Total RNA Samples Research Articles

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

Aquaporin 9 expression and its localization in normal skeletal myofiber

The examination was performed whether aquaporin (AQP) 9 is expressed in normal skeletal muscle at mRNA and protein levels. Gel electrophoresis of the reverse transcription-polymerase chain reaction (RT-PCR) product of total RNA samples of human normal muscles by oligonucleotide primers for human AQP9 showed a band of 221 basepairs, which corresponded to the basepair length between two primers of AQP9. The nucleotide sequence of RT-PCR product coincided with that of human AQP9. Immunoblot analysis revealed that the rabbit and sheep antibodies against the synthetic peptide of the C-terminal cytoplasmic domain of human AQP9 molecule reacted with a protein of approximately 30 kDa molecular weight in extracts of human normal skeletal muscles. Immunohistochemistry with our anti-AQP9 antibodies showed an immunoreaction at the myofiber surface of both type 1 and type 2 fibers with almost equal staining intensity in human skeletal muscles. The implication of AQP9 expression in skeletal myofibers was discussed.

Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer™ capillary electrophoresis system

RNA quality is critical to achieve valid results in microarray experiments and to save resources. The RNA integrity number (RIN) can be measured with minimal sample consumption by microfluidics-based capillary electrophoresis. To determine whether RIN can predict the qualitative outcome of microarray hybridization, we measured RIN in total RNA samples from 484 different experiments by the 2100 Bioanalyzer system and correlated with the percentage of present calls (%pc) of downstream oligonucleotide microarrays. The correlation coefficient for RNA and %pc in all 408 samples for which the bioanalyzer algorithm was able to produce an RIN was 0.475 (p < 0.05), ranging from 0.039 to 0.673 for different tissue- and assay-type subgroups. Multivariate analysis found RIN to be the best predictor of microarray quality as assessed by %pc, outperforming the 28S to 18S ratio. For a %pc threshold of 25% and 35%, we determined optimal cut points for RIN at 7.15 and 8.05, respectively. Using the suggested cut points, RIN can support the final decision whether a certain RNA sample is appropriate for successful microarray hybridization.

AKT1 E17 K pleckstrin homology domain mutation in urothelial carcinoma

The PI3 K/AKT pathway is frequently activated in human cancer. Recently, a G to A point mutation (E17 K) was found in the pleckstrin homology domain of AKT1. We aimed to explore this mutation in cases of urothelial carcinoma. Using chip-based matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer, AKT1 E17 K mutation was searched in 26 total RNA samples obtained from 26 patients known to have urothelial carcinoma. Mutation was found in one out of 26 (3.8%) patients – a 46 year old female with a low grade transitional cell carcinoma located to the lamina propria (Ta disease). Our finding is in line with previous studies showing AKT1 E17 K mutation to be rare. Yet, further studies are required to determine whether this mutation is indeed related to less aggressive disease and carries better prognosis.

Highly Sensitive Determination of microRNA Using Target‐Primed and Branched Rolling‐Circle Amplification

One-nucleotide differences in microRNAs (miRNAs) can be discriminated in an assay based on a branched rolling-circle amplification (BRCA) reaction and fluorescence quantification. With the proposed method miRNA can be detected at concentrations as low as 10 fM, and the miRNA in a total RNA sample of a few nanograms can be determined.

Quantification of Small Non‐Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

MicroRNAs (miRNAs) are highly conserved ∼22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3′-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW) species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant) differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues.

Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System

BackgroundWe assessed NanoString's nCounter™ Analysis System for its ability to quantify gene expression of forty-eight genes in a single reaction with 100 ng of total RNA or an equivalent amount of tissue lysate. In the nCounter™ System, multiplexed gene expression target levels are directly detected, without enzymatic reactions, via two sequence-specific probes. The individual mRNA is captured with one mRNA target sequence-specific capture probe that is used in a post-hybridization affinity purification procedure. The second mRNA target specific-sequence and fluorescent-labeled colored coded probe is then used in the detection with the 3-component complex separated on a surface via an applied electric field followed by imaging. We evaluated reproducibility, accuracy, concordance with quantitative RT-PCR, linearity, dynamic range, and the ability of the system to assay different inputs (matched samples of total RNA from Flash Frozen (FF) and Formalin Fixed Paraffin Embedded Tissues (FFPET), and crude tissue lysates (CTL)).FindingsThe nCounter™ Analysis System provided data equivalent to that produced by Taqman®-based assays for genes expressed within the ranges of the calibration curves (above ~0.5 mRNA copies per human cell based on an assumption of 10 pg of total RNA per cell). System response was linear over more than two orders of magnitude with typical CVs of ~6% for concentrations above 1 fM (105 molecules per mL). Profiling the industry-standard MAQC data set yielded correlation coefficients of >0.83 for intensity values and >0.99 for measured ratios. Ninety percent of nCounter™ ratio measurements were within 1.27–1.33 fold changes of the Taqman® data (0.34–0.41 in log2 scale) for FF total RNA samples.ConclusionThe nCounter™ Analysis System generated robust data for multi-gene expression signatures across three different sample preparation conditions.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Adamts-13 Gene Mutations and Transcription Analysis in Primary Antiphospholipid Syndrome.

Von Willebrand factor (vWF) is a large multimeric protein produced by endothelial cells and megakaryocytes. vWF mediates adhesion of plateletes at sites of vascular injury, and transports factor VIII. After secretion, vWF multimers are cleaved by ADAMTS-13 (A disintegrin –like and metalloprotease with thrombospondin type 1 motif 13) enzyme to smaller multimers. Decreased activity of ADAMTS-13 by autoantibodies, mutations and other etiologies causes thrombotic microangiopathy (intraluminal platelet thrombosis in the microvasculature, thrombocytopenia, and red blood cell fragmentation). Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent arterial and venous thrombosis and fetal losses associated with elevated levels of antiphospholipid antibodies. The pathology of small vessel thrombosis in APS patients has been shown to be related with thrombotic microangiopathy. Recently, Austin et al reported that ADAMTS-13 activity was reduced in patients with APS. They also showed an increase of large vWF multimers in plasma of those patients. In this study, we aimed to investigate ADAMTS-13 mutations which decrease the activity of enzyme and the quantitative gene transcrition rates in patients with primary APS and healthy controls. We evaluated 70 primary APS patients (male to female ratio was 25/45), and age and sex matched healthy controls. Exons 10+11,12,13+14 of ADAMTS-13 gene were amplified with the PCR assay for 365del, Q449stop C508Y mutations and P475S polymorphism analysis. PCR products were cleaved with restriction digestion enzymes and genotyped. C508Y mutation was screened by a DNA sequencer system. Total RNA samples were extracted from peripheral blood and cDNA samples were synthesized with reverse transcriptase. Original primers and probes were designed for the target gene ADAMTS-13 and the reference gene HPRT- 1. cDNA amounts of target and reference genes in patient and healthy control groups were determined by real-time PCR. We did not find C365del, Q449stop mutations and P475S polymorphism in the control group and the patients with APS (Table1.) We used Pfaffl method for transcription analysis and found that mRNA amounts of ADAMTS-13 was significantly decreased in APS group compared to helathy controls (Table 1). Our results may suggest that ADAMTS-13 gene is down regulated at the transcription stage in patients with APS. Transcription factors or existence of other mutation(s) at promotor region may explain this effect. This study demonstrated that ADAMTS 13- vWF related mechanisms may have important role in the development of thrombosis in APS patients.Table 1. Data of transcription analysis and mutations which affect the activity of ADAMTS13.C365delQ449stopP475SC508YADAMTS13 CP*HPRT1 CP*ADAMTS13 mRNA ratio (PAPS/ Control)APS n=70000033,7130,430,5Control n=100000032,2330,05* Cross Point

Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR

The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, beta-actin, and calcyphosine; reference genes found to be stably expressed under gamma-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and beta-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the C (T) values of single reference genes and the geometric mean of the C (T) values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the C (T) values of more than three reference genes validated in the same experimental setting.

Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or beta-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals.

