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Colorectal cancer (CRC) is ranked as one of the three most common cancers in the world. This research aims at exploring the molecular mechanisms of CRC. We used R language to analyze gene expression data from the Gene Expression Omnibus (GEO) database. By using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database, the function and pathway enrichment of differentially expressed genes were analyzed. The Protein-protein interaction (PPI) network was constructed by STRING and Cytoscape. A total of 205 differentially expressed genes (DEGs) were screened out. These differentially expressed genes mainly enriched in the terms of regulation of leukocyte chemo taxis, chemokine receptor binding and chemokine-mediated signaling pathway. The PPI network of differentially expressed genes was constructed. Seven hub genes (CCL2, TPM1, DES, CXCL12, MYLK, MYL9 and TPM2) were identified. We conclude that these 7 genes may be involved in the occurrence of CRC.

AimsTumour recurrence is inevitable in glioblastoma (GBM) and mostly noted in the peritumoural brain zone (PT). In our previous microarray-based study, we identified Myosin Light Chain 9 (MYL9) as a highly expressed gene in the PT of GBM. Therefore, we aimed to study the expression pattern and clinical significance of MYL9 in GBM.MethodsPatient samples included three retrospective cohorts: 25 GBM cases with differential biopsies of tumour core and PT, 62 retrospective cases of newly diagnosed GBM with survival information and 20 paired samples (newly diagnosed and recurrent GBM). All tumour tissues, archived as formalin fixed paraffin embedded blocks were retrieved and immunohistochemistry for MYL9 and IDH1 R132H was performed. MYL9 expression was correlated with patient prognosis in our cohort and in The Cancer Genome Atlas (TCGA) and Rembrandt cohorts. It was further evaluated in the 20 paired samples of GBM.ResultsMYL9 showed a cytoplasmic membranous staining of tumour cells. The staining pattern was variable and patchy within tumours. Higher MYL9 expression was associated with poor overall and progression-free survival in our and in TCGA and Rembrandt cohorts. The expression of MYL9 was higher in IDH1 R132H immunonegative cases.ConclusionsWe show MYL9 as a novel biomarker, variably expressed in GBM. The association of high MYL9 expression with poor prognosis in newly diagnosed GBM patients and increased expression in recurrent GBM is indicative of its role in conferring tumour aggressiveness.

This study was designed to identify the potential key protein interaction networks, genes, and correlated pathways in early-onset colorectal cancer (CRC) via bioinformatics methods. We selected microarray data GSE4107 consisting 12 patient’s colonic mucosa and 10 healthy control mucosa; initially, the GSE4107 were downloaded and analyzed using limma package to identify differentially expressed genes (DEGs). A total of 131 DEGs consisting of 108 upregulated genes and 23 downregulated genes of patients in early-onset CRC were selected by the criteria of adjusted P values

Embryonic development of skeletal muscle is a complex process that is important to the growth of skeletal muscle after birth. However, the mechanisms by which skeletal muscle growth and development in embryonic phase remain unclear. We have previously produced myostatin-knockout (MKO) Meishan pigs with double-muscle (DM) phenotype via zinc finger nucleases (ZFN) technology. To further investigate the molecular mechanisms involved in skeletal muscle growth and development, in this study, we performed an integrated analysis of transcriptome and proteome in longissimus dorsi muscle from myostatin wild type (MWT) and MKO pigs on 65 days post coitus. Results showed that, compared with MWT group, there were 438 differentially expressed genes (DEGs) and 66 differentially expressed proteins (DEPs) in MKO group. These DEGs and DEPs were mainly enriched in signaling pathways that are involved in skeletal muscle growth and development, glucose metabolism and apoptosis. Furthermore, we identified two key genes, Troponin T 1 (TNNT1) and Myosin regulatory light chain 9 (MYL9), which showed significant changes in both mRNA and protein levels with the similar changing trends in MKO group. It is thus speculated that TNNT1 and MYL9 may play an important role in skeletal muscle growth and development. Significance Our study analyzed some important regulatory genes and proteins during skeletal muscle growth and development, our results provided (1) a new insight to further understanding of the molecular mechanisms by which growth and development are regulated in porcine skeletal muscle, and (2) some possible molecular makers for improvement of meat quality in the animal husbandry and diagnosis of human muscle diseases in medicine.

