miR-433-3p Targets AJUBA to Inhibit Malignant Progression of Glioma.

INTRODUCTION: Glioma is the most aggressive and malignant type of tumors among primary intracranial tumors. miR-433-3p has been verified to be correlated with the formation and progression of many types of cancers. METHODS: In this study, the effects of miR-433-3p and AJUBA on the proliferation, migration, and invasion of glioma and the molecular mechanisms were investigated. We analyzed bioinformatics databases and conducted cell biology experiments to determine that compared with adjacent tissue and normal cells, the expression level of miR-433-3p in glioma tissue and cells was lower, while the expression level of AJUBA was higher. Overexpressing miR-433-3p could significantly inhibit the proliferation, migration, and invasion of glioma cells and promote cell apoptosis. RESULTS: In addition, after overexpressing miR-433-3p and AJUBA, it was found that overexpressing AJUBA could attenuate the inhibitory effect of overexpressing miR-433-3p on the proliferation, migration, and invasion of glioma cells, which suggested that miR-433-3p regulated the biological function of glioma by downregulating AJUBA expression. CONCLUSION: These results proved that miR-433-3p could target to inhibit the expression of AJUBA, thus inhibiting the biological function and malignant progression of glioma.

LINC01503/miR-342-3p facilitates malignancy in non-small-cell lung cancer cells via regulating LASP1.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world. LncRNA LINC01503 is a super-enhancer-driven long non-coding RNA that is dysregulated in several types of human cancer. However, its role in NSCLC remains unknown. METHODS: Thirty NSCLC patients were recruited between April 2012 and April 2016. Luciferase reporter assay, qRT-PCR, Cell Counting Kit-8 (CCK-8), Transwell migration assay, RNA pull-down assay, western blotting, 5-ethynyl-29-deoxyuridine (EdU) assays, and flow cytometry were utilized to characterize the roles and relationships among LINC01503, miR-342-3p, and LASP1 in NSCLC. The transplanted mouse model was built to examine their biological functions in vivo. RESULTS: We demonstrated that the expression of lncRNA LINC01503 and LIM and SH3 domain protein 1 (LASP1) were upregulated and miR-342-3p was downregulated in NSCLC samples and cell lines. Functional experiments revealed that inhibiting the expression of LINC01503 or over-expression of miR-342-3p inhibited NSCLC growth and metastasis both in vitro and in vivo. In addition, LINC01503 could bind to miR-342-3p and affect the expression of LASP1. CONCLUSION: These results provide a comprehensive analysis of the roles of LINC01503 as a competing endogenous RNA (ceRNA) in NSCLC progression.

Upregulation of LASP2 inhibits pancreatic cancer cell migration and invasion through suppressing TGF-ß-induced EMT.

LASP2 (LIM and SH3 protein 2), a member of the LIM-protein subfamily of the nebulin group, was first identified as a splice variant of the nebulin gene. In the past, investigators mainly focused on the impact of LASP2 on cardiac diseases because of its identification in the myocardium. Recently, several studies have reported that LASP2 is associated with the progression of various cancers. However, there have been no investigations on the expression and function of LASP2 in pancreatic cancer (PC). In this study, we performed the quantitative real-time polymerase chain reaction and Western blot analysis to detect the expression of LASP2 in PC tissues and cell lines. PC cells were transfected with LASP2 overexpression plasmid or the negative control in the presence or absence of tumor growth factor-ß (TGF-ß). The transwell assays were used to measure the effects of LASP2 on PC cell migration and invasion. The protein expression of epithelial-mesenchymal transition (EMT) markers was detected using Western blot assay. Our results demonstrated that LASP2 was downregulated in PC tissues and cell lines. In addition, upregulation of LASP2 inhibited the PC cell migration and invasion. We also found that LASP2 upregulation reversed TGF-ß-induced EMT in PC cells. Taken together, we provided novel evidence supporting the tumor-suppressor role of LASP2 in PC and suggested it as a potential therapeutic target in PC treatment.

SCL/TAL1: a multifaceted regulator from blood development to disease.

