severity.

Association of Gene Variants with Seasonal Variation in Muscle Strength and Aerobic Capacity in Elite Skiers.

Background: The training of elite skiers follows a systematic seasonal periodization with a preparation period, when anaerobic muscle strength, aerobic capacity, and cardio-metabolic recovery are specifically conditioned to provide extra capacity for developing ski-specific physical fitness in the subsequent competition period. We hypothesized that periodization-induced alterations in muscle and metabolic performance demonstrate important variability, which in part is explained by gene-associated factors in association with sex and age. Methods: A total of 34 elite skiers (20.4 ± 3.1 years, 19 women, 15 men) underwent exhaustive cardiopulmonary exercise and isokinetic strength testing before and after the preparation and subsequent competition periods of the World Cup skiing seasons 2015-2018. Biometric data were recorded, and frequent polymorphisms in five fitness genes, ACE-I/D (rs1799752), TNC (rs2104772), ACTN3 (rs1815739), and PTK2 (rs7460, rs7843014), were determined with specific PCR reactions on collected DNA. Relative percentage changes of cardio-pulmonary and skeletal muscle metabolism and performance over the two seasonal periods were calculated for 160 data points and subjected to analysis of variance (ANOVA) to identify hypothesized and novel associations between performance alterations and the five respective genotypes and determine the influence of age × sex. A threshold of 0.1 for the effect size (h2) was deemed appropriate to identify relevant associations and motivate a post hoc test to localize effects. Results: The preparation and competition periods produced antidromic functional changes, the extent of which varied with increasing importance for anaerobic strength, aerobic performance, cardio-metabolic efficiency, and cardio-metabolic/muscle recovery. Only peak RER (-14%), but not anaerobic strength and peak aerobic performance, and parameters characterizing cardio-metabolic efficiency, differed between the first and last studied skiing seasons because improvements over the preparation period were mostly lost over the competition period. A number of functional parameters demonstrated associations of variability in periodic changes with a given genotype, and this was considerably influenced by athlete "age", but not "sex". This concerned age-dependent associations between periodic changes in muscle-related parameters, such as anaerobic strength for low and high angular velocities of extension and flexion and blood lactate concentration, with rs1799752 and rs2104772, whose gene products relate to sarcopenia. By contrast, the variance in period-dependent changes in body mass and peak VO2 with rs1799752 and rs2104772, respectively, was independent of age. Likely, the variance in periodic changes in the reliance of aerobic performance on lactate, oxygen uptake, and heart rate was associated with rs1815739 independent of age. These associations manifested at the post hoc level in genotype-associated differences in critical performance parameters. ACTN3 T-allele carriers demonstrated, compared to non-carriers, largely different periodic changes in the muscle-associated parameters of aerobic metabolism during exhaustive exercise, including blood lactate and respiration exchange ratio. The homozygous T-allele carriers of rs2104772 demonstrated the largest changes in extension strength at low angular velocity during the preparation period. Conclusions: Physiological characteristics of performance in skiing athletes undergo training period-dependent seasonal alterations the extent of which is largest for muscle metabolism-related parameters. Genotype associations for the variability in changes of aerobic metabolism-associated power output during exhaustive exercise and anaerobic peak power over the preparation and competition period motivate personalized training regimes. This may help to predict and maximize the benefit of physical conditioning of elite skiers based on chronological characteristics and the polymorphisms of the ACTN3, ACE, and TNC genes investigated here.

Androgen receptor variant-7 regulation by tenascin-c induced src activation

BackgroundBone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC.MethodsProstate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation.ResultsUtilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells.ConclusionOverall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC.9BxMEZaJbqgytBg6QvviEaVideo

Dipotassium Glycyrrhizinate on Melanoma Cell Line: Inhibition of Cerebral Metastases Formation by Targeting NF-kB Genes-Mediating MicroRNA-4443 and MicroRNA-3620-Dipotassium Glycyrrhizinate Effect on Melanoma.

