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Results for “RNA interference”

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PubMed2004

RNA interference-induced reduction in CD98 expression suppresses cell fusion during syncytialization of human placental BeWo cells.

The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion has been studied in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using RNA interference. CD98 protein abundance (determined by Western blot) was decreased by 40-50% following double-stranded small interfering RNA transfection. Cell fusion (determined by quantitative flow cytometry) was similarly inhibited and human chorionic gonadotropin secretion was suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation.

Blotting, Western↗

PubMed2006

RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat.

Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.

Carotenoids↗

PubMed2004

RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells.

DNA methyltransferase 1 (DNMT1) is required to maintain DNA methylation patterns in mammalian cells, and is thought to be the predominant maintenance methyltransferase gene. Recent studies indicate that inhibiting DNMT1 protein expression may be a useful approach for understanding the role of DNA methylation in tumorigenesis. To this end, we used RNA interference to specifically down-regulate DNMT1 protein expression in NCI-H1299 lung cancer and HCC1954 breast cancer cells. RNA interference-mediated knockdown of DNMT1 protein expression resulted in >80% reduction of promoter methylation in RASSF1A, p16(ink4A), and CDH1 in NCI-H1299; and RASSF1A, p16(ink4A), and HPP1 in HCC1954; and re-expression of p16(ink4A), CDH1, RASSF1A, and SEMA3B in NCI-H1299; and p16(ink4A), RASSF1A, and HPP1 in HCC1954. By contrast, promoter methylation and lack of gene expression was maintained when these cell lines were treated with control small interfering RNAs. The small interfering RNA treatment was stopped and 17 days later, all of the sequences showed promoter methylation and gene expression was again dramatically down-regulated, indicating the tumor cells still were programmed for these epigenetic changes. We saw no effects on soft agar colony formation of H1299 cells 14 days after DNMT1 knockdown indicating that either these genes are not functioning as tumor suppressors under these conditions, or that more prolonged knockdown or other factors are also required to inhibit the malignant phenotype. These results provide direct evidence that loss of DNMT1 expression abrogates tumor-associated promoter methylation and the resultant silencing of multiple genes implicated in the pathogenesis of human lung and breast cancer.

Breast Neoplasms↗

PubMed2006

RNA interference-mediated inhibition of brain-derived neurotrophic factor expression increases cocaine's cytotoxicity in cultured cells.

Previous studies showed that cocaine exposure decreased brain-derived neurotrophic factor (BDNF) function and resulted in neuronal cell death. To investigate a role of BDNF in cocaine's cytotoxicity, an RNA interference (RNAi) approach was used. Transfection of neuroblastoma SK-N-AS cells or primary rat hippocampal neurons with the small double-stranded interfering RNA (siRNA) targeting BDNF mRNA, but not the scrambled siRNA, resulted in reductions in levels of BDNF mRNA and proteins by more than 70% in the transfected cells as compared with the control group, suggesting an RNAi-mediated, sequence-specific gene silencing. The results also showed that cocaine-induced cytotoxicity, assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide) assay, was more pronounced in the cells transfected with the siRNA than in the cells transfected with the scrambled siRNA or in the cells treated with Lipofectamine 2000 alone (the control group), suggesting that inhibition of BDNF expression enhances cocaine's cytotoxicity. Together with previous studies showing that cocaine suppresses BDNF expression, the present data suggest that the drug-induced reduction of BDNF productions may make neurons more vulnerable to cocaine's toxic effects and precipitate cocaine-induced central nervous system damages.

Animals↗

PubMed2006

RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells.

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.

Cell Line, Tumor↗

PubMed2005

RNA interference-mediated choline kinase suppression in breast cancer cells induces differentiation and reduces proliferation.

Choline kinase is overexpressed in breast cancer cells and activated by oncogenes and mitogenic signals, making it a potential target for cancer therapy. Here, we have examined, for the first time, the effects of RNA interference (RNAi)-mediated down-regulation of choline kinase in nonmalignant and malignant human breast epithelial cell lines using magnetic resonance spectroscopy (MRS) as well as molecular analyses of proliferation and differentiation markers. RNAi knockdown of choline kinase reduced proliferation, as detected by proliferating cell nuclear antigen and Ki-67 expression, and promoted differentiation, as detected by cytosolic lipid droplet formation and expression of galectin-3. The functional importance of RNAi-mediated choline kinase down-regulation on choline phospholipid metabolism was confirmed by the significant reduction of phosphocholine detected by MRS. These results strongly support the targeting of choline kinase in breast cancer cells with RNAi and show the potential ability of noninvasive MRS to detect and evaluate future treatments incorporating such strategies.

