Use of RNA­sequencing to detect abnormal transcription of the collagen α­2 (VI) chain gene that can lead to Bethlem myopathy.

Bethlem myopathy (BM) is an autosomal dominant or autosomal recessive disorder and is usually associated with mutations in the collagen VI genes. In the present study, the pathogenicity of a novel splice­site mutation was explored using RNA­sequencing in a family with suspected BM, and a myopathy panel was performed in the proband. The genetic status of all family members was confirmed using Sanger sequencing. Clinical data and magnetic resonance imaging (MRI) features were also documented. In silico analysis was performed to predict the effects of the splice mutation. RNA­sequencing and reverse transcription (RT)­PCR were used to assess aberrant splicing. Immunocytochemistry was conducted to measure collagen VI protein levels within the gastrocnemius and in cultured skin fibroblasts. The results revealed that three patients in the family shared a similar classic BM presentation. MRI revealed distinct patterns of fatty infiltration in the lower extremities. A novel splicing mutation c.736­1G>C in the collagen α­2 (VI) chain (COL6A2) gene was found in all three patients. In silico analysis predicted that the mutation would destroy the normal splice acceptor site. RNA­sequencing detected two abnormal splicing variants adjacent to the mutation site, and RT­PCR confirmed the RNA­sequencing findings. Furthermore, a defect in the collagen protein within cultured fibroblasts was detected using immunocytochemistry. The mutation c.736­1G>C in the COL6A2 gene caused aberrant splicing and led to premature termination of protein translation. In conclusion, these findings may improve our knowledge of mutations of the COL6A2 gene associated with BM and demonstrated that RNA­sequencing can be a powerful tool for finding the underlying mechanism of a disease­causing mutations at a splice site.

COL3A1, COL6A3, and SERPINH1 Are Related to Glucocorticoid-Induced Osteoporosis Occurrence According to Integrated Bioinformatics Analysis.

BACKGROUND Glucocorticoid-induced osteoporosis (GIOP) represents the most frequently seen type of secondary osteoporosis, a systemic skeleton disorder. Numerous factors are associated with GIOP occurrence, but there are no specific diagnostic and therapeutic biomarkers for GIOP so far. MATERIAL AND METHODS In this work, gene modules related to GIOP were screened through weighted gene coexpression network analysis. Moreover, protein-protein interaction (PPI) networks and gene set enrichment analysis (GSEA) were carried out for hub genes. In addition, microarray GSE30159 dataset was used as a training set to analyze gene expression within bone biopsy samples from patients with endogenous Cushing's syndrome with GIOP and from normal controls. GSE129228 was used as the test set for investigating the hub gene involvement within GIOP. RESULTS According to our results, the turquoise module showed clinical significance, and 10 genes (COL3A1, POSTN, COL6A3, COL14A1, SERPINH1, ASPN, OGN, THY1, NID2, and TNMD) were discovered to be the "real" hub genes within coexpression as well as PPI networks. GSEA showed that the interaction of extracellular matrix receptors together with the focal adhesion pathway had significant enrichment within samples with high COL3A1 and COL6A3 expression. After the results from both test and training sets were overlapped, SERPINH1 was also significantly altered between GIOP and normal control samples. CONCLUSIONS COL3A1, COL6A3, and SERPINH1 were identified to be the candidate biomarkers for GIOP.

Collagen Stiffness and Architecture Regulate Fibrotic Gene Expression in Engineered Adipose Tissue.

Adipose tissue (AT) has a dynamic extracellular matrix (ECM) surrounding adipocytes that allows for remodeling during metabolic fluctuations. During the progression of obesity, AT has increased ECM deposition, stiffening, and remodeling, resulting in a pro-fibrotic dysfunctional state. Here, the incorporation of ethylene glycol-bis-succinic acid N-hydroxysuccinimide ester (PEGDS) allows for control over 3D collagen hydrogel stiffness and architecture to investigate its influence on adipocyte metabolic and fibrotic function. Upon stiffening and altering ECM architecture, adipocytes did not alter their expression of key adipokines, leptin, and adiponectin. However, they do increase actin cytoskeletal fiber formation, pro-fibrotic gene expression, ECM deposition, and remodeling within a stiffer, 3D collagen hydrogel. For example, COL6A3 gene expression is upregulated approximately twofold, resulting in increased deposition of pericellular collagen VI alpha 3 surrounding adipocytes. Furthermore, inhibition of actin contractility results in a reversal of pro-fibrotic gene expression and ECM deposition, indicating that adipocytes are mediating mechanical cues through actin cytoskeletal networks. This study demonstrates that ECM stiffness and architecture plays a critical regulatory role in adipocyte fibrotic function and contributes to the overall pro-fibrotic dysfunctional state of AT during the progression of obesity and AT fibrosis.