Glycyrrhizic acid (GA), a natural compound isolated from licorice (Glycyrrhiza glabra), has exhibited anti-inflammatory and anti-tumor effects in vitro. Dipotassium glycyrrhizinate (DPG), a dipotassium salt of GA, also has shown an anti-tumor effect on glioblastoma cell lines, U87MG and T98G. The study investigated the DPG effects in the melanoma cell line (SK-MEL-28). MTT assay demonstrated that the viability of the cells was significantly decreased in a time- and dose-dependent manner after DPG (IC50 = 36 mM; 24 h). DNA fragmentation suggested that DPG (IC50) induced cellular apoptosis, which was confirmed by a significant number of TUNEL-positive cells (p-value = 0.048) and by PARP-1 [0.55 vs. 1.02 arbitrary units (AUs), p-value = 0.001], BAX (1.91 vs. 1.05 AUs, p-value = 0.09), and BCL-2 (0.51 vs. 1.07 AUs, p-value = 0.0018) mRNA compared to control cells. The proliferation and wound-healing assays showed an anti-proliferative effect on DPG-IC50-treated cells, also indicating an inhibitory effect on cell migration (p-values < 0.001). Moreover, it was observed that DPG promoted a 100% reduction in melanospheres formation (p-value = 0.008). Our previous microRNAs (miRs) global analysis has revealed that DPG might increase miR-4443 and miR-3620 expression levels. Thus, qPCR showed that after DPG treatment, SK-MEL-28 cells presented significantly high miR-4443 (1.77 vs. 1.04 AUs, p-value = 0.02) and miR-3620 (2.30 vs. 1.00 AUs, p-value = 0.01) expression compared to control cells, which are predicted to target the NF-kB, CD209 and TNC genes, respectively. Both genes are responsible for cell attachment and migration, and qPCR revealed significantly decreased CD209 (1.01 vs. 0.54 AUs, p-value = 0.018) and TNC (1.00 vs. 0.31 AUs, p-value = 2.38 × 10−6) mRNA expression levels after DPG compared to untreated cells. Furthermore, the migration of SK-MEL-28 cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) was attenuated by adding DPG by wound-healing assay (48 h: p-value = 0.004; 72 h: p-value = 7.0 × 10−4). In addition, the MMP-9 expression level was inhibited by DPG in melanoma cells stimulated by TPA and compared to TPA-treated cells (3.56 vs. 0.99 AUs, p-value = 0.0016) after 24 h of treatment. Our results suggested that DPG has an apoptotic, anti-proliferative, and anti-migratory effect on SK-MEL-28 cells. DPG was also able to inhibit cancer stem-like cells that may cause cerebral tumor formation.

Identification of novel therapeutic targets for Fuchs' endothelial corneal dystrophy based on gene bioinformatics analysis.

Fuchs’ endothelial corneal dystrophy (FECD) is a disease where progressive visual impairment occurs by the thickening of the Descemet’s membrane and the gradual degeneration and loss of corneal endothelial cells. This study aimed to investigate the key changes in gene expression associated with FECD and explore potential biomarkers and new therapeutic strategies for FECD. To explore the potential therapeutic targets of FECD, we downloaded the gene expression dataset GSE171830 from the Gene Expression Omnibus (GEO) database. A total of 303 differentially expressed genes (DEGs) were identified by the limma package. The enriched Gene Ontology (GO) annotations and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs mostly included the extracellular matrix organization, collagen-containing extracellular matrix, and the structural constituents of the extracellular matrix. Fifteen hub genes from the most significant module were ascertained by Cytoscape. Both collagen-containing extracellular matrix and extracellular matrix hit to ANXA1, VCAN, GPC3, TNC, IGFBP7, MATN3, and SPARCL1 genes in the GO cellular components. Among these genes, the expression of SPARCL1 was down-regulated in the FECD samples, whereas the expression of GPC3, MATN3, IGFBP7, TNC, VCAN, and ANXA1 was up-regulated in the FECD samples. Gene set enrichment analysis (GSEA) plots showed that among the 20,937 genes, SPARCL1 played an important role in three pathways, cytokine-cytokine receptor interaction, the TGF-beta signaling pathway, and antigen processing and presentation. The top three pathways enriched by the GPC3, MATN3, IGFBP7, TNC, VCAN, and ANXA1 genes were those for cytokine-cytokine receptor interaction, TGF-beta signaling, and RIG-I-like receptor signaling. In conclusion, the DEGs identified here might assist clinicians in understanding the pathogenesis of FECD. Furthermore, these identified biomarkers might serve as potential therapeutic targets for the treatment of FECD.

