Single-Cell Analysis Reveals Transcriptomic Features of Drug-Tolerant Persisters and Stromal Adaptation in a Patient-Derived EGFR-Mutated Lung Adenocarcinoma Xenograft Model.

INTRODUCTION: Targeted therapies require life-long treatment, as drug discontinuation invariably leads to tumor recurrence. Recurrence is mainly driven by minor subpopulations of drug-tolerant persister (DTP) cells that survive the cytotoxic drug effect. In lung cancer, DTP studies have mainly been conducted with cell line models. METHODS: We conducted an in vivo DTP study using a lung adenocarcinoma patient-derived xenograft tumor driven by an EGFR mutation. Daily treatment of tumor-bearing mice for 5 to 6 weeks with the EGFR inhibitor erlotinib markedly shrunk tumors and generated DTPs, which were analyzed by whole exome, bulk population transcriptome, and single-cell RNA sequencing. RESULTS: The DTP tumors maintained the genomic clonal architecture of untreated baseline (BL) tumors but had reduced proliferation. Single-cell RNA sequencing identified a rare (approximately 4%) subpopulation of BL cells (DTP-like) with transcriptomic similarity to DTP cells and intermediate activity of pathways that are up-regulated in DTPs. Furthermore, the predominant transforming growth factor-ß activated cancer-associated fibroblast (CAF) population in BL tumors was replaced by a CAF population enriched for IL6 production. In vitro experiments indicate that these populations interconvert depending on the levels of transforming growth factor-ß versus NF-κB signaling, which is modulated by tyrosine kinase inhibitor presence. The DTPs had signs of increased NF-κB and STAT3 signaling, which may promote their survival. CONCLUSIONS: The DTPs may arise from a specific preexisting subpopulation of cancer cells with partial activation of specific drug resistance pathways. Tyrosine kinase inhibitor treatment induces DTPs revealing greater activation of these pathways while converting the major preexisting CAF population into a new state that may further promote DTP survival.

A short peptide encoded by long non-coding RNA small nucleolar RNA host gene 6 promotes cell migration and epithelial-mesenchymal transition by activating transforming growth factor-beta/SMAD signaling pathway in human endometrial cells.

BACKGROUND: Endometrial dysfunction is closely correlated with the development of multiple severe gynecological disorders including intrauterine adhesion. Accumulating evidence supports that some long non-coding RNAs (lncRNAs) have peptide-coding potential. In this text, the peptide-coding ability of lncRNA SNHG6 was examined. Also, the effects of an SNHG6-encoded peptide on the viability and migration of human endometrial stromal cells (hESCs) and human endometrial epithelial cells (hEECs) and related molecular mechanisms were explored. METHODS: The peptide-encoding potential of SNHG6 was predicted by FuncPEP and getorf databases and validated by western blot assay. Cell viability was tested by cell counting kit-8 assay. Cell migratory ability was examined by wound healing and transwell migration assays. Protein levels of genes were measured by western blot assay. RESULTS: Prediction analysis suggested that SNHG6 had the potential peptide-coding ability and multiple open-reading frames (ORFs). Western blot validated that SNHG6 ORF#1 and ORF#2 could translate into short peptides. SNHG6 ORF#2 overexpression facilitated cell migration and epithelial-mesenchymal transition (EMT) in hESCs and hEECs, while these effects were abrogated by transforming growth factor-beta (TGF-ß)/SMAD signaling inhibitor GW788388. Moreover, GW788388 inhibited the increase of p-SMAD2 and p-SMAD3 levels induced by SNHG6 ORF#2 in hESCs. SNHG6 ORF#2-encoded peptide did not influence endometrial stromal and epithelial cell viability. CONCLUSIONS: LncRNA SNHG6 ORF#1 and ORF#2 could translate into small peptides and SNHG6 ORF#2 overexpression promoted cell migration and EMT by activating the TGF-ß/SMAD pathway in hESCs and hEECs, suggesting the potential roles of SNHG6-encoded peptides in the development of endometrial stromal and epithelial cells and related gynecological diseases.

