Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Knockdown of DIXDC1 Inhibits the Proliferation and Migration of Human Glioma Cells

DIX domain containing 1 (DIXDC1), the human homolog of coiled-coil-DIX1 (Ccd1), is a positive regulator of Wnt signaling pathway. Recently, it was found to act as a candidate oncogene in colon cancer, non-small-cell lung cancer, and gastric cancer. In this study, we aimed to investigate the clinical significance of DIXDC1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that DIXDC1 was overexpressed in glioma tissues and glioma cell lines. The expression level of DIXDC1 was evidently linked to glioma pathological grade and Ki-67 expression. Kaplan–Meier curve showed that high expression of DIXDC1 may lead to poor outcome of glioma patients. Serum starvation and refeeding assay indicated that the expression of DIXDC1 was associated with cell cycle. To determine whether DIXDC1 could regulate the proliferation and migration of glioma cells, we transfected glioma cells with interfering RNA-targeting DIXDC1; investigated cell proliferation with Cell Counting Kit (CCK)-8, flow cytometry assays, and colony formation analyses; and investigated cell migration with wound healing assays and transwell assays. According to our data, knockdown of DIXDC1 significantly inhibited proliferation and migration of glioma cells. These data implied that DIXDC1 might participate in the development of glioma, suggesting that DIXDC1 can become a potential therapeutic strategy for glioma.     

Decorin Mimic Inhibits Vascular Smooth Muscle Proliferation and Migration

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.     

环孢霉素A抑制NIT-1胰岛β细胞增殖和胰岛素分泌

为研究免疫抑制剂环孢霉素A对NIT-1胰岛β细胞增殖和胰岛素分泌的影响,体外培养NIT-1胰岛β细胞,用不同浓度的环孢霉素A处理48h和72h,MIT法检测NIT-1胰岛β细胞的增殖情况,放射免疫测定法(RIA)检测胰岛素释放。实验结果显示环孢霉素A处理48h和72h可显著抑制细胞的增殖和胰岛素的释放,可见免疫抑制剂环孢霉素A对NIT-1胰岛β细胞有直接的细胞毒作用。     

MicroRNA-125b Down-regulates Matrix Metallopeptidase 13 and Inhibits Cutaneous Squamous Cell Carcinoma Cell Proliferation, Migration, and Invasion

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.     

STAT2的小分子干扰RNA抑制HeLa细胞增殖

新近研究发现STAT2基因具有致瘤性.前期研究发现:多种肿瘤组织和细胞系高表达STAT2,因此为进一步研究STAT2基因在肿瘤发生发展中的功能,利用RNA基因沉默技术,降低STAT2基因在宫颈癌HeLa细胞系中的内源表达水平,采用XTT实验、软琼脂集落形成实验以及裸鼠体内成瘤实验等研究策略,发现沉默STAT2基因可抑制...     

Cyclin D1 Inhibits Cell Proliferation through Binding to PCNA and Cdk2

Cyclin D1 is known as a promoting factor for cell growth. We previously showed, however, that the expression of cyclin D1 increases markedly in senescent human fibroblastsin vitro.Here we investigate whether the overexpression of cyclin D1 inhibits cell proliferation. Colony formation after transfection with the cyclin D1 expression vector was repressed in NIH-3T3, TIG-1, CHO-K1, and HeLa cells, compared with those with mock and cyclin E expression vectors. A transient transfection assay demonstrated that the overexpression of cyclin D1 inhibited DNA synthesis of TIG-1 cells. The complexes of cyclin D1 with PCNA and cdk2 increased remarkably in senescent cells, compared with young counterparts. Excessive glutathioneS-transferase (GST)–cyclin D1 inhibited DNA replication and repressed cdk2-dependent kinase activityin vitro.DNA synthesis of NIH-3T3 transfectants with PCNA or cdk2 expression vectors was not inhibited by the overexpression of cyclin D1. These results indicate that an excessive level of cyclin D1 represses cell proliferation by inhibiting DNA replication and cdk2 activity through the binding of cyclin D1 to PCNA and cdk2, as it does in senescent cells.     

Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.     

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

Background

Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model.

Methodology/Principal Findings

We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate.

Conclusions/Significance

This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.     

COX10-AS1 Facilitates Cell Proliferation and Inhibits Cell Apoptosis in Glioblastoma Cells at Post-Transcription Level

Neurochemical Research - Glioblastoma (GBM) is an invasive cancer with poor prognosis in patients. Researching on molecular functions in GBM has attracted more and more attention. Actin gamma 1...     