Breast Neoplasms↗

PubMed2003

A CDC45 homolog in Arabidopsis is essential for meiosis, as shown by RNA interference-induced gene silencing.

CDC45 is required for the initiation of DNA replication in yeast and cell proliferation in mammals and functions as a DNA polymerase alpha loading factor in Xenopus. We have cloned a CDC45 homolog from Arabidopsis whose expression is upregulated at the G1/S transition and in young meiotic flower buds. One-third of Arabidopsis 35S:CDC45 T1 RNA interference lines are partially to completely sterile, and the proportion of sterile plants is increased by using a dmc1 promoter. T1 plants have decreased levels of the CDC45 transcript and contain 21- to 23-bp RNA fragments specific to the CDC45 gene. T2 transgenic lines, in which small RNA fragments are still present, were used to analyze S-phase entry by 5-bromodeoxyuridine incorporation, which was not altered compared with that in the wild type. However, microarray data show that other cell cycle genes are upregulated or downregulated. T2 plants also have highly reduced fertility. The severity of the phenotype is correlated with the levels of the CDC45 transcript and small RNA fragments. Severe chromosome fragmentation arising during meiosis, which is not the result of a defect in the repair of SPO11-induced double strand breaks, leads to abnormal chromosome segregation and defective pollen and ovule development.

Arabidopsis↗

PubMedDate not supplied

Antisense- and RNA interference-based therapeutic strategies in allergy.

Modern therapeutic methods for manipulation of gene expression in allergic diseases have been receiving increased attention in the emerging era of functional genomics. With the growing application of gene silencing technologies, pharmacological modulation of translation represents a great advance in molecular therapy for allergy. Several strategies for sequence-specific post-transcriptional inhibition of gene expression can be distinguished: antisense oligonucleotides (AS-ONs), ribozymes (RZs), DNA enzymes (DNAzymes), and RNA interference (RNAi) triggered by small interfering RNAs (siRNAs). Potential anti-mRNA drugs in asthma and other allergic disorders may be targeted to cell surface receptors (adenosine A1 receptor, high-affinity receptor Fc-epsilon RI-alpha, cytokine receptors), adhesion molecules and ligands (ICAM-1, VLA-4), ion channels (calcium-dependent chloride channel-1), cytokines and related factors (IL-4, IL-5, IL-13, SCF, TNF-alpha, TGF-beta1), intracellular signal transduction molecules, such as tyrosine-protein kinases (Syk, Lyn, Btk), serine/ threonine-protein kinases (p38 alpha MAPkinase, Raf-1), non-kinase signaling proteins (RasGRP4), and transcription factors involved in Th2 differentiation and allergic inflammation (STAT-6, GATA-3, NF-kappaB). The challenge to scientists is to determine which of the candidate targets warrants investment of time and resources. New-generation respirable AS-ONs, external guide sequence ribozymes, and RNA interference-based therapies have the potential to satisfy unmet needs in allergy treatment, acting at a more proximal level to a key etiopathogenetic molecular process, represented by abnormal expression of genes. Moreover, antisense and siRNA technologies imply a more rational design of new drugs for allergy.

Animals↗

PubMed2005

In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein.

Multidrug resistance (MDR) remains a major obstacle to successful chemotherapeutic treatment of cancer and can be caused by overexpression of P-glycoprotein, the MDR1 gene product. To further validate a knockdown approach for circumventing MDR, we developed a P-glycoprotein inhibition strategy using short hairpin RNA interference (shRNAi) and now show efficacy and target specificity in vivo. Two of eight tested shRNAi constructs targeted against human MDR1 mRNA inhibited expression of P-glycoprotein by >90%, whereas control shRNAi had no effect. Ablation of P-glycoprotein in cells stably transduced with retroviral-mediated shRNAi was documented by Western blot and functionally confirmed by increased sensitivity of MDR1-transfected cells toward the cytotoxic drugs vincristine, paclitaxel, and doxorubicin as well as by transport of (99m)Tc-Sestamibi. shRNAi-mediated down-regulation of P-glycoprotein transport activity both in cultured cells and in tumor implants in living animals could be followed by direct noninvasive bioluminescence imaging using the Renilla luciferase fluorophore, coelenterazine, a known P-glycoprotein transport substrate. Furthermore, after somatic gene transfer by hydrodynamic infusion of a MDR1-Firefly luciferase (MDR1-FLuc) fusion construct into mouse liver, the effect of shRNAi delivered in vivo on P-glycoprotein-FLuc protein levels was documented with bioluminescence imaging using d-luciferin. ShRNAi against MDR1 reduced bioluminescence output of the P-glycoprotein-FLuc reporter 4-fold in vivo compared with mice treated with control or scrambled shRNAi. Targeted down-regulation of a somatically transferred P-glycoprotein-eGFP fusion reporter also was observed using fluorescence microscopy. Our results show that shRNAi effectively inhibited MDR1 expression and function in cultured cells, tumor implants and mammalian liver, documenting the feasibility of a knockdown approach to reversing MDR in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.