Salmonella pathogenicity island 1 knockdown confers protection against myocardial fibrosis and inflammation in uremic cardiomyopathy via down-regulation of S100 Calcium Binding Protein A8/A9 transcription.

BACKGROUND/AIM: Uremic cardiomyopathy (UCM) is a characteristic cardiac pathology that is commonly found in patients with chronic kidney disease. This study dissected the mechanism of SPI1 in myocardial fibrosis and inflammation induced by UCM through S100A8/A9. METHODS: An UCM rat model was established, followed by qRT-PCR and western blot analyses of SPI1 and S100A8/A9 expression in myocardial tissues. After alterations of SPI1 and S100A8/A9 expression in UCM rats, the blood specimens were harvested from the cardiac apex of rats. The levels of creatine phosphokinase-MB (CK-MB), blood creatinine, blood urea nitrogen (BUN), and inflammatory cytokines (interleukin [IL]-6, IL-1ß, and tumor necrosis factor-α [TNF-α]) were examined in the collected blood. Collagen fibrosis was assessed by Masson staining. The expression of fibrosis markers [transforming growth factor (TGF)-ß1, α-smooth muscle actin (SMA), Collagen 4a1, and Fibronectin], IL-6, IL-1ß, and TNF-α was measured in myocardial tissues. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were conducted to test the binding relationship between SPI1 and S100A8/A9. RESULTS: S100A8/A9 and SPI1 were highly expressed in the myocardial tissues of UCM rats. Mechanistically, SPI1 bound to the promoter of S100A8/A9 to facilitate S100A8/A9 transcription. S100A8/A9 or SPI1 knockdown reduced myocardial fibrosis and inflammation and the levels of CK-MB, blood creatinine, and BUN, as well as the expression of TGF-ß1, α-SMA, Collagen 4a1, Fibronectin, IL-6, TNF-α, and IL-1ß in UCM rats. CONCLUSION: SPI1 knockdown diminished S100A8/A9 transcription, thus suppressing myocardial fibrosis and inflammation caused by UCM.

Integration of transcriptomics and metabolomics reveals pathways involved in MDSC supernatant attenuation of TGF-ß1-induced myofibroblastic differentiation of mesenchymal stem cells.

Overexposure to transforming growth factor b1 (TGF-ß1) induces myofibroblastic differentiation of mesenchymal stem cells (MSCs), which could be attenuated by myeloid-derived suppressor cell (MDSC) supernatant. However, the promyofibroblastic effects of TGF-ß1 and the antimyofibroblastic effects of MDSC supernatant in MSCs have not been fully elucidated. To further clarify the latent mechanism and identify underlying therapeutic targets, we used an integrative strategy combining transcriptomics and metabolomics. Bone marrow MSCs were collected 24 h following TGF-ß1 and MDSC supernatant treatment for RNA sequencing and untargeted metabolomic analysis. The integrated data were then analyzed to identify significant gene-metabolite correlations. Differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) were assessed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for exploring the mechanisms of myofibroblastic differentiation of MSCs. The integration of transcriptomic and metabolomic data highlighted significantly coordinated changes in glycolysis/gluconeogenesis and purine metabolism following TGF-ß1 and MDSC supernatant treatment. By combining transcriptomic and metabolomic analyses, this study showed that glycolysis/gluconeogenesis and purine metabolism were essential for the myofibroblastic differentiation of MSCs and may serve as promising targets for mechanistic research and clinical practice in the treatment of fibrosis by MDSC supernatant.

Hsa-microRNA-27b-3p inhibits hepatocellular carcinoma progression by inactivating transforming growth factor-activated kinase-binding protein 3/nuclear factor kappa B signalling.

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p (hsa-miR-27b) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear. METHODS: The expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin (F-actin) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b. To detect the impacts of hsa-miR-27b on nuclear factor kappa B (NF-кB) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 (TAB3), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (IHC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (OncoAd) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC. RESULTS: The expression of hsa-miR-27b was lower in HCC than in adjacent non-tumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NF-κB by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b. The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3. Recombinant OncoAd with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) OncoAd in vivo and in vitro. CONCLUSIONS: By targeting TAB3, hsa-miR-27b acted as a tumour suppressor by inactivating the NF-кB pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC.