A new single nucleotide polymorphism affects the predisposition to thoracic ossification of the posterior longitudinal ligament.

BACKGROUND: Thoracic ossification of the posterior longitudinal ligament (T-OPLL) is one of the common factors that cause thoracic spinal stenosis, which results in intractable myelopathy and radiculopathy. Our previous study first reported rs201153092A site mutation in the collagen 6A1 (COL6A1) gene as a potentially pathogenic locus for T-OPLL. We aimed to determine whether the rs201153092A site mutation causes abnormal expression of the COL6A1 in Han Chinese patients with T-OPLL and whether this locus is also associated with cervical-OPLL. METHODS: Peripheral blood was collected from a total of 60 patients with T-OPLL disease (30 patients carrying the rs201153092A site mutation in COL6A1 and 30 wild-type patients) and 400 northern Chinese individuals (200 cervical-OPLL patients and 200 control subjects) using the Sequenom system. The expression of COL6A1 was analyzed by enzyme-linked immunosorbent assay, reverse transcription-quantitative polymerase chain reaction, and Western blotting. RESULTS: rs201153092A mutation resulted in markedly increased COL6A1 gene expression levels in peripheral blood samples. The allele frequency and genotype frequency results showed that this locus is no difference between cervical-OPLL patients and controls. CONCLUSIONS: The rs201153092A site mutation of COL6A1 can significantly increase the expression of COL6A1. The COL6A1 gene rs201153092A site polymorphism is a potential pathogenic mutation in T-OPLL disease, which may be only associated with the occurrence of T-OPLL.

Fra-2-expressing macrophages promote lung fibrosis in mice.

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with Type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections and Fra-2 transgenic mice (Fra-2tg) exhibit spontaneous lung fibrosis. Here we show that Bleomycin-induced lung fibrosis is attenuated upon myeloid-inactivation of Fra-2 and aggravated in Fra-2tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is up-regulated in three lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2-dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine pro-fibrotic activity of macrophages. Importantly, ColVI knock-out mice (KO) and ColVI-KO bone marrow chimeras are protected from Bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2tg mice and in the Bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and co-localize in lung macrophages. Therefore, the Fra-2/ColVI pro-fibrotic axis is a promising biomarker and therapeutic target for lung fibrosis, and possibly other fibrotic diseases.

Collagen Type VI Alpha 3 Chain Promotes Epithelial-Mesenchymal Transition in Bladder Cancer Cells via Transforming Growth Factor ß (TGF-ß)/Smad Pathway.

BACKGROUND Collagen type VI alpha 3 chain (COL6A3) has been proven to be a biomarker in the occurrence and development of bladder cancer, which is the most common malignant tumor in the urinary system. This study aimed to explore the effect and molecular mechanism of COL6A3 on EMT in vitro induced by TGF-ß/Smad in bladder carcinoma. MATERIAL AND METHODS There were 42 patients included in the Kaplan-Meier survival analysis. A cell counting kit-8 (CCK-8) assay and an angiogenesis assay were used to measure cell proliferation and tube formation, respectively. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) were conducted for the proteins and mRNAs expression. RESULTS COL6A3 was highly expressed in tissues and cells of bladder cancer. COL6A3 silencing could inhibit the cell proliferation and angiopoiesis. In addition, COL6A3 silencing obviously suppressed the levels of matrix metalloproteinase-2 (MMP2), Matrix metalloproteinase-9 (MMP9), and vimentin. On the contrary, the levels of epithelium-specific cell-cell adhesion molecule (E-cadherin) and tumor inhibitor of metalloproteinase-1 (TIMP-1) were significantly increased. Furthermore, we found that COL6A3 silencing reduced the activity of p-Smad2, p-Smad3, and transforming growth factor ß (TGF-ß). CONCLUSIONS COL6A3 could influence the viability and angiogenesis of bladder cancer cells. COL6A3 may have a certain relationship with the TGF-ß/Smad-induced EMT process.

FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.