Ingested food is received, mixed, and ground into chyme by distinct gastric motility patterns. Diabetes impairs gastric muscle function, but the mechanisms underlying diabetes-induced gastric muscle dysfunction are unknown. Here, we compared the expression and phosphorylation of Ca2+sensitization and contractile proteins in human gastric muscles from obese nondiabetic and diabetic patients. We also compared the spontaneous phasic contractions and the contractile responses evoked by electrical field stimulation of cholinergic motor neurons. Fundus and antrum muscles were obtained from sleeve gastrectomies and were used in in vitro myobath contractile studies and for capillary electrophoresis and immunodetection ofγ-actin, CPI-17, pT38-CPI-17, MYPT1, pT853-MYPT1, pT696-MYPT1, myosin light chain (MYL9), pS19-MYL9, myosin light chain kinase (MYLK), protein phosphatase-1δ(PP1δ), and Rho-associated kinase (ROCK2). In diabetic fundus muscles, MYLK, ROCK2, and PP1δexpression was unchanged; MYPT1 and CPI-17 expression was decreased; and the pT853/MYPT1 and pT38/CPI-17 ratios, but not the pT696/MYPT1 ratio, were increased. Although MYL9 expression was increased, the pS19/MYL9 ratio was unchanged in diabetic fundus muscles. In diabetic antrum muscles, MYLK and MYL9 expression was unchanged, but ROCK2, CPI-17, and PP1δexpression was decreased. The pT38/CPI-17 ratio was unchanged, while the pS19/MYL9, pT853/MYPT1, and pT696/MYPT1 ratios were decreased, consistent with the reduced ROCK2 expression. The frequencies of spontaneous phasic contractions from nondiabetic and diabetic gastric fundus and antrum muscles did not significantly differ from each other, regardless of age, sex, or diabetic status. The fold increases in the contractions of diabetic fundus and antrum muscles in response to increased frequencies of electrical field stimulation were significantly lower compared to nondiabetic fundus and antrum muscles. The altered contractile responses and the protein expression and phosphorylation in gastric muscles of obese patients with diabetes illustrate the importance of understanding how smooth muscle Ca2+sensitization mechanisms contribute to gastric motility.

Mechanical strain associated with the expanding uterus correlates with increased preterm birth rates. Mechanical signals result in a cascading network of protein phosphorylation events. These signals direct cellular activities and may lead to changes in contractile phenotype and calcium signaling. In this study, the complete phospho-proteome of uterine smooth muscle cells subjected to mechanical strain for 5 min was compared to un-strained controls. Statistically significant, differential phosphorylation events were annotated by Ingenuity Pathway Analysis to elucidate mechanically induced phosphorylation networks. Mechanical strain leads to the direct activation of ERK1/2, HSPB1, and MYL9, in addition to phosphorylation of PAK2, vimentin, DOCK1, PPP1R12A, and PTPN11 at previously unannotated sites. These results suggest a novel network reaction to mechanical strain and reveal proteins that participate in the activation of contractile mechanisms leading to preterm labor.

Summary CD69 has been known as an early activation marker of lymphocytes; whereas, recent studies demonstrate that CD69 also has critical functions in immune responses. Early studies using human samples revealed the involvement of CD69 in various inflammatory diseases including asthma. Moreover, murine disease models using Cd69−/− mice and/or anti-CD69 antibody (Ab) treatment have revealed crucial roles for CD69 in inflammatory responses. However, it had not been clear how the CD69 molecule contributes to the pathogenesis of inflammatory diseases. We recently elucidated a novel mechanism, in which the interaction between CD69 and its ligands, myosin light chain 9, 12a and 12b (Myl9/12) play a critical role in the recruitment of activated T cells into the inflammatory lung. In this review, we first summarize CD69 function based on its structure and then introduce the evidence for the involvement of CD69 in human diseases and murine disease models. Then, we will describe how we discovered CD69 ligands, Myl9 and Myl12, and how the CD69-Myl9 system regulates airway inflammation. Finally, we will discuss possible therapeutic usages of the blocking Ab to the CD69-Myl9 system.