BMP2 inhibits cell proliferation by downregulating EZH2 in gastric cancer.

Gastric cancer is among the most common gastrointestinal malignancies. Recent studies have suggested that bone morphogenetic protein-2 (BMP2) is related to the development and progression of various cancers. Meanwhile, evidence suggests that BMP2 might lead to epigenetic changes in gastric cancer. Thus, we investigated whether BMP2 plays a role in the development of gastric cancer via epigenetic regulation. Cell viability, colony formation, and cell cycle assays were performed to assess the effect of recombinant human BMP2 (rhBMP2) in gastric cancer cells. LDN-193189 and Noggins were used as antagonists of the canonical BMP-SMAD signaling pathway. The protein levels were determined using a western blot analysis. Lentiviral vectors with EZH2 shRNA or EZH2 overexpression were used to mediate the role of EZH2 and the relationship between BMP2 and EZH2 in gastric cancer. We found that rhBMP2 inhibits cell proliferation by arresting the cell cycle in HGC-27 and SNU-216 gastric cancer cells. Neither LDN-193189 nor Noggins, antagonists of the canonical BMP-SMAD signaling pathway, can reverse the effect of rhBMP2 on gastric cancer. Molecularly, rhBMP2 downregulates the expression of EZH2 and H3K27me3, leading to increases in P16 and P21 and decreases in CDK2, CDK4, and CDK6. Altogether, in this study, we demonstrate that BMP2 serves as a tumor suppressor in gastric cancer cells by downregulating EZH2 and H3K27me3 through the non-SMAD BMP pathway, suggesting that BMP2 might be a new therapeutic target for gastric cancer treatment. Abbreviations: BMP: bone morphogenetic protein; TGF-ß: transforming growth factor-beta; EZH2: enhancer of zeste homolog 2; H3K27me3: trimethylation histone H3 lysine 27; HRECs: human retinal endothelial cells; PcG: polycomb group; PRC: polycomb repressive complexes.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Circ-POLR3A accelerates TGF-ß2-induced promotion in cell viability, migration, and invasion of lens epithelial cells via miR-31/TXNIP signaling cascade.

Posterior capsular opacification (PCO) is the major complication after cataract surgery and can result in secondary vision loss. Circular RNAs (circRNAs) are reported to play critical regulatory roles in multiple cell biological processes. The most common working mechanism of circRNAs is by acting as microRNA sponges. Here, we analyzed the role and mechanism of circRNA RNA polymerase III subunit A (POLR3A) in PCO. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell motility was assessed by transwell and wound healing assays. Dual-luciferase reporter and RNA-pull-down assays were performed to verify the interaction between microRNA-31 (miR-31) and circ-POLR3A or thioredoxin interacting protein (TXNIP). PCO cell model was established by treating SRA01/04 cells with transforming growth factor-ß2 (TGF-ß2). We found that TGF-ß2 enhanced SRA01/04 cell viability, migration, and invasion abilities. Circ-POLR3A expression was upregulated in PCO tissues and TGF-ß2-induced SRA01/04 cells. TGF-ß2 promoted the viability and motility of SRA01/04 cells largely by upregulating circ-POLR3A. Circ-POLR3A negatively regulated the miR-31 level by directly interacting with it. Circ-POLR3A absence-induced influences in TGF-ß2-induced SRA01/04 cells were partly reversed by silencing miR-31. miR-31 is directly bound to the 3'-untranslated region of TXNIP. TXNIP overexpression largely attenuated miR-31 overexpression-mediated effects in TGF-ß2-induced SRA01/04 cells. Circ-POLR3A could elevate the protein expression of TXNIP by sponging miR-31. Exosomes were involved in mediating the delivery of circ-POLR3A in SRA01/04 cells. In conclusion, circ-POLR3A contributed to TGF-ß2-induced promotion of cell viability, migration, and invasion of SRA01/04 cells by targeting miR-31/TXNIP axis.