SCL/TAL1 (stem cell leukemia/T-cell acute lymphoblastic leukemia [T-ALL] 1) is an essential transcription factor in normal and malignant hematopoiesis. It is required for specification of the blood program during development, adult hematopoietic stem cell survival and quiescence, and terminal maturation of select blood lineages. Following ectopic expression, SCL contributes to oncogenesis in T-ALL. Remarkably, SCL's activities are all mediated through nucleation of a core quaternary protein complex (SCL:E-protein:LMO1/2 [LIM domain only 1 or 2]:LDB1 [LIM domain-binding protein 1]) and dynamic recruitment of conserved combinatorial associations of additional regulators in a lineage- and stage-specific context. The finely tuned control of SCL's regulatory functions (lineage priming, activation, and repression of gene expression programs) provides insight into fundamental developmental and transcriptional mechanisms, and highlights mechanistic parallels between normal and oncogenic processes. Importantly, recent discoveries are paving the way to the development of innovative therapeutic opportunities in SCL+ T-ALL.

Trypanosoma carassii infection in goldfish (Carassius auratus L.): changes in the expression of erythropoiesis and anemia regulatory genes.

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1ß. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Long non-coding RNA LINC00672 contributes to p53 protein-mediated gene suppression and promotes endometrial cancer chemosensitivity.

Thousands of long intergenic non-protein coding RNAs (lincRNAs) have been identified in mammals in genome-wide sequencing studies. Some of these RNAs have been consistently conserved during the evolution of species and could presumably function in important biologic processes. Therefore, we measured the levels of 26 highly conserved lincRNAs in a total of 176 pairs of endometrial carcinoma (EC) and surrounding non-tumor tissues of two distinct Chinese populations. Here, we report that a lincRNA, LINC00672, which possesses an ultra-conserved region, is aberrantly down-regulated during the development of EC. Nevertheless, LINC00672 is a p53-targeting lincRNA acting along with heterogeneous nuclear ribonucleoproteins as a suppressive cofactor, which locally reinforces p53-mediated suppression of LASP1, an evolutionarily conserved neighboring gene of LINC00672 and putatively associated with increased tumor aggressiveness, during anti-tumor processes. LINC00672 overexpression could lower the levels of LASP1 and slow the development of malignant phenotypes of EC both in vitro and in vivo Moreover, LINC00672 significantly increased the 50% inhibitory concentration of paclitaxel in EC cells and increased the sensitivity of xenograft mice to paclitaxel. These findings indicate that LINC00672 can influence LASP1 expression as a locus-restricted cofactor for p53-mediated gene suppression, thus impacting EC malignancies and chemosensitivity to paclitaxel.

Specific Soft-Tissue Invasion and LMP1 Expression Are Potential Indicators of Extranodal NK/T Cell Lymphoma, Nasal Type.

BACKGROUND Extranodal NK/T cell lymphoma, nasal type (ENKTL-NT) is difficult to distinguish from nasal polyps and inverted papilloma, leading to its high misdiagnosis ratio. The aim of this study was to investigate its potential prognostic indicators. MATERIAL AND METHODS Kaplan-Meier method was used to calculate overall survival (OS) rate. Cox proportional hazards regression was used to analyze risk ratios (ORs) with 95% confidence intervals (CIs). RESULTS Nasal ala infiltration and nasal floor thickness >2.0 mm or nasal septum thickness >2.5 mm were potential prognostic factors for OS (p=0.0323 and 0.0072, respectively). Cox proportional-hazards regression indicated that high LMP1 expression and the nasal floor thickness >2.0 mm or nasal septum thickness >2.5 mm were the independent risk factors for poor OS of ENKTL-NT (HR=3.0655, p=0.028; HR=2.3650, p=0.0452, respectively). In the subgroup analysis, the OS rate was lower when the nasal floor thickness >2.0 mm or nasal septum thickness >2.5 mm in the patients who had high expression of LMP1 (p=0.0651), whereas high LMP1 expression increased the risk of worse prognostic outcome in patients with deep infiltration thickness. Thus, high LMP1 expression may contribute to the tissue invasion of ENKTL-NT. CONCLUSIONS Any patient with nasal ala soft-tissue invasion, nasal floor thickness >2.0 mm/nasal septum thickness >2.5 mm on CT imaging or high LMP1 expression should prompt immediate histopathologic diagnosis to rule out ENKTL-NT in clinical practice.