BACKGROUND: In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. METHODS: Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-ß1 (TGF-ß1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. RESULTS: FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. CONCLUSIONS: These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

Silencing of COL1A2, COL6A3, and THBS2 inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3k-Akt signaling pathway.

This study explores the effect of COL1A2, COL6A3, and THBS2 gene silencing on proliferation, migration, invasion, and apoptosis of gastric cancer cells through the PI3K-Akt signaling pathway. The gastric cancer microarray expression data (GSE19826, GSE79973, and GSE65801) was analyzed. Gastric cancer tissues and corresponding adjacent normal tissues were extracted from patients. Positive expression rate of PI3K, Akt, and p-Akt was measured with immunohistochemistry. Two cell lines, BGC-823 and SGC-7901, were transfected and cells were grouped into blank, negative control, COL1A2-shRNA, COL6A3-shRNA, and THBS2-shRNA groups. Expressions of COL1A2, COL6A3, and THBS2 in gastric cancer cells transfected with corresponding silencing sequences were evaluated by RT-qPCR and Western blot. MTT assay, Transwell, and cell scratch tests were conducted to evaluate cell proliferation, invasion, and migration capacity, respectively. Flow cytometry was used to evaluate cell cycle distribution and apoptosis. The positive expression of PI3K, Akt, and p-Akt was higher in gastric cancer tissues compared with adjacent normal tissues, and the mRNA expression of COL1A2, COL6A3, and THBS2 was increased in gastric cancer tissues. Akt, p-Akt, and PI3K expression drastically decreased in cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences. Cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences exhibited promoted apoptosis but inhibited proliferation, migration, and invasion. This study demonstrates that COL1A2, COL6A3, and THBS2 gene silencing inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3K-Akt signaling pathway.

Hirschsprung's disease, Down syndrome, and missing heritability: too much collagen slows migration.

Hirschsprung's disease (HSCR) causes functional intestinal obstruction due to the absence of the enteric nervous system (ENS) in the distal bowel and is usually diagnosed shortly after birth or during childhood. While several genetic and nongenetic factors have been linked to HSCR, the underlying mechanisms that prevent ENS precursors from colonizing distal bowel during fetal development are not completely understood in many affected children. In this issue of the JCI, Soret and colleagues identify a new mechanism that causes HSCR-like disease in mice and involves deposition of excess collagen VI in the intestine by migrating ENS precursors as they colonize fetal bowel. Remarkably, their findings may explain some of the so-called missing heritability of HSCR and suggest a mechanism for increased HSCR incidence in children with Down syndrome (trisomy 21).

A collagen VI-dependent pathogenic mechanism for Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR--named Holstein--that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.

Immunohistochemical Evaluation of Idiopathic Epiretinal Membranes and In Vitro Studies on the Effect of TGF-ß on Müller Cells.

PURPOSE: The purpose of this study was to investigate the presence of type VI collagen and glial cells in idiopathic epiretinal membrane (iERM) and the role of TGF-ß in the expression of collagens and α-smooth muscle actin (α-SMA) in retinal Müller cells. METHODS: Idiopathic ERM samples from vitrectomy were analyzed for glial acidic fibrillary protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), α-SMA, and type VI collagen using flat-mount immunohistochemistry. To study intracellular collagen expression in relation to cellular phenotype, spontaneously immortalized human Müller cells (MIO-M1) were treated with TGF-ß1 for 48 hours, and the expression of α-SMA and intracellular type I, II, IV, and VI collagens was studied by using immunocytology. Findings in Müller cells were compared with those in fetal lung fibroblasts and newborn skin fibroblasts. RESULTS: A colocalization of GFAP/CRALBP and GFAP/α-SMA was found in iERM, indicating a dynamic process of activation of retinal Müller cells in vivo. Transforming growth factor-ß1 induced up-regulation of α-SMA stress fibers in retinal Müller cells and both types of fibroblasts in vitro. The intracellular staining intensity of type I, II, and VI collagens was decreased in retinal Müller cells containing α-SMA stress fibers, whereas the intracellular staining intensity of type I and VI collagens in both types of fibroblasts was not affected. CONCLUSIONS: Type VI collagen and activated retinal Müller cells are present in iERM. Transforming growth factor-ß1 induces an up-regulation of α-SMA stress fibers in retinal Müller cells and fibroblasts and appears to have a cell-specific effect on intracellular collagen expression.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

Collagen VI and hyaluronan: the common role in breast cancer.