ACTIVATION OF MK2 IN CROHN’S DISEASE-ASSOCIATED FIBROSIS: NEW TARGET FOR ANTI-FIBROTIC THERAPY

Abstract INTRODUCTION Fibrosis is the major aggressive complication of Crohn’s disease (CD), causing intestinal obstruction. No targeted therapies are currently available to revert CD-associated fibrosis. While inflammatory mechanisms in CD have been extensively investigated, understanding the pathogenesis of fibrosis is relatively limited. Mesenchymal cells are believed to be the major effectors in profibrotic processes in CD. There is a dramatic increase in α-SMA expressing subsets of mesenchymal cells, also known as myofibroblasts (MF), and this is considered to be among the main hallmarks of profibrotic changes in CD. Thus, targeting the profibrotic reprogramming/increase of α-SMA+ MFs and/or fibrosis-driving signaling could lead to the development of novel anti-fibrotic approaches in CD. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream substrate of p38 and is known to regulate pro-inflammatory cytokines. Moreover, MK2 is a regulator of downstream signaling in the profibrotic non-canonical TGF-β1/p38 pathway. However, the role of MK2 in CD-associated fibrosis and profibrotic reprogramming of MFs is not known. We hypothesize that hyperactivation of MK2 is critical to the profibrotic reprogramming of MF in CD and has potential as an anti-fibrotic therapeutic target. METHODS Human normal- and CD-derived MFs were analyzed using RNAseq, cytokine/chemokine multiplex array, FACS, and confocal microscopy. Chronic IL-10 KO colitis murine models relevant to CD were used in this study. RESULTS Increased expression of MK2 within α-SMA+ mesenchymal cells was observed in stricturing CD. Ex vivo treatment of the CD tissues with MK2 inhibitor PF-3644022 (10 μM) abrogated MK2 activity and decreased expression of fibrosis-related genes. An increase in MK2 activity within intestinal MFs was observed in vivo in IL-10KO murine colitis. Therapeutic use of the MK2 inhibitor PF-3644022 (0.2 mg/kg, daily for 10 days) in vivo significantly decreased the degree of intestinal fibrosis as measured by collagen deposition and reduced the expression of fibrosis-related genes. When compared to the WT murine-derived MFs, MK2-/- MFs showed low basal and TGF-β1 induced levels of pro-fibrotic genes expression in vitro. Using primary human MFs, we found that MK2 activity was associated with hallmark profibrotic genes in CD-MFs. Inhibition of MK2 activity with PF-3644022 reduced TGF-β1-induced overexpression of Col1α2, α-SMA, TNC, Fn1, and TGF-β1 genes. Profibrotic responses in MFs through SMAD2/3 and p38, but not MAPK signaling were mediated by MK2. CONCLUSIONS Our data provide evidence that activation of MK2 drives profibrotic reprogramming of mesenchymal cells in CD and it may be an attractive therapeutic target for the development of novel targeted strategies against CD-associated fibrosis.

The long non-coding RNA ET-20 mediates EMT by impairing desmosomes in breast cancer cells.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFβ-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

The expression of tenascin-C in neural stem/progenitor cells is stimulated by the growth factors EGF and FGF-2, but not by TGF\u03b21

Neural stem/progenitor cells (NSPCs) rely on internal and external cues determining their lineage decisions during brain development. The progenitor cells of the embryonic mammalian forebrain reside in the ventricular and subventricular zones of the lateral ventricles, where they proliferate, generate neurons and glial cells, and respond to external cues like growth factors. The extracellular matrix (ECM) surrounds NSPCs and influences the cell fate by providing mechanical scaffold, trophic support, and instructive signals. The ECM molecule tenascin-C (Tnc) is expressed in the proliferative zones of the developing forebrain and involved in the proliferation and maturation of NSPCs. Here, we analyzed the regulation of the Tnc gene expression by NSPCs cultivated under the influence of different growth factors. We observed that the epidermal growth factor (EGF) and the fibroblast growth factor (FGF)-2 strongly increased the expression of Tnc, whereas the transforming growth factor (TGF)β 1 had no effect on Tnc gene expression, in contrast to previous findings in cell cultures of neural and non-neural origin. The stimulation of the Tnc gene expression induced by EGF or FGF-2 was reversible and seen in constantly treated as well as short term stimulated NSPC cultures. The activation depended on the presence of the respective receptors, which was slightly different in cortical and striatal NSPC cultures. Our results confirm the influence of extracellular stimuli regulating the expression of factors that form a niche for NSPCs during embryonic forebrain development.

Endometrial gene expression profiling of recurrent implantation failure after in vitro fertilization.