Suppression of LIM and SH3 Domain Protein 1 (LASP1) Negatively Regulated by Androgen Receptor Delays Castration Resistant Prostate Cancer Progression.

BACKGROUND: LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti-tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO). METHODS: A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies. RESULTS: The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4-2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts. CONCLUSIONS: These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309-320, 2017. © 2016 Wiley Periodicals, Inc.

Paving the path for invasion: The polyedric role of LASP1 in cancer.

Although usually referred to as a structural actin-binding protein, LIM and SH3 domain-containing protein may actually be dynamically involved in the control of a wide spectrum of cellular processes, by virtue of its interaction with several molecular partners. Alongside being ubiquitously expressed in physiological conditions, LIM and SH3 domain-containing protein is overexpressed in a growing number of human cancers, in which it may actively contribute to their aggressiveness by promoting cell proliferation and migration. In view of the recent findings, implicating the protein in cancer progression, we discuss here the most relevant discoveries highlighting the role of this versatile protein in various human tumors. The correlation between LIM and SH3 domain-containing protein expression levels in cancer and the poor outcome and metastatic behavior of tumors denotes the clinical significance of this protein and hints its potential value as a new cancer prognostic or even diagnostic biomarker. This may be decisive not only to optimize existing pharmacological regimes but also to delineate novel, more efficacious therapeutic and/or preventive approaches.

PDLIM2 suppression efficiently reduces tumor growth and invasiveness of human castration-resistant prostate cancer-like cells.

BACKGROUND: Although PDLIM2 gene may have a context-dependent role in various human malignancies and can be a potential therapeutic target, only a limited number of in vitro studies addressed the molecular functions of PDLIM2 in prostate cancer. Here, we aimed to explore the role of PDLIM2 and the effect of the PDLIM2 gene suppression on oncogenic phenotypes of human castration-resistant prostate cancer (CRPC)-like cells. METHODS: We used human CRPC-like cell lines (PC3, DU145, and C4-2B) for our experiments. Transcription levels of PDLIM2 and relevant genes were measured by real time-PCR and protein expression was analyzed by western blot. Cell viability, proliferation, clonogenic growth, and tumor sphere formation were examined after a specific inhibition of PDLIM2 using RNA interference. Flow cytometry was used to examine apoptotic cell death and cell cycle disturbances. Wound healing and transwell migration assays were performed to investigate the invasion capabilities of CRPC-like cells. Additionally, key oncogenic signaling pathways were examined using western blot. Lastly, we evaluated the in vivo efficacy of PDLIM2 suppression on tumor growth of human CRPC xenografts in mice. RESULTS: We observed a significant enhancement of PDLIM2 expression in human CRPC-like cell lines, while a specific inhibition of PDLIM2 reduced cell viability and proliferation due to apoptotic cell death. Conversely, PDLIM2 overexpression significantly reduced cell proliferation compared to the negative control in androgen-sensitive LNCaP cells. Moreover, PDLIM2 suppression led to a decrease of clonogenic growth and tumor sphere formation in three-dimensional cultures with the G2/M cell cycle arrest in human CRPC-like cells. PDLIM2 inhibition also attenuated cellular migration and invasion capabilities of human CRPC-like cells, and reduced the expression of mesenchymal marker. Among several oncogenic signaling pathways, only the MAPK/ERK signaling cascade was decreased by PDLIM2 inhibition and reciprocally, ERK inhibition down-regulated PDLIM2 expression. Importantly, PDLIM2 inhibition remarkably compromised tumor growth in a human CRPC xenograft model. CONCLUSION: In summary, the suppression of PDLIM2 significantly reduced such oncogenic phenotypes as proliferation, clonogenicity, invasiveness, and tumor cell growth in human CRPC-like cells both in vitro and in vivo, indicating that PDLIM2 may be considered a novel therapeutic target gene for treating human CRPC.

Planar cell polarity gene expression correlates with tumor cell viability and prognostic outcome in neuroblastoma.