Gene expression microarrays are widely used to investigate molecular targets in cancers, including lung cancer. In this study, we analyzed online non-small cell lung cancer (NSCLC) microarray databases, to screen the key genes and pathways related to NSCLC by bioinformatics analyses. And then, the expression levels of two selected genes in the down-regulated co-pathways, myosin light chain kinase (MYLK) and myosin regulatory light chain 9 (MYL9), were determined in tumor, paired paraneoplastic, and normal lung tissues. First, gene set enrichment analysis and meta-analysis were conducted to identify key genes and pathways that contribute to NSCLC carcinogenesis. Second, using the total RNA and protein extracted from lung cancer tissues (n = 240), adjacent non-cancer tissues (n = 240), and normal lung tissues (n = 300), we examined the MYLK and MYL9 expression levels by quantitative real-time PCR and Western blot. Finally, we explored the correlations between mRNA and protein expressions of these two genes and the clinicopathological parameters of NSCLC. Fifteen up-regulated and nine down-regulated co-pathways were observed. A number of differentially expressed genes (CALM1, THBS1, CSF3, BMP2, IL6ST, MYLK, ROCK2, IL3RA, MYL9, PPP2CA, CSF2RB, CNAQ, GRIA2, IL10RA, IL10RB, IL11RA, LIFR, PLCB4, and RAC3) were identified (P 0.01) in the down-regulated co-pathways. The expression levels of MYLK and MYL9, which act downstream of the vascular smooth muscle contraction signal pathway and focal adhesion pathway, were significantly lower in cancer tissue than those in the paraneoplastic and normal tissues (P 0.05). Moreover, the expression levels of these two genes in stages III and IV NSCLC were significantly increased, when compared to stages I and II, and expressions levels in NSCLC with lymphatic metastasis were higher than that without lymphatic metastasis (P 0.05). Additionally, significant lower expression levels of the two genes were found in smokers than in nonsmokers (P 0.05). In contrast, gender, differentiated degrees, and pathohistological type appeared to have no impact on these gene expressions (P 0.05). These findings suggested that low MYLK and MYL9 expressions might be associated with the development of NSCLC. These genes may be also relevant to NSCLC metastasis. Future investigations with large sample sizes needed to verify these findings.

Many lines of evidence have suggested that estrogen plays important roles not only in the initiation and proliferation of breast cancer, but also in cancer metastasis. However, the mechanistic basis of the latter events is poorly understood. In addition, recent studies have suggested that myocardin-related transcription factor A (MRTF-A) might be related to cancer metastasis. However, as reports are contradictory, certain of its roles still remain confusing. In the present study, we showed that excessive 17β-estradiol could promote the migration of MCF-7 breast cancer cells and up-regulate the expression of MRTF-A, myosin regulatory light chain 9 (MYL9), and cysteine-rich angiogenic inducer 61 (CYR61). Overexpression of MRTF-A significantly promoted the migration of MCF-7 cells through its transactivation effects on MYL9 and CYR61 genes, while RNA interference-mediated knockdown of MRTF-A strongly inhibited transcription and expression of the target genes and reduced the migration ability of MCF-7 cells. These results provided novel evidence supporting the metastasis-promoting functions of MRTF-A, and implied that MRTF-A might be a switch for the estrogen pathway to change its proliferation-promoting roles into migration-stimulating roles in breast cancer.

Cardiovascular diseases, such as ischemic heart disease and stroke, represent the number one cause of death worldwide.1 In most cases, atherosclerosis and hypertension constitute the underlying causes for cardiovascular disease because it leads to arterial occlusion and impaired cardiac function, respectively. Therapeutic compensation of atherosclerotic artery occlusion by bypass grafting, angioplasty, or stenting bears the risk of intimal hyperplasia and subsequent restenosis.2 In general, a prerequisite for any pathological and physiological vascular remodeling process is the phenotypic switch of vascular smooth muscle cells (VSMCs).3 Even in adult blood vessels, VSMCs retain a remarkable plasticity that is essential for any changes in the vessel wall architecture. Contractile VSMCs representing the majority of VSMCs in healthy vessels guarantee maintenance of vascular tone and thereby the systemic blood pressure and blood flow by controlling the blood vessel diameter. As outlined in the Figure, a plethora of humoral factors, such as platelet-derived growth factor-BB and biomechanical stimuli, especially chronic changes in blood flow and wall stress, is capable to trigger the phenotypic switch of VSMCs from a quiescent, contractile differentiated state to a synthetic, proliferative, and dedifferentiated state.3,4 Figure. The effect of microRNA (miR)-633 on vascular homeostasis and remodeling. Major stimuli, transcription factors, and miRs that are involved in the differentiation and phenotypic switch of vascular smooth muscle cells (VSMCs) are depicted. miR-143/miR-145 promotes the contractile state by inhibiting suppressors of myocardin/serum response factor (SRF) activity (eg, ETS domain-containing protein-1 [Elk-1] and Kruppel-like factor [KLF] 4/5). Interestingly, myocardin activity seems to be regulated by an intrinsic loop because it stabilizes the expression of miR-145, whereas miR-143 seems to inhibit myocardin expression. miR-633 is a novel player with a dual effect on the VSMC phenotype not only by attenuating proliferation, migration, and proteolytic activity, but also by limiting their …