Molecular Evolution of Transforming Growth Factor-ß (TGF-ß) Gene Family and the Functional Characterization of Lamprey TGF-ß2.

The transforming growth factor-ßs (TGF-ßs) are multifunctional cytokines capable of regulating a wide range of cellular behaviors and play a key role in maintaining the homeostasis of the immune system. The TGF-ß subfamily, which is only present in deuterostomes, expands from a single gene in invertebrates to multiple members in jawed vertebrates. However, the evolutionary processes of the TGF-ß subfamily in vertebrates still lack sufficient elucidation. In this study, the TGF-ß homologs are identified at the genome-wide level in the reissner lamprey (Lethenteron reissneri), the sea lamprey (Petromyzon marinus), and the Japanese lamprey (Lampetra japonica), which are the extant representatives of jawless vertebrates with a history of more than 350 million years. The molecular evolutionary analyses reveal that the lamprey TGF-ß subfamily contains two members representing ancestors of TGF-ß2 and 3 in vertebrates, respectively, but TGF-ß1 is absent. The transcriptional expression patterns show that the lamprey TGF-ß2 may play a central regulatory role in the innate immune response of the lamprey since it exhibits a more rapid and significant upregulation of expression than TGF-ß3 during lipopolysaccharide stimuli. The incorporation of BrdU assay reveals that the lamprey TGF-ß2 recombinant protein exerts the bipolar regulation on the proliferation of the supraneural myeloid body cells (SMB cells) in the quiescent and LPS-activated state, while plays an inhibitory role in the proliferation of quiescent and activated leukocytes in lampreys. Furthermore, caspase-3/7 activity analysis indicates that the lamprey TGF-ß2 protects SMB cells from apoptosis after serum deprivation, in contrast to promoting apoptosis of leukocytes. Our composite results offer valuable clues to the origin and evolution of the TGF-ß subfamily and imply that TGF-ßs are among the most ancestral immune regulators in vertebrates.

Confirmation of association of TGFBI p.Ser591Phe mutation with variant lattice corneal dystrophy.

PURPOSE: To provide the initial confirmation of the c.1772C>T (p.Ser591Phe) mutation in the transforming growth factor-ß-induced (TGFBI) gene as being associated with variant lattice corneal dystrophy (LCD). METHODS: Ophthalmologic examination of the proband was performed with slit lamp biomicroscopy. Saliva was collected as a source of DNA for screening all 17 exons of TGFBI, after which three family members were selectively screened for variants in exon 13. Rosetta-based structure prediction was used to calculate changes in TGFBI protein (TGFBIp) stability secondary to the c.1772C>T (p.Ser591Phe) missense mutation. RESULTS: Slit lamp examination of the 38-year-old proband revealed a clear cornea right eye and unilateral, discrete, and branching lattice lines in the anterior and mid-stroma of the central cornea left eye. Screening of TGFBI in the proband revealed a heterozygous missense mutation in exon 13 (c.1772C>T (p.Ser591Phe)) that was also identified in her affected mother but not in her brother or maternal grandmother. Calculated energy change in Rosetta (ΔΔG) for the TGFBIp variant p.Ser591Phe was 23.5, indicating a thermodynamic destabilization resulting from energetic frustration. CONCLUSIONS: The p.Ser591Phe mutation in TGFBI is associated with an unilateral variant of LCD. Rosetta-predicted stability changes indicate that the p.Ser591Phe variant is destabilizing, which is consistent with other observations for LCD-causing mutations.

Transforming growth factor-beta (TGF-ß) in prostate cancer: A dual function mediator?