BACKGROUND: The non-canonical Wnt/Planar cell polarity (PCP) signaling pathway is a major player in cell migration during embryonal development and has recently been implicated in tumorigenesis. METHODS: Transfections with cDNA plasmids or siRNA were used to increase and suppress Prickle1 and Vangl2 expression in neuroblastoma cells and in non-tumorigenic cells. Cell viability was measured by trypan blue exclusion and protein expression was determined with western blotting. Transcriptional activity was studied with luciferase reporter assay and mRNA expression with real-time RT-PCR. Immunofluorescence stainings were used to study the effects of Vangl2 overexpression in non-tumorigenic embryonic cells. Statistical significance was tested with t-test or one-way ANOVA. RESULTS: Here we show that high expression of the PCP core genes Prickle1 and Vangl2 is associated with low-risk neuroblastoma, suppression of neuroblastoma cell growth and decreased Wnt/ß-catenin signaling. Inhibition of Rho-associated kinases (ROCKs) that are important in mediating non-canonical Wnt signaling resulted in increased expression of Prickle1 and inhibition of ß-catenin activity in neuroblastoma cells. In contrast, overexpression of Vangl2 in MYC immortalized neural stem cells induced accumulation of active ß-catenin and decreased the neural differentiation marker Tuj1. Similarly, genetically modified mice with forced overexpression of Vangl2 in nestin-positive cells showed decreased Tuj1 differentiation marker during embryonal development. CONCLUSIONS: Our experimental data demonstrate that high expression of Prickle1 and Vangl2 reduce the growth of neuroblastoma cells and indicate different roles of PCP proteins in tumorigenic cells compared to normal cells. These results suggest that the activity of the non-canonical Wnt/PCP signaling pathway is important for neuroblastoma development and that manipulation of the Wnt/PCP pathway provides a possible therapy for neuroblastoma.

ZNF185 inhibits growth and invasion of lung adenocarcinoma cells through inhibition of the akt/gsk3ß pathway.

Zinc finger (ZNF) proteins, a diverse family of proteins, have multiple biological functions in cancer. Increased expression of ZNF185 has been involved in the regulation of tumor growth and metastasis. However, the function and underlying mechanisms of ZNF185 in the tumorigenesis of lung adenocarcinoma (LAC) remain unclear. The protein expression of ZNF185 was examined in human LAC tissues by immunohistochemical assay. After lentiviral vector-mediated ZNF185 overexpression was infected into the LAC cell lines (A549 and LETPα-2), cell growth and invasive potential were respectively evaluated by MTT and Transwell assays. We found that the protein expression of ZNF185 was significantly downregulated in LAC tissues compared with the adjacent non-cancerous tissues (ANCT) (37.10% vs 58.06%, P=0.015), and was negatively correlated with the lymph node metastasis of the LAC patients (P=0.005). Furthermore, overexpression of ZNF185 reduced cell proliferation and invasion in LAC cells, followed by the downregulation of p-AKT, p-GSK3ß, VEGF and MMP-9 expression. Taken together, our findings indicate that the decreased expression of ZNF185 is linked to the tumor metastasis in human LAC patients, and ZNF185 overexpression inhibits the growth and invasion of LAC cells through inhibition of the AKT/GSK3ß signaling, suggesting that ZNF185 may represent a potential therapeutic target for the treatment of LAC.

BCL6--regulated by AhR/ARNT and wild-type MEF2B--drives expression of germinal center markers MYBL1 and LMO2.

Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.

Lmo2 induces hematopoietic stem cell-like features in T-cell progenitor cells prior to leukemia.

LIM domain only 2 (Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells (HSCs) and early T-cell precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the HSC. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.

Up-regulated FHL1 expression maybe involved in the prognosis of Hirschsprung's disease.