Objective Myosin light chain 9 (MYL9) is necessary for cytoskeletal dynamics and experimental metastasis, but its expression in esophageal squamous cell carcinoma (ESCC) has not been addressed. We investigated the expression pattern and clinical significance of MYL9 in patients with ESCC. Methods We examined MYL9 expression using quantitative real-time PCR and western blotting in NE1 immortalized esophageal epithelial cells, ESCC cell lines, and paired ESCC tissues. MYL9 protein in 136 primary ESCC tissues and other types of solid tumor was detected using immunohistochemistry. The association between MYL9 expression and clinical parameters and survival was evaluated by statistical analysis. Results MYL9 was significantly upregulated in the ESCC cell lines as compared with NE1 cells. In the paired ESCC samples, MYL9 mRNA and protein expression was not significantly different between lesion tissues and the matched adjacent noncancerous tissues. In ESCC tissue, both intratumoral and peritumoral stroma were positive for MYL9. In the 136 ESCC samples, high MYL9 expression in the tumor cells significantly correlated with histological differentiation (p = 0.028), recurrence (p = 0.01), and vital status (p < 0.01). Patients with high MYL9 expression in the tumor cells had poorer overall survival (OS) and recurrence-free survival. Multivariate analysis revealed that high MYL9 expression in tumor cells was an independent and significant risk factor affecting OS after curative treatment (hazard ratio = 2.254, 95% confidence interval = 1.347–3.771, p = 0.002). Conclusions MYL9 expression might be a promising prognostic marker and therapeutic target in ESCC.

Cell division is strictly regulated by a diversity of proteins and lipids to ensure proper duplication and segregation of genetic material and organelles. Here we report a novel role of the putative lipid transporter ACAT-related protein required for viability 1 (Arv1) during telophase. We observed that the subcellular localization of Arv1 changes according to cell cycle progression and that Arv1 is recruited to the cleavage furrow in early telophase by epithelial protein lost in neoplasm (EPLIN). At the cleavage furrow Arv1 recruits myosin heavy chain 9 (MYH9) and myosin light chain 9 (MYL9) by interacting with IQ-motif-containing GTPase-activating protein (IQGAP1). Consequently the lack of Arv1 delayed telophase-progression, and a strongly increased incidence of furrow regression and formation of multinuclear cells was observed both in human cells in culture and in follicle epithelial cells of egg chambers of Drosophila melanogaster in vivo. Interestingly, the cholesterol-status at the cleavage furrow did not affect the recruitment of either IQGAP1, MYH9 or MYL. These results identify a novel function for Arv1 in regulation of cell division through promotion of the contractile actomyosin ring, which is independent of its lipid transporter activity.

Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4(+) T helper 2 cells play critical roles in the pathogenesis of allergic airway inflammation, and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention through inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and Myl12 are new functional ligands for CD69. Blockade of CD69-My19/12 interaction ameliorates allergic airway inflammation in ovalbumin-induced and house dust mite-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of My19/12 was increased and that platelet-derived My19/12 localized to the luminal surface of blood vessels and formed intravascular netlike structures. Analysis of nasal polyps of eosinophilic chronic rhinosinusitis patients revealed that My19/12 expression was increased in inflammatory lesions and was distributed within net-like structures in the intravascular space. In addition, we detected My19/12 in perivascular spaces where many CD69(+) cells were positioned within My19/12 structures. Thus, CD69-My19/12 interaction is a key event in the recruitment of activated CD69(+) T cells to inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases. Refereed/Peer-reviewed

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34+ was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.