Transforming growth factor-beta (TGF-ß) is a member of a family of secreted cytokines with vital biological functions in cells. The abnormal expression of TGF-ß signaling is a common finding in pathological conditions, particularly cancer. Prostate cancer (PCa) is one of the leading causes of death among men. Several genetic and epigenetic alterations can result in PCa development, and govern its progression. The present review attempts to shed some light on the role of TGF-ß signaling in PCa. TGF-ß signaling can either stimulate or inhibit proliferation and viability of PCa cells, depending on the context. The metastasis of PCa cells is increased by TGF-ß signaling via induction of EMT and MMPs. Furthermore, TGF-ß signaling can induce drug resistance of PCa cells, and can lead to immune evasion via reducing the anti-tumor activity of cytotoxic T cells and stimulating regulatory T cells. Upstream mediators such as microRNAs and lncRNAs, can regulate TGF-ß signaling in PCa. Furthermore, some pharmacological compounds such as thymoquinone and valproic acid can suppress TGF-ß signaling for PCa therapy. TGF-ß over-expression is associated with poor prognosis in PCa patients. Furthermore, TGF-ß up-regulation before prostatectomy is associated with recurrence of PCa. Overall, current review discusses role of TGF-ß signaling in proliferation, metastasis and therapy response of PCa cells and in order to improve knowledge towards its regulation, upstream mediators of TGF-ß such as non-coding RNAs are described. Finally, TGF-ß regulation and its clinical application are discussed.

TRPV2 Promotes Cell Migration and Invasion in Gastric Cancer via the Transforming Growth Factor-ß Signaling Pathway.

BACKGROUND: Transient receptor potential vanilloid 2 (TRPV2) is a highly Ca2+-permeable ion channel that is involved in a number of cellular processes. It is expressed in various human cancers; however, the role of TRPV2 in gastric cancer (GC) remains poorly understood. METHODS: TRPV2 gene expression was knocked down in GC cell lines by small-interfering RNA (siRNA), and the biological roles of TRPV2 in the proliferation, migration, and invasion of GC cells were then investigated. The gene expression profile of GC was elucidated using a microarray analysis. TRPV2 expression in tumor tissue sections was analyzed by immunohistochemistry. RESULTS: The migration and invasion abilities of GC cells were inhibited by the knockdown of TRPV2. Moreover, the microarray assay revealed that TRPV2 was associated with the transforming growth factor (TGF)-ß signaling pathway. Immunohistochemical staining showed that the strong expression of TRPV2 correlated with lymphatic invasion, venous invasion, pathological T (pT), pathological N (pN), and a poor prognosis in GC patients. CONCLUSIONS: TRPV2 appeared to promote tumor migration and invasion via the TGF-ß signaling pathway, and the strong expression of TRPV2 was associated with a worse prognosis in GC patients.

Up-regulation of TGFBI and TGFB2 in the plasma of gestational diabetes mellitus patients and its clinical significance.

BACKGROUND: Gestational diabetes mellitus (GDM) reflects a deficiency in the relative need for insulin during pregnancy, as well as temporary metabolic stress in the placenta and fetus. Our study aimed to research the potential diagnostic value of transforming growth factor-beta-induced protein ig-h3 (TGFBI) and transforming growth factor beta-2 proprotein (TGFB2) for GDM patients. METHODS: Online database Gene Expression Omnibus (GEO) was used to screen for different expressed genes (DEGs) associated with GDM. Meanwhile, KEGG and GO were used to analyze the molecular functions as well as pathways of enriched DEGs. One hundred ten pregnant women diagnosed with GDM and 110 healthy controls were enrolled, of whose placenta and fasting venous blood samples were collected. mRNA expression levels were determined by real-time quantitative polymerase chain reaction (RT-qPCR), and fasting blood glucose (FBG) was measured by the clinical lab of hospital. Furthermore, receiver operating characteristics curve (ROC) analysis was performed to evaluate the sensitivity and specificity of detection indexed in the placenta and plasma of GDM patients. Finally, Pearson and Spearman analysis was used for the correlation analysis. RESULTS: After GEO data analysis, TGFBI and TGFB2 were identified as the most significantly up-regulated genes of GDM. TGFBI and TGFB2 expressions in placenta and plasma samples of GDM patients were in line with bioinformatic analysis. Meanwhile, the area under the curve (AUC) of TGFBI in the placenta and plasma for the diagnosis of GDM were 0.8783 (95% CI, 0.8281 to 0.9284) and 0.7832 (95% CI, 0.7215 to 0.8449) while for TGFB2 were 0.9225 (95% CI, 0.8829 to 0.9621) and 0.8961 (95% CI, 0.8526 to 0.9396). Besides, levels of TGFBI along with TGFB2 in the placenta were positively correlated with that in the plasma of GDM patients. Furthermore, both TGFBI and TGFB2 expressions in the plasma were positively correlated with FBG levels of the GDM patients. CONCLUSIONS: TGFBI and TGFB2 were up-regulated in the placenta and plasma of GDM patients, and TGFBI and TGFB2 in the plasma are potent to be diagnostic markers for the GDM.