Silencing gene expression by RNA interference (RNAi) can provide insight into gene function but requires efficient delivery of small interfering RNAs (siRNAs) into cells. Introduction of exogenous nucleic acids can be especially difficult in cultured pancreatic islets. This article describes a method for making recombinant adenoviruses that efficiently drive expression of siRNAs in islet beta-cells and a beta-cell-derived cell line. Transduction with a virus expressing an siRNA specific for GLUT2 reduced GLUT2 mRNA and protein levels by 80% in the INS-1-derived beta-cell line, 832/13, and GLUT2 protein levels by >90% in primary rat islets. Another virus expressing an siRNA specific for glucokinase (GK) caused 80% suppression of GK mRNA and 50% suppression of GK protein levels in 832/13 cells. These experiments validate recombinant adenoviral RNAi vectors as a useful tool for suppression of the expression of specific genes in pancreatic islets and beta-cell lines. Advantages of this approach include 1) the high efficiency of adenovirus-mediated gene transfer in insulinoma cell lines and rat islets and 2) the rapidity with which RNAi constructs can be prepared and tested relative to stable-transfection strategies.

Adenoviridae↗

PubMed2005

Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach.

The intragenic heterogeneity encountered in many dominant disease-causing genes represents a significant challenge with respect to development of economically viable therapeutics. For example, 25% of autosomal dominant retinitis pigmentosa is caused by over 100 different mutations within the gene encoding rhodopsin, each of which could require a unique gene therapy. We describe here an RNA interference (RNAi)-based mutation-independent approach, targeting as an example murine rhodopsin. Native transcripts are suppressed by a single RNAi molecular species, whereas transcripts from replacement genes engineered at degenerate third-codon wobble positions are resistant to suppression. We demonstrate suppression of murine rhodopsin transcript by up to 90% with full concomitant expression of replacement transcript and establish the validity of this approach in cell culture, retinal explants, and mouse liver in vivo.

Animals↗

PubMed2005

RNA interference-mediated silencing of the acetyl-CoA-carboxylase-alpha gene induces growth inhibition and apoptosis of prostate cancer cells.

Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)-mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-alpha (ACC-alpha), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-alpha gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-alpha or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumor-associated lipogenesis and suggest that in addition to FAS, ACC-alpha is a potential target for cancer intervention.

Acetyl-CoA Carboxylase↗

PubMed2006

RNA interference-mediated silencing of mitotic kinesin KIF14 disrupts cell cycle progression and induces cytokinesis failure.

KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression.

Adenosine Triphosphatases↗

PubMed2004

Effects of RNA interference-mediated silencing of gamma-secretase complex components on cell sensitivity to caspase-3 activation.

Familial Alzheimer's disease mutations in the presenilin 1 gene (PSEN1) have been previously shown to potentiate caspase activation and apoptosis in transfected cells and transgenic mice. However, the mechanism underlying this effect is not known. We set out to determine whether cellular sensitivity to caspase activation could be affected by modulating presenilin 1 (PS1) processing. PS1 processing was altered using RNA interference (RNAi) aimed at silencing the expression of the genes encoding the four components of the gamma-secretase complex, PSEN1, APH-1, PEN-2, and nicastrin. RNAi for these genes was carried out in naive H4 human neuroglioma cells, as well as H4 cell lines overexpressing either wild-type PSEN1 or the Familial Alzheimer's disease mutant PSEN1-Delta9 (PS1-mutant), that were induced to undergo apoptosis. In wild-type PSEN1 cells, RNAi for PEN-2, as expected, increased levels of full-length PS1 (PS1-FL) and decreased PS1 endoproteolysis. This was accompanied by potentiated caspase-3 activation in response to an apoptotic stimulus. In contrast, nicastrin RNAi, which only decreased levels of PS1-amino-terminal fragment and did not affect PS1-FL levels, had no effect on caspase-3 activation during apoptosis. Surprisingly, in the PS1-mutant cells, RNAi for PEN-2 (and APH-1) did not increase but instead reduced the levels of PS1-FL deleted for exon 9. In turn, this was accompanied by attenuated caspase-3 activation in response to an apoptotic stimulus. Finally, in naive H4 cells, PSEN1 RNAi also attenuated caspase-3 activation in response to an apoptotic stimulus. Collectively, these findings indicate that cellular sensitivity to caspase activation correlates with overall PS1 protein levels, particularly with levels of FL-PS1.