BACKGROUND: In a subset of patients with Hirschsprung's disease (HSCR), gastrointestinal motor dysfunction persisted long after surgical correction. Gastrointestinal motility is achieved through the coordinated activity of the enteric nervous system, interstitial cells of Cajal, and smooth muscle (SMC) cells. Inhibition of four-and-a-half LIM protein-1 (Fhl1) expression by siRNA significantly decreases pulmonary artery SMCs migration and proliferation. Furthermore when up-expressing FHL1 in atrial myocytes, K (+) current density markedly increases, therefore changing myocytes' response to an electrical stimulus. However whether FHL1 in colon SMCs (the final effector organ) influences intestinal motility in HSCR patients has not been clarified. METHODS: FHL1 mRNA and protein expressions were analyzed in 32 HSCR colons and 4 normal colons. RESULTS: Smooth muscle layers were thicken and disorganized in HSCR. FHL1 was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the ganglionic colon. FHL1 mRNA relative expression level in aganglionic colons was 1.06 ± 0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83 ± 0.09. FHL1 expression level in ganglionic colon (1.66 ± 0.30) or aganglionic colon (1.81 ± 0.35) was significantly higher than that in normal colons (P=0.045 and P=0.041, respectively). Meanwhile, we found FHL1 expression in aganglionic colon was slightly stronger than that in ganglionic colon (P=0.036). CONCLUSION: These data suggested that up-regulated FHL1 in smooth muscle in HSCR might be associated with intestinal wall remodeling in HSCR and might be one of the risk factors for gastrointestinal motor dysfunction.

Alk is a transcriptional target of LMO4 and ERα that promotes cocaine sensitization and reward.

Previously, we showed that the mouse LIM-domain only 4 (Lmo4) gene, which encodes a protein containing two zinc-finger LIM domains that interact with various DNA-binding transcription factors, attenuates behavioral sensitivity to repeated cocaine administration. Here we show that transcription of anaplastic lymphoma kinase (Alk) is repressed by LMO4 in the striatum and that Alk promotes the development of cocaine sensitization and conditioned place preference, a measure of cocaine reward. Since LMO4 is known to interact with estrogen receptor α (ERα) at the promoters of target genes, we investigated whether Alk expression might be controlled by a similar mechanism. We found that LMO4 and ERα are associated with the Alk promoter by chromatin immunoprecipitation and that Alk is an estrogen-responsive gene in the striatum. Moreover, we show that ERα knock-out mice exhibit enhanced cocaine sensitization and conditioned place preference and an increase in Alk expression in the nucleus accumbens. These data define a novel regulatory network involved in behavioral responses to cocaine. Interestingly, sex differences in several behavioral responses to cocaine in humans and rodents have been described, and estrogen is thought to mediate some of these differences. Our data suggest that estrogen regulation of Alk may be one mechanism responsible for sexually dimorphic responses to cocaine.

Transforming growth factor-ß1-induced transcript 1 protein, a novel marker for smooth muscle contractile phenotype, is regulated by serum response factor/myocardin protein.

Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-ß1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-ß-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-ß-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.

Role of hepatitis B virus X protein in regulating LIM and SH3 protein 1 (LASP-1) expression to mediate proliferation and migration of hepatoma cells.

BACKGROUND: Hepatitis B virus X protein (HBx) has been shown to be responsible for the development of hepatocellular carcinoma (HCC) caused by Hepatitis B virus infection. However, its potential effect on the progression of hepatocellular carcinoma remains yet unclear. LIM and SH3 protein 1 (LASP-1), a focal adhesion protein, is expressed in an up-regulation manner in the HCC tissues. LASP-1 plays an important role in the regulation of proliferation and migration of HCC. In this study, we investigated the effect of LASP-1 involved in HBx-related tumor progression. METHODS: LASP-1 levels in the HBx stable transfected HepG2 and Huh-7 cells were detected by RT-PCR and western blot analysis. The cellular localization of LASP-1 was assessed by immunofluorescence analysis. The activity of phosphatidylinositol 3-kinase (PI3-K) pathway was demonstrated by western blot assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against LASP-1. The proliferation and migration ability of cells were evaluated by cell viability assay and plate clone formation assay. The migration ability of cells was detected by transwell assay and wound healing assay. RESULTS: RT-PCR and western blot analysis indicated the expression of LASP-1 was increased in the stable HBx-expressing cells compared with the control cells. Immunofluorescence study revealed that the distributions of LASP-1 in HepG2-HBX cells were mainly in pseudopods and the cytoplasm while they were mainly localized in the cytoplasm of HepG2-Mock cells. The cellular localizations of LASP-1 in Huh-7-HBX cells were in the perinuclear fractions while they were mainly localized in the cytoplasm of Huh-7-Mock cells. The upregulation of LASP-1 was inhibited after treatment with LY294002, PI3-K pathway inhibitor. Overexpression of LASP-1 in the stable HBx-expressing cells enhanced the proliferation and migration ability of hepatocellular cells. siRNA-mediated LASP-1 knowdown in the stable HBx-expressing cells significantly suppressed hepatocellular cells proliferation and migration. CONCLUSIONS: These results demonstrated that HBx could upregulate LASP-1 through PI3-K pathway to promote the proliferation and migration of hepatoma cells.