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high P_K⁺/P_Na⁺ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K⁺ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.Gliomas are the most common primary tumors of the central nervous system. These tumors arise either from astrocytes or their progenitor cells (1). Gliomas are divided into four grades based on the degree of malignancy. Glioblastoma multiforme (GBM),² Grade IV, is the most frequently occurring, most invasive, and has the worst prognostic outcome with a median survival of approximately one year from diagnosis (2).We have previously reported the presence of an amiloride-sensitive current in glioblastoma cells that is not seen in normal astrocytes or low grade gliomas (3). Amiloride is a potassium sparing diuretic that inhibits sodium channels composed of subunits from the epithelial sodium channel (ENaC)/Degenerin (Deg) family. Amiloride-sensitive Na⁺ channels are essential for the regulation of Na⁺ transport into cells and tissues throughout the body. These channels are found in all body tissues; from epithelia, endothelia, osteoblasts, keratinocytes, taste cells, lymphocytes, and brain (4). Apart from the ENaCs, the ENaC/Deg family also includes acid-sensing ion channels (ASICs) which have been found predominantly in neurons (4–6). Primary malfunctions of ENaC/Deg family members underlie or are involved in the pathophysiology of several human diseases such as salt-sensitive hypertension (7, 8), pseudohypoaldosteronism type I (7), cystic fibrosis (9), chronic airway diseases (10, 11), and flu (12).The ENaC/Deg family subunits share the same structural topology. They all have short intracellular N and C termini, two transmembrane spanning domains, and a large extracellular cysteine-rich loop (4, 5). There are five ENaC subunits termed α, β, γ, δ, and ϵ. Functional ion channels arise from a multimeric assembly of these subunits. The prototypical ENaC channel of the collecting duct principal cell is thought to be αβγENaC (13, 14). The α-ENaC subunit appears to be the core conducting element, whereas the β- and γ-ENaC subunits are associated with trafficking and insertion of the channel in the cell membrane (13, 15, 16). ASICs are homologous to ENaCs and are most prevalently expressed in the brain and nervous system (17–19), although they are also found in the retina (20–22), testes (23), pituitary gland (24), lung epithelia (22), and bone and cartilage (25). Four ASIC genes have been identified so far, ASIC1–4. Of these, ASIC1–3 has multiple splice variants (19, 22). The crystal structure of chicken ASIC1 has revealed it to be a homotrimer (26). ASICs differ from their ENaC counterparts in that they are transiently activated by extracellular acid (19) and are much less sensitive to inhibition by amiloride (27, 28). Also ASIC1 is inhibited with high affinity by psalmotoxin 1 (PcTX-1), a 40-amino acid peptide found in the venom of the West Indies tarantula, Psalmopoeus Cambridgei (29). ASICs, because they are activated by acidic pH, have been suggested to play a role in chemical pain associated with increased tissue acidification as occurs in ischemia (30, 31). They have also been implicated in touch sensation (32), taste (33), fear-conditioning (6), and learning and memory (34).Our laboratory has proposed that ENaC/Deg channels underlie the basally activated cation current measured in high grade glioma cells (3). We hypothesize that the channels forming this current pathway are composed of a mixture of ASIC and ENaC subunits. RNA profiling of a large number of GBM-derived cell lines and freshly resected tumors have revealed the presence of a myriad of ASIC/ENaC components (3). The basally active current seen in GBM cells can be significantly reduced by amiloride or benzamil (a higher affinity amiloride analog), both of which are inhibitors of the ENaC/Deg family of ion channels (3). PcTX1, a selective ASIC1 blocker, also effectively abolishes the basally active GBM current (35). We have previously shown that ENaC and ASIC subunits can form cross-clade interactions in a heterologous expression system (36). This study aims to probe the composition of the novel ENaC/Deg heteromer in a glioma cell line, D54-MG. Our study postulates that a change in GBM cell electrophysiological properties after subunit knockdown would be indicative of that subunit being a part of the GBM channel. We have sequentially knocked down different ENaC/Deg subunits from the D54-MG glioma cells and measured amiloride-sensitive whole cell current using patch clamp. We found that knocking down various ENaC/Deg subunits significantly reduced the whole cell patch clamp current in glioma cells and changed the resting Na⁺/K⁺ permeability of the these cells. After subunit knockdown, glioma cells showed a reduced cell migration as compared with control cells, consistent with our hypothesis that ENaC/Deg subunits play an important role in glioma cell pathophysiology.     