CD3 is a complex of polypeptides which form part of the T cell receptor. Normal human peripheral pan T cells, express not only CD3, but the mRNA for myosin regulatory light chains MYL9, MYL12A, and MYL12B are also significantly expressed. In the Jurkat wild strain, an acute T cell leukemia cell line, CD3 on the surface and MYL9 mRNA are not expressed, while both MYL12A mRNA and MYL12B mRNA are expressed. Jurkat-I, a new clone was established by the transfection of the MYL9 gene into the Jurkat wild strain. As a result, the level of CD3 expressed on the surface of Jurkat-I cells was significantly higher than those in the Jurkat wild strain. Phorbol 12-myristate 13-acetate increased the surface CD3 levels in Jurkat wild strain cells without resulting in MYL9 gene expression, indicating that protein kinase C is partially involved in the expression of CD3 on the surface. These results suggest that surface CD3 expression proceeds through both MYL9-dependent and MYL9-independent pathways (i.e. the protein kinase C- dependent pathway) in Jurkat cells.

Phagocytosis of immunoglobulin G-opsonised particles takes place via Fcγ receptor ligation, leading to uptake through an actin-dependent mechanism. Myosin regulatory light chains have previously been reported to control contractility during uptake through the Fcγ receptor. In this study, we show that p21-activated kinase 4 contributes to Fcγ receptor-mediated uptake downstream of actin cup formation by regulating phosphorylation of myosin regulatory light chain. siRNA-mediated knockdown of p21-activated kinase 4 leads to reduced myosin regulatory light chain phosphorylation at Serine 19, with a corresponding reduction in phospho-myosin regulatory right chain localised to bound immunoglobulin G-opsonised red blood cells. p21-activated kinase 4 phosphorylates myosin light chain 9 at Serine 19 in vitro and RNA interference against myosin light chain 9 implicates this isoform, but not myosin light chain 12A or 12B, in Fcγ receptor-mediated uptake. Taken together, these data indicate that p21-activated kinase 4 regulates regulatory myosin light chain phosphorylation and myosin contractility during FcγR-mediated phagocytosis.

Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region −729/−542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations.

Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

Gastric cancer (GC) is a prevalent cancer, which remains incurable, and therefore requires an alternative treatment method. Celecoxib is a nonsteroidal anti-inflammatory drug that targets cyclooxygenase-2, and exhibits anticancer effects. The present study aimed to investigate the anti-GC mechanism of celecoxib using bioinformatics methods. Gene expression datasets {"type":"entrez-geo","attrs":{"text":"GSE56807","term_id":"56807"}}GSE56807 (GC tissues and normal gastric tissues) and {"type":"entrez-geo","attrs":{"text":"GSE54657","term_id":"54657"}}GSE54657 (celecoxib-treated and non-treated human GC epithelial AGS cells) were downloaded from the Gene Expression Omnibus database. Two groups of differentially expressed genes (DEGs) were identified using limma package in R language. The criterion for {"type":"entrez-geo","attrs":{"text":"GSE56807","term_id":"56807"}}GSE56807 was a false discovery rate of

This study aimed to identify differentially expressed genes (DEGs) of unruptured intracranial aneurysms (IAs) and provide beneficial information for early diagnosis and treatment of IAs. The gene expression profile GSE26969 from the Gene Expression Omnibus database was downloaded, which included six human IA samples: three intracranial arterial aneurysm samples and three normal superficial temporal artery samples (control). Based on these data, we identified the DEGs between normal and disease samples with packages in the R language. The selected DEGs were further analyzed using bioinformatic methods. First, the STRING software was used to search co-expression relationships among DEGs, and the most important hub gene was found. We then used the plugins of the Cytoscape software, Mcode and Bingo, to conduct a module analysis. Next, pathways of the module genes were annotated using the FuncAssociate program. Compared with the control group, we obtained 169 DEGs in total, and by mining a module with the hub gene MYH11, we retrieved the ACTA2, MYH11, MYLK, and MYL9 genes, which were all in the module and were most significantly related to vascular smooth muscle contraction. We hypothesize that the genes identified here can be beneficial for early diagnosis and treatment of IAs.