Recurrent implantation failure (RIF) is diagnosed when good-quality embryos repeatedly fail to implant after transfer in several in vitro fertilization (IVF) treatment cycles. Different expression profiles in maternal mRNAs could be referring to many diseases including RIF. This study aimed to reveal significantly dysregulated selected genes expression between healthy fertile women and RIF patients in the implantation window days of the natural menstrual cycle. MME, WWC1, TNC, and FOXP3 genes were chosen as target genes regarding their possible relations with the implantation process. Pathways with these genes were identified and the relationship between these pathways and RIF was investigated. In this study, the endometrial biopsy samples were collected in the secretory phase (cycle day 20-24) of the menstrual cycle from RIF patients (n = 34) and healthy fertile controls (n = 34). After "Pathway and network-oriented GWAS analysis" (PANOGA) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG) pathway analysis; "Membrane Metalloendopeptidase" (MME), "WW and C2 Domain Containing 1" (WWC1), "Tenascin C" (TNC) and "Forkhead Box P3" (FOXP3) genes were chosen as target genes by regarding their possible relation with implantation process. Detection of differences in mRNA expressions between the control group and RIF patients has been performed with the droplet digital PCR (ddPCR) method. Results of the study showed that MME and WWC1 genes expression levels are significantly (p < 0,05) up-regulated 4.9 and 5.2 times respectively and TNC gene expression level is significantly (p < 0,05) down-regulated 9 times in the RIF samples compared to the control group. However, no statistically significant difference was observed between the patient group and the control group in the expression of the FOXP3 gene (p < 0.05). Changes are observed in the expression of the renin-angiotensin system pathway in which the MME gene is involved in the implantation process. The increase in MME gene expression can be speculated to cause implantation failure by restricting the invasion of trophoblast cells. Increasing WWC1 gene expression in the Hippo signaling pathway inhibits "Yes-associated protein 1" (YAP) expression, which is a transcriptional cofactor. Inhibition of YAP protein expression may impair the implantation process by causing the failure of endometrial decidualization. The TNC gene is located in the focal adhesion pathway and this pathway reduces cell adhesion on the endometrial surface to facilitate the attachment of the embryo to the endometrium. The reason for implantation failure might be that the intercellular connections are not suitable for implantation as a result of decreased expression of the focal adhesion pathway in which the TNC gene is effective. Considering the relations between the pathways of the target genes and the implantation process, changes in the expression of target genes might be a cause of RIF.

The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

Exploring the Correlation between TNC Gene and Osteoporosis

Introduction: Osteoporosis is one of the main causes of bone fractures in old age. The examination of osteoporosis in the elderly is very complicated due to the heterogeneity of the aging process. This study aimed to investigate the correlation of the TNC gene in the patients with an inherited and very rare osteoporosis syndrome. The importance of this study was the identification of a specific biomarker for the osteoporosis process in a monogenic disease. Methods: In this case-control study, the patients with Osteogenesis Imperfecta were used as a simple and monogenic model to investigate the possibility of TNC gene role in osteoporosis. For this purpose, TNC protein network and its related biological pathways were firstly evaluated by bioinformatic analysis. Then, skin biopsies were taken from 3 patients with inherited osteoporosis syndrome called Osteogenesis Imperfecta and two healthy individuals. After culturing the biopsy, fibroblast cells were isolated from keratinocyte cells. Then, the total RNA of each sample was extracted from two different passages and cDNA was synthesized. Subsequently, the expression of TNC gene was measured by Real-time PCR in healthy and patient cells using various replicates. Results: TNC network proteins are significantly associated with the biological pathways involved in the ossification process. The expression of the TNC gene was assessed in wild type and patient cells. Finally, our results confirmed a significant increase in the expression of TNC in the patients’ compared to wild types’ cells by using technical and biological replicates (p:0.005, p:0.007) and Student t-test. In this study, Excel and GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA) were used. Conclusion: Increased expression of TNC gene in a monogenic osteoporosis syndrome can indicate the potential role of this gene in the process of bone loss and introduce this gen as a new biomarker for osteoporosis. The study on rare Osteogenesis Imperfecta syndrome is a simple and monogenic model to investigate the heterogeneous osteoporosis. Therefore, the expression of the candidate gene needs to be evaluated and confirmed in an appropriate statistical population composed of patients with osteoporosis.

Suppression of Hematopoietic Activity in Tenascin-C–Deficient Mice

AbstractTenascin-C (TN-C), a member of the extracellular matrix (ECM) glycoprotein family, is expressed on the surface of stromal cells in the hematopoietic system or lymphoid organs. Recently, TN-C–deficient mutant mice produced by TN-C gene targeting through homologous recombination were shown to develop normally, although TNs have been reported to play important roles in organogenesis and carcinogenesis. In the present study, we found that colony-forming capacity of bone marrow (BM) cells was considerably lower in TN-C–deficient mice (a decrease of ∼35% from control), although their mononuclear cell count and BM architecture showed no significant difference from those of normal mice. Furthermore, in long-term BM culture in vitro, hematopoietic cell production (a decrease of ∼40% in Dexter's condition and of ∼65% in Whitlock-Witte's condition from control), colony-forming capacity of the produced cells (a decrease of ∼60% from control), and longevity of the cultures were markedly lower in the TN-C–deficient mice than in control mice, whereas hematopoiesis in the TN-C–deficient mutant mice was sustained. The addition of TN-C glycoprotein to long-term BM cultures of TN-C–deficient mice clearly induced the recovery of hematopoietic cell production and colony-forming capacity of hematopoietic progenitor cells. Thus, these results provide direct evidence that an ECM glycoprotein component, TN-C, plays a relevant role in hematopoiesis through interactions between stromal cells and hematopoietic progenitor cells.