Altered Expression of Extracellular Vesicles miRNAs from Primary Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-ß2.

Primary open-angle glaucoma (POAG) is tightly related with extracellular matrix (ECM) remodeling of human trabecular meshwork cells (HTMCs). Transforming growth factor-ß2 (TGF-ß2) can induce ECM remodeling. The aim of the study was to investigate the microRNAs (miRNAs) expression changes of extracellular vesicles (EVs) derived from HTMCs treated with TGF-ß2. EVs were isolated from HTMCs supernatant cultured for 24 h with TGF-ß2. The morphology of EVs pellets was examined by transmission electron microscopy. Nanoparticle tracking analysis used to demonstrate the particle size distribution. Total EVs RNAs were extracted for subsequent miRNA gene chip analysis to investigate differentially expressed miRNAs between the controls and treatment cells. Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict potential target and validate possible functions of the miRNAs. There were 23 miRNAs upregulated and 3 miRNAs downregulated and 469,102, and 94 GO terms involved in biological processes, cellular components, and molecular function for the possible functions of the 26 miRNAs. These findings indicate that TGF-ß2 may alter EVs miRNAs expression to participate in the pathogenesis of POAG. They may provide significant information for potential biomarkers for POAG diagnosis and treatment.

Gastric Acid and Pepsin Work Together in Simulated Gastric Acid Inhalation Leading to Pulmonary Fibrosis in Rats.

BACKGROUND The clinical association between gastroesophageal reflux disease (GERD) and idiopathic pulmonary fibrosis (IPF) has been known for many years, but it is still unclear. The present study investigated the association between experimentally simulated aspiration and pulmonary fibrosis. MATERIAL AND METHODS A total of 120 male Sprague-Dawley rats were randomly divided into a negative control group, a bleomycin group, and 3 simulated aspiration groups. The bleomycin group was administered a one-time intratracheal injection of bleomycin, whereas the 3 simulated aspiration groups were treated either with an intratracheal instillation of gastric fluid combined with pepsin, with pepsin alone, or with hydrochloric acid, all twice a week, and the negative control group was administered normal saline twice a week. Lung tissues were collected to evaluate pathological changes and the mRNA expression levels of connective tissue growth factor (CTGF), type I collagen, and transforming growth factor. RESULTS The results demonstrated that the degree of fibrosis in the early stage was low in each of the 3 simulated aspiration groups, but gradually increased over time. The expression levels of the downstream factor of fibrosis, CTGF, and type I collagen also reflected this trend. CONCLUSIONS The study demonstrates that aspiration of gastric contents can cause pulmonary fibrosis, and mixed aspiration of pepsin and gastric fluid can accelerate this process. This study provides strong evidence in support of a potential association between human GERD and IPF.