The aim of this study was to investigate the associations of myosin light chain (MYL9) downregulation with tumor progression and prognosis in patients with prostate cancer (PCa). MYL9 protein expression in human PCa and non-cancerous prostate tissues was detected by Western blot and immunohistochemistry analyses, which was validated by microarray-based Taylor data at mRNA level. Then, the associations of MYL9 expression with clinicopathological features and clinical outcome of PCa patients were statistically analyzed. Both Western blot and immunohistochemistry analyses found that MYL9 expression was significantly decreased (both P < 0.001) in PCa tissues compared with those in non-cancerous prostate tissues. In addition, MYL9 was mainly expressed in the cytoplasm of stromal cells of prostate tissues, and the decreased expression of MYL9 in PCa tissues was significantly correlated with the older age of patients (P = 0.011), the higher Gleason score (P < 0.001), the advanced pathological stage (P = 0.002), the presence of metastasis (P < 0.001) and PSA failure (P = 0.001). Furthermore, both univariate and multivariate analyses showed that the downregulation of MYL9 was an independent predictor of shorter overall survival (P = 0.026 and P = 0.009, respectively) and biochemical recurrence-free survival (P = 0.001 and P = 0.002, respectively). Our data strongly confirmed for the first time that the decreased expression of MYL9 may play an important role in tumor progression of PCa. More importantly, the downregulation of MYL9 may efficiently predict both overall and biochemical recurrence-free survivals in PCa patients.

International audience; Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Endometriosis is a gynecological disease defined as the presence of endometrial tissue outside the uterine cavity, which is caused by various factors. Proteomic analysis of two sets of eutopic endometrial cells collected from the menstrual blood of females with (n=6; n=3) or without (n=6; n=3) endometriosis was performed to identify novel potential biomarkers for endometriosis. The data revealed that samples from endometriosis patients had stem cell characteristics, as they had higher mRNA expression levels of octamer-binding transcription factor 4 (Oct-4), C-X-C chemokine receptor type 4 (CXCR4), SRY-box containing gene 2 (SOX2) and mesen- chymal-epithelial transition factor (MET) compared with that of the normal controls. Three proteins, collapsin response mediator protein 2 (CRMP2), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) and myosin regulatory light polypeptide 9 (MYL9), were simultaneously identified from the two sets of samples from females with or without endometriosis by two-dimensional electrophoresis (2-DE). A difference in CRMP2 expression was confirmed with western blotting. Taken together, the results suggest that CRMP2 plays a role in the pathogenesis of endometriosis.

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events. Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p < 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value < 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors. Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. Disclosures No relevant conflicts of interest to declare.

Background Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels. Methodology/Principal Findings We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzye A reductase, possess pleiotropic effects that have been extended to modulation of various cellular behaviors. This study aimed at examining whether atorvastatin (AVN) modulates cell growth, adhesion, migration, and contraction of cultured human retinal pigment epithelium (RPE) cells. The in vitro effects of AVN on human RPE cells was analyzed in terms of cell proliferation, cell cycle, cell adhesion, migration, and contraction assays. The modulatory effect of AVN on TGF-β2-triggered signaling was determined by Western blotting detection. AVN at submicromolar dose exhibited no prominent morphological alteration and cytotoxicity, whereas it elicited cytostatic effect at concentrations higher than 1 μM. Cell cycle analysis showed that AVN induced growth arrest in both G1 and G2/M phases. AVN at 1 μM or higher concentrations significantly suppressed RPE cell adhesion. Cell migration and 3D collagen contraction assays showed that AVN significantly suppressed RPE cell migration and contractility, respectively. Mechanistically, AVN treatment transiently up-regulated phosphorylation of Akt, ERK1/2, and p38 MAPK, whereas down-regulated that of JNK1. Intriguingly, AVN pretreatment prominently attenuated the TGF-β2-mediated non-Smad signaling, including Akt, ERK1/2, p38 MAPK, and JNK1 phosphorylation. Besides, it directly reduced constitutive level of myosin regulatory light chain peptide MYL9 and mitigated the TGF-β2-induced phosphorylation of myosin phosphatase-targeting subunit 1, MYPT1. These in vitro findings strongly suggest that AVN possesses pleiotropic function on RPE cells, including anti-proliferation, anti-adhesion, anti-migration as well as anti-contraction. In conclusion, AVN treatment may be considered a useful therapy for proliferative vitreoretinal diseases.

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC–MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.