Collagen VI and hyaluronan are widely distributed extracellular matrix macromolecules that play a crucial role in tissue development and are highly expressed in cancers. Both hyaluronan and collagen VI are upregulated in breast cancer, generating a microenvironment that promotes tumour progression and metastasis. A growing number of studies show that these two molecules are involved in inflammation and angiogenesis by recruiting macrophages and endothelial cells, respectively. Additionally, collagen VI induces epithelial-mesenchymal transition that is correlated to increased synthesis of hyaluronan in mammary cells. Hyaluronan has also a specific role in cellular functions that depends mainly on the size of the polymer, whereas the effect of collagen VI in tumour progression may be the result of the intact molecule or the C5 peptide of α3(VI) chain, known as endotrophin. Collectively, these findings strongly support the parallel role of these molecules in tumour progression and suggest that they may be used as prognostic factors for the breast cancer treatment.

Transcriptome analysis reveals differential splicing events in IPF lung tissue.

Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of the transcriptome through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. We sequenced messenger RNA from 8 IPF lung samples and 7 healthy controls on an Illumina HiSeq 2000, and found evidence for substantial differential gene expression and differential splicing. 873 genes were differentially expressed in IPF (FDR<5%), and 440 unique genes had significant differential splicing events in at least one exonic region (FDR<5%). We used qPCR to validate the differential exon usage in the second and third most significant exonic regions, in the genes COL6A3 (RNA-Seq adjusted pval = 7.18e-10) and POSTN (RNA-Seq adjusted pval = 2.06e-09), which encode the extracellular matrix proteins collagen alpha-3(VI) and periostin. The increased gene-level expression of periostin has been associated with IPF and its clinical progression, but its differential splicing has not been studied in the context of this disease. Our results suggest that alternative splicing of these and other genes may be involved in the pathogenesis of IPF. We have developed an interactive web application which allows users to explore the results of our RNA-Seq experiment, as well as those of two previously published microarray experiments, and we hope that this will serve as a resource for future investigations of gene regulation in IPF.

G-CSF prevents progression of diabetic nephropathy in rat.

BACKGROUND: The protective effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated in a variety of renal disease models. However, the influence of G-CSF on diabetic nephropathy (DN) remains to be examined. In this study, we investigated the effect of G-CSF on DN and its possible mechanisms in a rat model. METHODS: Otsuka Long-Evans Tokushima Fatty (OLETF) rats with early DN were administered G-CSF or saline intraperitoneally. Urine albumin creatinine ratio (UACR), creatinine clearance, mesangial matrix expansion, glomerular basement membrane (GBM) thickness, and podocyte foot process width (FPW) were measured. The levels of interleukin (IL)-1ß, transforming growth factor (TGF)-ß1, and type IV collagen genes expression in kidney tissue were also evaluated. To elucidate the mechanisms underlying G-CSF effects, we also assessed the expression of G-CSF receptor (G-CSFR) in glomeruli as well as mobilization of bone marrow (BM) cells to glomeruli using sex-mismatched BM transplantation. RESULTS: After four weeks of treatment, UACR was lower in the G-CSF treatment group than in the saline group (p<0.05), as were mesangial matrix expansion, GBM thickness, and FPW (p<0.05). In addition, the expression of TGF-ß1 and type IV collagen and IL-1ß levels was lower in the G-CSF treatment group (p<0.05). G-CSFR was not present in glomerular cells, and G-CSF treatment increased the number of BM-derived cells in glomeruli (p<0.05). CONCLUSIONS: G-CSF can prevent the progression of DN in OLETF rats and its effects may be due to mobilization of BM cells rather than being a direct effect.

Adipose tissue collagen and inflammation in nonobese Asian Indian men.