Potential regulatory role of circular RNA in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive type of interstitial pneumonia with unknown causes, poor prognosis and no effective therapy available. Circular RNAs (circRNAs), which serve as potential therapeutic targets and diagnostic biomarkers for certain diseases, represent a recent hotspot in the field of RNA research. In the present study, a total of 67 significantly dysregulated circRNAs were identified in the plasma of IPF patients by using a circRNA microarray. Among these circRNAs, 38 were upregulated, whereas 29 were downregulated. Further validation of the results by polymerase chain reaction analysis indicated that Homo sapiens (hsa)_circRNA_100906, hsa_circRNA_102100 and hsa_circRNA_102348 were significantly upregulated, whereas hsa_circRNA_101225, hsa_circRNA_104780 and hsa_circRNA_101242 were downregulated in plasma samples of IPF patients compared with those in samples from healthy controls. The majority of differentially expressed circRNAs were generated from exonic regions. The host genes of the differentially expressed circRNAs were involved in the regulation of the cell cycle, adherens junctions and RNA transport. The competing endogenous RNA (ceRNA) network of the circRNAs/micro(mi)RNAs/mRNAs indicated that circRNA­protected mRNA participated in transforming growth factor­ß1, hypoxia­inducible factor­1, Wnt, Janus kinase, Rho­associated protein kinase, vascular endothelial growth factor, mitogen­activated protein kinase, Hedgehog and nuclear factor κB signalling pathways or functioned as biomarkers for pulmonary fibrosis. Furthermore, luciferase reporter assays confirmed that hsa_circRNA_100906 and hsa_circRNA_102348 directly interact with miR­324­5p and miR­630, respectively, which were downregulated in IPF patients. The present study provided a novel avenue for exploring the underlying molecular mechanisms of IPF disease.

Aicar effect in early neuronal development.

The neurological manifestations of Lesch-Nyhan disease (LND) have been attributed to the effect of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency on nervous system development. An increase has been reported in the levels of 5-aminoimidazole-4-carboxamide-1-ß-D-ribotide (AICAR) and its triphosphate form ZTP in the red blood cells of patients with LND. AICAR accumulation in the brain has been hypothesized as the cause of some of the neurological symptoms of patients with LND. In this study, we examined the effect of AICAR on the differentiation of neurons in the well-established human NTERA-2 cl.D1 (NT2/D1) embryonic carcinoma neurogenesis model. NT2/D1 cells were differentiated along neuroectodermal lineages after exposure to 10-µM retinoic acid (RA), with or without the addition of 25-µM AICAR to the culture medium. The effect of AICAR on RA differentiation were examined through changes in the expression of genes essential to neuronal differentiation, as well as genes from the Wnt/ß-catenin, transforming growth factor beta (TGFß) and sonic hedgehog (SHH) pathways. Results: RA-induced differentiation in the NT2/D1 cells significantly increased the expression of MAP2, NRG1, NRP1, NRP2, NEUROG1 and EN1 genes (genes linked to neural differentiation) compared with undifferentiated NT2/D1 cells. We found that AICAR increased the expression of the SHH gene and the WNT2 and WNT7B genes but did not influence the expression of genes whose overexpression characterize early neurodevelopmental processes. Conclusion: The relevance of the AICAR related changes in the SHH and Wnt/ß-catenin pathway genes expression in the physiopathology of LND warrants further exploration.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-ß1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer.

PURPOSE: Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients' survival with CRC. MATERIALS AND METHODS: The expression of EGFR ligands, including heparin binding epidermal growth factor-like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH. RESULTS: Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands‒/EGFR+ group showed a significant association with the worst disease-free survival (DFS; p=0.018) and overall survival (OS; p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis. CONCLUSION: Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

Genomic diagnostics leading to the identification of a TFG-ROS1 fusion in a child with possible atypical meningioma.

Meningiomas are rare in children. They are highly complex, harboring unique clinical and pathological characteristics, and many occur in patients with neurofibromatosis type 2. Hereby, we present a case of a two-year-old boy presented with a diagnostically challenging intraventricular tumor. It was incompletely resected 6 times over 14 months but kept progressing and was ultimately deemed unresectable. Histologically, the tumor was initially classified as schwannoma, but extensive international review concluded it was most likely an atypical meningioma, WHO grade II. Comprehensive genomic profiling revealed a TFG-ROS1 fusion, suggesting that ROS1-signaling pathway alterations were driving the tumor growth. In light of this new information, the possibility of a diagnosis of inflammatory myofibroblastic tumor was considered; however the histopathological results were not conclusive. This specific molecular finding allowed the potential use of precision medicine and the patient was enrolled in the AcSé phase 2 trial with crizotinib (NCT02034981), leading to a prolonged partial tumor response which is persisting since 14 months. This case highlights the value of precision cancer medicine in children.