Isolation of an elicitor-stimulated 5-epi-aristolochene synthase gene (gPEAS1) from chili pepper (Capsicum annuum)

Phytoalexins play an important role in the inducible defense responses of plants against diseases caused by fungi. Some enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key intermediate in the biosynthesis of terpenes. It may be converted into several cyclic and some acyclic sesquiterpenic derivatives, sterol precursors, or to geranylgeranyl pyrophosphate according to the requirements of the plant cells. Specific cyclization reactions of FPP are catalyzed by particular sesquiterpene cyclases. The 5-epi-aristolochene synthase (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3′-end specific regions of tobacco and Hyoscyamus muticus inducible sesquiterpene cyclase genes were used with the system for rapid amplification of cDNA ends (3′-RACE) technique to prepare pepper EAS-cDNA fragments (PEAS-cDNA). Three specific PEAS-cDNAs were isolated (PEAS1, PEAS18, and PEAS55). Northern blots of total RNA samples from pepper plant tissues challenged with cellulase or Phytophthora capsici showed different expression levels of the respective transcripts. PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5–8 members was demonstrated by Southern blot.

Promotion of BACE1 mRNA Alternative Splicing Reduces Amyloid β-Peptide Production

Production of the amyloid beta-peptide (Abeta) via sequential proteolytic cleavage of the amyloid precursor protein by beta- and gamma-secretases is strongly implicated in the pathogenesis of Alzheimer disease. The beta-secretase that executes the first cleavage event is a transmembrane aspartyl protease known as beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). BACE1 pre-mRNA is alternatively spliced through the use of alternative splice sites in exons 3 and 4, although the significance of these splicing events is unclear. Here, we quantitatively measured relative levels of BACE1 transcripts and identified a novel splice variant of BACE1. We found a subtle but significant difference in BACE1 splicing between brain and pancreas, indicating the cellular environment can affect BACE1 alternative splicing. Furthermore, we have shown that BACE1 proteins translated from alternatively spliced transcripts have dramatically reduced beta-secretase activity and promotion of BACE1 alternative splicing reduces Abeta production. These findings illustrate the importance of BACE1 alternative splicing in affecting the level of Abeta produced in cells and suggest that targeting regulation of BACE1 alternative splicing is a potential therapeutic strategy for lowering beta-secretase activity.

UP-REGULATED THROMBOXANE PRODUCTION IN THE RAT LIVER WITH BILIARY OBSTRUCTION DOES NOT CONTRIBUTE TO PROMOTE HEPATIC INJURY

This study sought to determine whether in vivo inhibition of thromboxane A2 (TXA2) action contribute to attenuate hepatic damage after bile duct ligation (BDL). Male Wistar rats were assigned to sham operation or BDL. At the time of operation, infusion pump with saline, ozagrel natrium (TXA2 synthase inhibitor), or SQ29548 (TXA2 receptor antagonists) was implanted in the abdominal cavity. Plasma alanine aminotransferase, aspartate aminotransferase, hyaluronic acid, and total bilirubin levels were measured at 4 days after the operation. The levels of plasma TXB2, a stable metabolite of TXA2, were significantly increased after BDL. Gene expression of TXA2 synthase was also significantly upregulated in the liver. Nonetheless, either an inhibition of TXA2 synthesis by ozagrel natrium or a blockade of TXA2 receptor by SQ29548 has no effect in every measured parameter related to hepatic function. These results indicated that despite a highly increased production in the liver, TXA2 is not directly related to the hepatic injury in BDL rats.

Improved microarray gene expression profiling of virus-infected cells after removal of viral RNA

BackgroundSensitivity and accuracy are key points when using microarrays to detect alterations in gene expression under different conditions. Critical to the acquisition of reliable results is the preparation of the RNA. In the field of virology, when analyzing the host cell's reaction to infection, the often high representation of viral RNA (vRNA) within total RNA preparations from infected cells is likely to interfere with microarray analysis. Yet, this effect has not been investigated despite the many reports that describe gene expression profiling of virus-infected cells using microarrays.ResultsIn this study we used coronaviruses as a model to show that vRNA indeed interferes with microarray analysis, decreasing both sensitivity and accuracy. We also demonstrate that the removal of vRNA from total RNA samples, by means of virus-specific oligonucleotide capturing, significantly reduced the number of false-positive hits and increased the sensitivity of the method as tested on different array platforms.ConclusionWe therefore recommend the specific removal of vRNA, or of any other abundant 'contaminating' RNAs, from total RNA samples to improve the quality and reliability of microarray analyses.

Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues

Histopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens.

Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses

The tobacco hornworm Manduca sexta is widely used as a model organism to investigate the biochemical basis of insect physiological processes but little transcriptome information is available. To get a broad view of the larval hemolymph proteins, particularly those related to immunity, we synthesized and sequenced cDNA fragments from a mixture of eight total RNA samples: fat body and hemocytes from larvae injected with killed bacteria, fat body, hemocytes, integument and trachea from naïve larvae, and fat body and hemocytes from wandering larvae. Using massively parallel pyrosequencing, we obtained 95,458 M. sexta expressed sequence tags (ESTs) at an average size of 185 bp per read. A majority of the sequences (69,429 reads) could be assembled into 7231 contigs with an average size of 300 bp, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only ∼8% (606) of the contigs matched known M. sexta cDNA sequences, representing 186 of the 375 unique NCBI entries. The remaining 6625 contigs represented newly discovered cDNA segments from this well studied biochemical model insect. A search of the 7231 contigs using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity ( E-value <1×10 −5). These included 218 previously unknown M. sexta sequences coding for putative defense molecules such as pattern recognition receptors, serine proteinases, serpins, Spätzle, Toll-like receptors, intracellular signaling molecules, and antimicrobial peptides.

MicroRNA expression profiling using microarrays

Microarray technology is a powerful high-throughput tool capable of monitoring the expression of thousands of small noncoding RNAs at once within tens of samples processed in parallel in a single experiment. To conduct a genome-wide analysis of miRNA expression of normal and disease samples, such as cancer, and to distinguish expression signatures associated with diagnosis, prognosis and therapeutic interventions, we have developed a unique miRNA microarray assay on a CodeLink platform. The miRNA array consists of 4,104 probes printed in duplicate. This array can simultaneously profile more than 1,500 mature miRNAs and their corresponding precursors from 474 human and 373 mouse miRNA genes. The full protocol details of the miRNA microarray assay developed by our group are described here, including miRNA oligo probe design, array fabrication and miRNA target preparation (by reverse transcription of total RNA), target-probe hybridization on array, signal detection and data analysis. The assay is simple, can be easily standardized and allows the reproducible profiling of up to 24 total RNA samples within 24 h.

Identification of Differentially‐Expressed MicroRNAs in the Paraventricular Nucleus (PVN) Following Myocardial Infarction (MI)

Alterations in gene regulation in central cardiovascular regulatory regions such as the PVN may play an important role in cardiovascular homeostasis. Given recent evidence suggesting that changes in miRNA expression may be involved in cardiovascular disease, we hypothesized that miRNA expression in the PVN is altered following MI. Microarray analyses for miRNA expression was performed on total RNA samples (n=3) isolated from PVN punches pooled from 5 brains of MI or sham mice using Sangers miRbase V8.0 array specific for mouse. Fifteen miRNAs were differentially regulated (&gt;1.5 fold change between MI and sham mice; 11 up‐regulated, 4 down‐regulated in MI mice). MI‐induced alterations in the expression of mir‐29(a,b,c), mmu‐mir‐9 and ‐9*, and mmu‐mir‐199b were statistically significant (p&lt;0.05; n=3). Up‐regulation of mmu‐mir29c expression was confirmed by real time PCR, and was increased 10‐fold in MI vs. sham mice (p&lt;0.01, n=3). Sequence analysis using miRBase revealed likely interactions of the mmu‐mir‐29 family of micoRNAs with Fos, Agtr2, and MTMR7 genes, some of the highly and specifically expressed genes in neuronal cells. Further studies for confirming the binding of miRNAs to postulated mRNAs are in progress. These studies suggest that differential expression of miRNAs following MI may contribute to changes in gene expression in the PVN involved in MI‐induced heart failure.

Alternate Cyclin D1 mRNA Splicing Modulates p27KIP1 Binding and Cell Migration

Cyclin D1 is an important cell cycle regulator, but in cancer its overexpression also increases cellular migration mediated by p27 KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N terminus as the canonical cyclin D1a isoform but a distinct C terminus. In this study we show that these different isoforms have unique properties with regard to the cellular migration function of cyclin D1. Although they displayed little difference in transcriptional co-repression assays on idealized reporter genes, microarray cDNA expression analysis revealed differential regulation of genes, including those that influence cellular migration. Additionally, whereas cyclin D1a stabilized p27 KIP1 and inhibited RhoA-induced ROCK kinase activity, promoting cellular migration, cyclin D1b failed to stabilize p27 KIP1 or inhibit ROCK kinase activity and had no effect on migration. Our findings argue that alternate splicing is an important determinant of the function of cyclin D1 in cellular migration.