OBJECTIVE: Obesity is thought to be the driving force for activation of adipose tissue (AT) collagen production and inflammation as well as systemic insulin resistance. The objective of this study was to determine whether these AT abnormalities can be found independent of obesity in the presence of systemic insulin resistance. RESEARCH DESIGN AND METHODS: Thirty-eight normoglycemic men (14 Asian Indians and 24 white) were enrolled in the study and matched for age, body mass index, and total body fat. Subjects underwent anthropometric measurement, total body fat determination by underwater weighing, euglycemic-hyperinsulinemic clamps, and abdominal sc AT biopsy for mRNA extraction and gene expression determination. Fasting blood was collected for adipokine measurements. RESULTS: Both groups were matched for age, body mass index, and percentage of total body fat. Subcutaneous abdominal AT mRNA expression was significantly higher for Col6a3 as well as genes associated with inflammation, CD68, MAC1, and MCP1 in Asian Indians compared with whites. Asian Indian men had significantly lower rates of glucose disposal and lower plasma adiponectin concentration. Plasma high-sensitivity C-reactive protein levels showed a trend towards higher levels in Asian Indian men. CONCLUSIONS: Increased col6a3 and macrophage infiltration in AT along with increased systemic insulin resistance is present independent of body fat content in young Asian Indian men, thus suggesting that AT dysfunction associates with systemic insulin resistance regardless of AT mass.

[Morphological features of placenta and expression of collagen-6, MMP-1 and TIMP-1 in placental membranes of women with premature rupture of membranes].

Morphological and immunohistochemical investigation of the women's afterbirth with premature rupture of membranes (PROM) and well-time ruptures of placental membranes has been carried out. Inflammatory infiltration of membranes and placental deficiency were higher at patients with PROM. The absence of inflammatory changes in placental tissue during growth of anhydrous gap was demonstrated. The optical density and square of MMP-1 expression were higher and TIMP-1 was lower in membranes of PROM-group than in the control one. No significant changes were found in expression of collagen-6.

Identification of the up-regulation of TP-alpha, collagen alpha-1(VI) chain, and S100A9 in esophageal squamous cell carcinoma by a proteomic method.

Esophageal squamous cell carcinoma (ESCC) is one of the most common primary malignant tumor of digestive tract. However, the early diagnosis and molecular mechanisms that underlie tumor formation and progression have been progressed less. To identify new biomarkers for ESCC, we performed a comparative proteomic research. Isobaric tags for relative and absolute quantitation-based proteomic method was used to screen biomarkers between ESCC and normal. 802 non-redundant proteins were identified, 39 of which were differentially expressed with 1.5-fold difference (29 up-regulated and 10 down-regulated). Through Swiss-Prot and GO database, the location and function of differential proteins were analyzed, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc. Among the differentially expressed proteins, TP-alpha, collagen alpha-1(VI) chain and S100A9 were verified to be upregulated in 77.19%, 75.44% and 59.65% of ESCC by immunohistochemistry and western-blot. Diagnostic value of these three proteins was validated. These results provide new insights into ESCC biology and potential diagnostic and therapeutic biomarkers, which suggest that TP-alpha, collagen alpha-1(VI) chain and S100A9 are potential biomarkers of ESCC, and may play an important role in tumorigenesis and development of ESCC.

Integrin α3 blockade enhances microtopographical down-regulation of α-smooth muscle actin: role of microtopography in ECM regulation.

Development of functional engineered matrices for regenerative therapies can benefit from an understanding of how physical cues at the microscale affect cell behavior. In this work, we use microfabricated systems to study how stiffness and microscale topographical cues in the form of "micropegs" affect extracellular matrix synthesis. Previous work from our lab has shown that microtopographical cues in 2D and 3D systems decrease cellular proliferation and regulate matrix synthesis. In this work, the combined role of stiffness and topography on ECM synthesis is investigated in a 2D micropeg system. These studies show that fibroblasts cultured on polydimethylsiloxane (PDMS) substrates with micropegs have reduced expression of collagen type I (Col I) and collagen type VI (Col VI) compared to fibroblasts cultured on flat substrates. In addition, cells on micropegged substrates exhibit down-regulation of other important regulators of ECM synthesis such as α-smooth muscle actin (α-SMA), and integrin α3 (Int α3). Interestingly, this effect is dependent on the contractility and adhesion of the cells. When cultured in the presence of RhoA kinase (ROCK) and myosin light chain kinase (MLCK) inhibitors, no significant differences in the expression of collagen, α-SMA, Int α3, and TGFB1 are observed. Additionally, disruptions in cell adhesion prevent microtopographical regulation of ECM synthesis. When using an antibody to block the extracellular domain of Int α3, no differences in the expression of collagen are observed and blocking Int α3 results in enhanced down-regulation of α-SMA on the stiffer micropegged substrates. These findings demonstrate that regulation of extracellular matrix production by cells on a synthetic substrate can be guided via physical cues at the microscale, and add to the body of knowledge on the role of integrin-mediated mechanotransduction.