SARS冠状病毒核衣壳蛋白抑制胞质分裂和细胞增值

目的:SARS冠状病毒(SARS-CoV)的核衣壳蛋白(N蛋白)能够结合病毒RNA形成螺旋状的核衣壳。在3种不同的细胞系中分别表达SARS-CoVN蛋白,研究它对转染细胞生长的影响。方法:将重组质粒pEGFP-N和pEGFP(作为对照)分别转染人胚肾HEK293细胞、成纤维细胞3T3、人宫颈癌HeLa细胞,通过激光共聚焦显微镜、荧光显微镜观察SARS-CoVN蛋白在细胞内的定位以及细胞的生长变化。结果:SARS-CoVN蛋白能定位于细胞质,并不像其他冠状病毒N蛋白那样能够定位到细胞核。同时发现SARS-CoVN蛋白能够诱导形成多核细胞,多核细胞的百分率可达33.9%。结论:SARS-CoVN蛋白抑制胞质分裂,延缓细胞生长。     

鼠源休眠蛋白具有抑制内皮细胞增殖和诱导细胞凋亡的活性

内皮抑素 (endostatin)是最近发现的一种具有抑制血管生长的蛋白质。休眠蛋白 (restin)是内皮抑素的同源蛋白质 ,最早由Ramchandran等人发现 ,它来源于胶原蛋白XV的羧端非胶原蛋白结构域 (NC1)。为了研究鼠源休眠蛋白对内皮细胞生长的影响 ,利用RT PCR从鼠肌肉中扩增出它的基因 ,克隆入原核表达载体pQE32。诱导后 ,重组蛋白质以包涵体形式高效表达 ,表达量约占菌体总蛋白质的 6 0 %～ 70 %。重组蛋白质经纯化复性后 ,可以特异地抑制bFGF刺激的牛主动脉内皮细胞的增殖 ,但是休眠蛋白的抑制活性比内皮抑素活性稍低。流式细胞仪检测发现 ,休眠蛋白可以引起内皮细胞的细胞周期的改变 ,并且引起细胞凋亡     

MicroRNA-146a抑制胶质瘤细胞增殖的研究

目的:检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法:应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果:miR-146a在胶质瘤组织中表达明显降低(P<0.01),相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低(P<0.01),仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论:miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。     

慢病毒介导RNA干扰HPIP蛋白抑制肿瘤细胞增殖

目的:建立高效稳定的造血相关的PBX相互作用蛋白质(HPIP)小干扰RNA(siRNA)细胞导入方法,检测HPIP的表达对肿瘤细胞生长增殖的影响。方法:构建人HPIP慢病毒siRNA干扰载体,将重组质粒转染人胚肾293T细胞,通过实时定量RT-PCR及Western印迹分析检测Lenti-H1 HPIP siRNA的干扰效果;将Lenti-H1 HPIPsiRNA与4个包装质粒共同转染293T细胞,包装成慢病毒后,感染宫颈癌HeLa和肝癌HepG2细胞,经嘌呤霉素筛选2周后,收集细胞进行Western印迹检测;用结晶紫实验检测其对肿瘤细胞生长增殖的影响。结果:构建的Lenti-H1 HPIP siRNA能有效抑制HPIP的表达;结晶紫实验显示,慢病毒介导的HPIP siRNA导致细胞增殖的显著抑制。结论:慢病毒介导的HPIP敲减能明显抑制肿瘤细胞的增殖,HPIP可能是一个潜在的肿瘤治疗新靶点。     

肝门部胆管癌临床诊断与放疗疗效分析

目的：探讨不同手术方式治疗肝门部胆管癌的效果及其对术后放疗的影响。方法：回顾性分析我科自2008年6月至2012年6月间收治的60例肝门部胆管癌患者的临床资料,根据手术方式不同,将所选病例分为两组,其中26例接受根治性切除术,34例患者采用姑息性手术进行治疗。对比不同手术方式下放疗后患者生存情况。结果：围手术期死亡1例。两组患者各有2例患者失访。随访的23例根治手术患者1、3、5年间的生存率为19（82．6％）、10（43．5％）、2（8．7％）;32例姑息性手术患者1、3、5年间的生存率为15（46．9％）、4（12．4％）、0（0％）。根治性手术后患者1、3、5年间的生存率显著性高于姑息性手术治疗的患者,差异具有统计学意义（P〈0．05）。结论：通过术前影像学诊断,选择合理的手术方式,联合术后放疗,可有效延长肝门部胆管癌患者的生存时间。