We have reported on a patient with thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb-IIIa activation, associated with a heterozygous mutation in transcription factor CBFA2 (core binding factor A2, RUNX1 or AML1). To obtain insights into the abnormal platelet mechanisms and CBFA2-regulated genes, we performed platelet expression profiling in four control subjects and the patient using the Affymetrix U133 GeneChips. In the patient, 298 probe sets were significantly downregulated at least 2-fold. MLC regulatory polypeptide (MYL9 gene) was decreased approximately 77-fold; this is an important finding because agonist-stimulated MLC phosphorylation is decreased in patient platelets. Genes downregulated > or = 5-fold include those involving calcium binding proteins (CABP5), ion transport (sodium/potassium/Ca exchanger, SLC24A3), cytoskeletal/microtubule proteins (erythrocyte membrane protein band 4.1-like 3, EPB41L3; tropomyosin 1, TPM1; tubulin, alpha 1, TUBA1), signaling proteins (RAB GTPase activating protein 1-like, RABGAP1L; beta3-endonexin, ITGB3 BP) and chemokines (platelet factor 4 variant 1, PF4V1; chemokine CXCL5, CXCL5). These and other downregulated genes are relevant to the patient's platelet defects in function and production. These studies provide the first proof of concept that platelet expression profiling can be applied to obtain insights into the molecular basis of inherited platelet defects.

Background: Ewing sarcoma (ES) is an aggressive and metastatic tumor of bone and soft tissue that primarily affects children and adolescents. The primary cause of tumor-related death in patients with ES is metastatic disease. In epithelial-derived cancers metastasis is dependent on phenotypic transition of cells from epithelial- to mesenchymal-like states (EMT). In addition, Rho signaling pathways are primary mediators of cancer cell motility and can induce cell migration and invasion, in part through transcriptional activation of MKL/SRF target genes. The mechanisms of metastasis in non-epithelial tumors such as ES remain to be elucidated. Objective: We sought to determine whether phenotypic plasticity between non-invasive and invasive states is also a feature of ES and to determine the molecular mechanism underlying this transition. Methods: ES cell lines were subjected to serum deprivation and phenotypic differences measured using scratch assays, transwell studies, and the xCelligence system. qRT-PCR, Western blot and immunofluorescence were used to quantitate or visualize changes in gene and protein expression, respectively. Results: Serum deprivation induced ES cells to rapidly adopt a more aggressive phenotype, reflected by changes in cell shape and increased motility, while proliferation was inhibited. Serum replenishment rapidly reverted these phenotypic changes. Mechanistically, markers of EMT were largely unaffected indicating that the observed phenotypic plasticity in ES cells was not mediated by classic EMT-MET transitions. Instead, activation of Rho-MKL signaling was apparent as evidenced by nuclear localization of MKL and upregulation of the metastasis-associated MKL/SRF target gene MYL9 in serum-deprived conditions. Exposure of ES cells to a small molecule antagonist of Rho-MKL signaling inhibited ES cell motility. Conclusions: Serum deprivation causes reversible transformation of ES cells to a more migratory phenotype. This is associated with nuclear translocation of MKL and induction of MYL9, a gene that encodes a regulatory light chain required for myosin stability. We propose a model of ES metastasis in which changes to the microenvironment that result in growth-factor deprivation (e.g. increased tumor size or cytotoxic therapy) induce expression of Rho-MKL transcriptional targets that contribute to cell migration and invasion. The Rho-MKL axis may thus be a novel therapeutic target for prevention of ES metastasis. Citation Format: Merlin Airik, Melanie Krook, Christopher Scannell, Andrew Haak, Richard Neubig, Elizabeth Lawlor. Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2013-5030

RUNX1 (also known as CBFA2 or AML1) is a transcription factor that plays a major role in hematopoiesis. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood87:1368–76, 1996). The patient platelets showed impaired phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-𝛉. This was associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood103: 948–54, 2004). Platelet transcript profiling showed a striking downregulation of myosin light chain 9 (MYL9) by ~77-fold relative to normal platelets (Sun et al, J. Thromb Haemost.5: 146–54, 2007). Myosin light chains (MLCs) play an important role in platelet responses to activation, in platelet biogenesis, and are involved in cellular processes such as cytokinesis, cell adhesion, cell contraction, cell migration. We have addressed the hypothesis that MYL9 is a direct transcriptional target of RUNX1. Studies were performed in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. To determine endogenous interaction of RUNX1 with MYL9 promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. These studies revealed RUNX1 binding to MYL9 chromatin at −742/−529 bp upstream of the ATG codon. TFSEARCH revealed four RUNX1 sites within this region. We performed electrophoretic mobility shift assay (EMSA) using probes containing each of the RUNX1 motifs and PMA-treated nuclear extracts from HEL cells. With each probe, protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. This protein binding was not competed by oligos containing mutations in the specific RUNX1 sites. No binding was noted directly to the mutant probes. To further corroborate our findings, we performed transient-ChIP analysis where wild type luciferase reporter construct −691/+4 and constructs with each of the RUNX1 sites individually mutated were transiently transfected into HEL cells. ChIP was performed using these cells and anti-RUNX1 antibody, and the expression analyzed by PCR amplification with a forward primer from MYL9 promoter sequence and reverse primer from luciferase vector sequence. Amplification was observed with immunoprecipitated wild type construct but not with any of the mutant constructs. Thus, RUNX1 interacts in vivo with MYL9 promoter, and the multiple RUNX1 sites interact with each other, as also shown for other genes. To test the functional relevance, the wild type construct −691/+4 containing all 4 RUNX1 sites or mutant constructs with each site individually deleted were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Deletion of RUNX1 site 1, 2, 3 or 4 caused ~60–90% reduction in the activity indicating that each site was functional. Lastly, siRNA mediated knock down of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and MYL9 protein. Conclusions: Our results provide the first evidence that MYL9 gene is transcriptionally regulated by RUNX1. They provide evidence for the presence of multiple RUNX1 sites in MYL9 promoter, as also observed in other genes. Moreover, these studies provide a cogent mechanism for the MYL9 transcript downregulation and the impaired MLC-phosphorylation we have previously described in association with RUNX1 haplodeficiency.