Genetic variants associated with rotator cuff tearing utilizing multiple population-based genetic resources

The etiology of rotator cuff tearing is likely multifactorial, including a potential genetic predisposition. The purpose of the study was to identify genetic variants associated with rotator cuff tearing utilizing the UK Biobank (UKB) cohort, confirm variants using a separate genetic database, and evaluate tissue expression of genes with associated variants following rotator cuff tearing using RNA sequencing. Genome-wide association study (GWAS): A GWAS was performed using data from UKB with 5701 cases of rotator cuff injury. RNA sequencing analyses: rotator cuff biopsies were obtained from 24 patients with full-thickness rotator cuff tears who underwent arthroscopic rotator cuff repair (cases) and 9 patients who underwent open reduction internal fixation for a proximal humerus fracture (controls). Total RNA was extracted and differential gene expression was measured by RNAseq for genes with variants associated with rotator cuff tearing. The results of the UKB GWAS identified 3 loci that reached genome-wide statistical significance: 2 loci on chromosome 7 in GLCCI1 (rs4725069; P = 5.0E-09) and THSD7A (rs575224171; P = 5.3E-09), and 1 locus on chromosome 2 in ZNF804A (rs775583810; P = 3.9E-09). The association with rotator cuff injury of the GLCCI1 single-nucleotide polymorphism (SNP; rs4725069) was confirmed in the Kaiser Permanente Research Bank cohort (P = .008). Twenty previously reported SNPs in 12 genes were evaluated using summary statistics from the UKB GWAS, which confirmed 3 SNPs in TNC with rotator cuff injury (rs1138545, rs72758637, and rs7021589; all P < .0024). Of 17 genes with variants associated with rotator cuff injury (14 previously from literature plus 3 new genes from current UKB GWAS), TIMP2, Col5A1, TGFBR1, and TNC were upregulated (P < .001 for all) and THSD7A was downregulated (P = .005) in tears vs. controls in the RNA sequencing data set. The UKB GWAS has identified 3 novel loci associated with rotator cuff tearing (ZNF804A, GLCCI1, THSD7A). Expression of the THSD7A gene was significantly downregulated in rotator cuff tears vs. controls supporting a potential functional role. Three previously reported SNPs in the TNC gene were validated in the UKB GWAS, supporting a role for this gene in rotator cuff tearing. Finally, TIMP2, Col5A1, TGFBR1, and TNC genes were found to have significantly upregulated tissue expression in cases vs. controls supporting a biologic role in tearing for these genes.

Microcurrent and adipose-derived stem cells modulate genes expression involved in the structural recovery of transected tendon of rats.

Tendon injuries are common and have a high incidence of re-rupture that can cause loss of functionality. Therapies with adipose-derived stem cells (ASC) and the microcurrent (low-intensity electrical stimulation) application present promising effects on the tissue repair. We analyzed the expression of genes and the participation of some molecules potentially involved in the structural recovery of the Achilles tendon of rats, in response to the application of both therapies, isolated and combined. The tendons were distributed in five groups: normal (N), transected (T), transected and ASC (C) or microcurrent (M) or with ASC, and microcurrent (MC). Microcurrent therapy was beneficial for tendon repair, as it was observed a statistically significant increase in the organization of the collagen fibers, with involvement of the TNC, CTGF, FN, FMDO, and COL3A1 genes as well as PCNA, IL-10, and TNF-α. ASC therapy significantly increased the TNC and FMDO genes expression with no changes in the molecular organization of collagen. With the association of therapies, a significant greater collagen fibers organization was observed with involvement of the FMOD gene. The therapies did not affect the expression of COL1A1, SMAD2, SMAD3, MKX, and EGR1 genes, nor did they influence the amount of collagen I and III, caspase-3, tenomodulin (Tnmd), and hydroxyproline. In conclusion, the application of the microcurrent isolated or associated with ASC increased the organization of the collagen fibers, which can result in a greater biomechanical resistance in relation to the tendons treated only with ASC. Future studies will be needed to demonstrate the biological effects of these therapies on the functional recovery of injured tendons.