Glomerular expression of hydrogen peroxide-inducible clone-5 in human and rat progressive mesangial proliferative glomerulonephritis.

BACKGROUND/AIMS: Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor-ß(1) (TGF-ß(1))- and hydrogen peroxide (H(2)O(2))-inducible focal adhesion protein that may be necessary for maintaining the myofibroblastic phenotype in pathological scar formation. To investigate the involvement of Hic-5 in the pathogenesis of glomerulonephritis (GN), we examined the glomerular expression of Hic-5 in human and rat GN as well as the regulation of Hic-5 by TGF-ß(1) in vitro. METHODS AND RESULTS: Immunohistochemical analyses showed that the expression of Hic-5 was increased in mesangial cells (MCs) in human mesangial proliferative GN. Hic-5 expression was significantly correlated not only with the levels of α-smooth muscle actin (α-SMA) and TGF-ß(1), the accumulation of extracellular matrix, and the number of glomerular cells, but also with the urinary protein level in patients with GN. Glomerular Hic-5 expression increased in parallel with α-SMA expression in a rat model of mesangial proliferative GN. Combined therapy with an angiotensin type I receptor blocker and an antioxidant in this model improved the histology and the expression of Hic-5 and α-SMA. TGF-ß(1) upregulated Hic-5 and α-SMA protein levels in human cultured MCs. CONCLUSION: Our findings suggest that Hic-5 is involved in changes in the MC phenotype to produce abnormal extracellular matrix remodeling in GN.

Expression pattern of Mical-1 in the temporal neocortex of patients with intractable temporal epilepsy and pilocarpine-induced rat model.

Mical-1 is a novel F-actin-disassembly factor that is critical in actin reorganization. It provides a molecular conduit through which actin reorganizes-a hallmark of cell morphological changes, including axon navigation. However, whether Mical-1 is involved in the epileptogenesis remains unknown. Here, we investigate Mical-1 expression pattern in patients with intractable temporal lobe epilepsy (TLE) and pilocarpine-induced rat model. We used double-labeled immunoflurescence, immunohistochemistry, and Western blotting to assess the location and expression of Mical-1 in temporal neocortex of patients with intractable TLE, and the expression pattern of Mical-1 at different time point in the hippocampus and temporal lobe cortex of the pilocarpine-induced rat model. Double-labeled immunofluorescence showed that Mical-1 was coexpressed with neuron-specific enolase (NSE) in the cytoplasm of neurons in temporal neocortex of patients with TLE and hippocampus of rat model. Faint and scattered immunoreactivity for Mical-1 in the neuron of temporal neocortex in TLE group, but strong immunoreactivity for Mical-1 was shown in control subjects. To quantitatively evaluate the Mical-1 immunoreactivity, we measured the mean optical density (OD) of Mical-1. In the hippocampus of pilocarpine-induced rat model, the OD values transient increased at 6 h after seizure then decreased from 1 day to 14 days, and returned to a subnormal level at 60 days. The lowest level of Mical-1 expression occurred at 14 days after seizure in the hippocampus. In the temporal lobe cortex of rat model, the OD values decreased at all time point after kindling compared to the normal group. Furthermore, our Western blot analysis confirmed these expression patterns of Mical-1 from latent stage to chronic stage. Our results indicate that in patients with TLE and pilocarpine-induced rat model, the expression of Mical-1 were followed a downtrend from the latent stage to chronic stage after seizure evoke. Thus, as an effect factor participated in F-actin disassemble, Mical-1 may associate with inner pathophysiological modulation in epilepsy.