We have previously reported (Blood, 87:1368, 1996) a patient with a mild thrombocytopenia, and markedly abnormal aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and activation of GPIIb-IIIa complex. We recently showed (Blood 2004, 103: 948–954) in this patient a heterozygous mutation in hematopoietic transcription factor CBFA2 (Core Binding factor A2, also called AML1) and decreased platelet PKC-theta. We postulated that platelet expression profiling would provide insights into the pathophysiological mechanisms and gene expression changes resulting from a CBFA2 mutation. We performed platelet expression profiling in 4 control subjects (6 profiles, 2 individuals profiled twice) and the patient (profiled twice, 10 months apart) using the Affymetrix microarray Human U133A and B GeneChips (total ~44000 probe sets). ~10,000 probe sets (genes and ESTs) are expressed in human platelets. Iterative comparisons of data from each individual platelet profiles had correlation coefficients (r) > 0.85. Using GeneSpring® analytical software we selected genes that showed at least 1 present call in the 8 profiles, and p2 were considered as significant changes. In the patient, 298 probe sets were significantly down regulated and 29 significantly upregulated. We focused on genes showing a > 5-fold decrease (fold change, fc < = 0.20). There was a striking ~75 fold decrease (fc = 0.013; p=0.00022) in myosin regulatory light chain polypeptide (MYL9 gene). Other down-regulated genes included calcium binding protein 5 (CABP5, fc = 0.02; a protein with similarity to calmodulin), Na/K/Ca exchanger member 3 (SLC24A3, fc = 0.11), b3-endonexin (ITGB3BP, fc = 0.01), platelet factor 4 variant 1 (PF4V1, fc = 0.03), chemokine CXCL5 (ENA-78) (fc = 0.20), tropomyosin I (alpha) (TPM1, fc = 0.13) and arachidonate 12-lipoxygenase (ALOX-12, fc = 0.22). The expression array findings were validated by decreased platelet mRNA levels on real time PCR or by immunoblotting (platelet PF4). Several down-regulated genes are directly relevant to the patient’s platelet defect. Agonist-stimulated MLC phosphorylation is impaired in patient platelets. The strikingly decreased expression of MYL9 with normal expression of MLC kinase (MLCK, by array and real time PCR) suggests that the blunted myosin phosphorylation arises from decreased MYL9 than due to decreased MLCK. Myosin light chains play a role in diverse processes including in cell contractility, mobility and cytokinesis, and are relevant to impaired platelet function and production. The decreased expression of the Na/K/Ca exchanger and b3-endonexin 3 (both implicated in activation of GPIIb-IIIa) provide mechanisms for the diminished GPIIb-IIIa activation. Chemokine CXCL5 and PF4V1 are expressed in megakaryocytes, and the genes are colocalized to “chemokine cluster” on chromosome 4. 12-lipoxygenase, a major platelet enzyme, regulates arachidonic acid metabolism and production of the 12-hydroxy eicosanoids. This is the first proof of concept that platelet expression profiling can be applied to obtain new insights into the molecular basis of inherited platelet dysfunction. Further studies will provide new information on the genes regulated by CBFA2, and on mechanisms regulating platelet production and activation.