Amyloid Precursor Protein Secretases↗

PubMed2005

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo.

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.

Animals↗

PubMed2006

Acute myelogenous leukemia-derived SMAD4 mutations target the protein to ubiquitin-proteasome degradation.

Disruption of transforming growth factor-beta (TGFB1/TGF-beta) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-beta signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with protein instability; however, the precise mechanism by which mutated Smad4 proteins undergo rapid degradation remains to be elucidated. A missense mutation of the SMAD4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Delta483-552) have been identified in acute myelogenous leukemia. It is not known whether protein instability of these SMAD4 mutants is one of the contributors to TGF-beta signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia-derived SMAD4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that beta-transducin-repeat-containing protein 1 (beta-TrCP1), an F-box protein in the ubiquitin E3 ligase Skp1-Cullin-F-box protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated SMAD4 proteins. In addition, small interference RNA (siRNA)-triggered endogenous beta-TrCP1 suppression increased the protein expression level of both overexpressed SMAD4 mutants and endogenous mutated SMAD4 protein in acute myelogenous leukemia cells. These data suggest that mutated SMAD4 proteins undergo rapid degradation in acute myelogenous leukemia cells via SCF(beta-TrCP1) E3 ligase-mediated protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation.

Animals↗

PubMed2007

The Fanconi anemia (FA) pathway confers glioma resistance to DNA alkylating agents.

DNA alkylating agents including temozolomide (TMZ) and 1,3-bis[2-chloroethyl]-1-nitroso-urea (BCNU) are the most common form of chemotherapy in the treatment of gliomas. Despite their frequent use, the therapeutic efficacy of these agents is limited by the development of resistance. Previous studies suggest that the mechanism of this resistance is complex and involves multiple DNA repair pathways. To better define the pathways contributing to the mechanisms underlying glioma resistance, we tested the contribution of the Fanconi anemia (FA) DNA repair pathway. TMZ and BCNU treatment of FA-proficient cell lines led to a dose- and time-dependent increase in FANCD2 mono-ubiquitination and FANCD2 nuclear foci formation, both hallmarks of FA pathway activation. The FA-deficient cells were more sensitive to TMZ/BCNU relative to their corrected, isogenic counterparts. To test whether these observations were pertinent to glioma biology, we screened a panel of glioma cell lines and identified one (HT16) that was deficient in the FA repair pathway. This cell line exhibited increased sensitivity to TMZ and BCNU relative to the FA-proficient glioma cell lines. Moreover, inhibition of FA pathway activation by a small molecule inhibitor (curcumin) or by small interference RNA suppression caused increased sensitivity to TMZ/BCNU in the U87 glioma cell line. The BCNU sensitizing effect of FA inhibition appeared additive to that of methyl-guanine methyl transferase inhibition. The results presented in this paper underscore the complexity of cellular resistance to DNA alkylating agents and implicate the FA repair pathway as a determinant of this resistance.

Antineoplastic Agents, Alkylating↗

PubMed2006

Alpha 8 integrin expression is required for maintenance of the smooth muscle cell differentiated phenotype.

OBJECTIVE: Vascular smooth muscle cell (VSMC) de-differentiation is a prerequisite for migration from the tunica media to the intima after vascular injury. Integrin cell adhesion molecules participate in VSMC phenotype modulation. Alpha 8 beta 1 integrin is a differentiation marker of VSMCs and its knockdown heightens migration. In the present study, we examined whether or not alpha 8 integrin is required for the maintenance of VSMC differentiated phenotype. METHODS: Alpha 8 integrin in rat VSMC was knocked down by short interference RNA (siRNA) targeting alpha 8 integrin in comparison to a non-silencing siRNA. Cytoskeletal and morphological changes in VSMC were examined by immunofluorescence staining. The expression of phenotype-dependent markers was analyzed by immunoblotting. RESULTS: Alpha 8 integrin gene silencing evoked drastic changes in characteristics of the VSMC differentiated phenotype, including VSMC morphology, actin fibre organization, focal adhesion assembly and the expression of phenotype-dependent markers in favor of de-differentiation. Then, we investigated whether or not phenotype modulation induced by alpha 8 integrin gene silencing could be reversed by an inducer of VSMC differentiation. Transforming growth factor-beta (TGF-beta) failed to upregulate smooth muscle-myosin heavy chain as well as the assembly of parallel actin fibres in VSMCs transfected by siRNA-alpha 8. In addition, TGF-beta-induced vinculin localization at the tip of the cells was impaired by alpha 8 integrin gene silencing. CONCLUSION: These data suggest that alpha 8 integrin expression is required for maintenance of the VSMC differentiated phenotype, a state that is crucial for non-motile VSMCs.

Actins↗