RBM24 controls cardiac QT interval through CaMKIIδ splicing.

Calcium/calmodulin-dependent kinase II delta (CaMKIIδ) is the predominant cardiac isoform and it is alternatively spliced to generate multiple variants. Variable variants allow for distinct localization and potentially different functions in the heart. Dysregulation of CaMKIIδ splicing has been demonstrated to be involved in the pathogenesis of heart diseases, such as cardiac hypertrophy, arrhythmia, and diastolic dysfunction. However, the mechanisms that regulate CaMKIIδ are incompletely understood. Here, we show that RNA binding motif protein 24 (RBM24) is a key splicing regulator of CaMKIIδ. RBM24 ablation leads to the aberrant shift of CaMKIIδ towards the δ-C isoform, which is known to activate the L-type Ca current. In line with this, we found marked alteration in Ca2+ handling followed by prolongation of the ventricular cardiac action potential and QT interval in RBM24 knockout mice, and these changes could be attenuated by treatment with an inhibitor of CaMKIIδ. Importantly, knockdown of RBM24 in human embryonic stem cell-derived cardiomyocytes showed similar electrophysiological abnormalities, suggesting the important role of RBM24 in the human heart. Thus, our data suggest that RBM24 is a critical regulator of CaMKIIδ to control the cardiac QT interval, highlighting the key role of splicing regulation in cardiac rhythm.

RNA-binding protein RBM24 represses colorectal tumourigenesis by stabilising PTEN mRNA.

BACKGROUND: RNA-binding motif protein 24 (RBM24) functions as a splicing regulator, which is critical for organ development and is dysregulated in human cancers. Here, we aim to uncover the biological function of RBM24 in colorectal tumourigenesis. METHODS: Xenograft tumour model, Rbm24 knockout and Apcmin/+ mouse models were utilised. Colorectal cancer cells overexpressing or silencing RBM24 were established. RNA immunoprecipitation (RIP) assay was conducted to detect protein-RNA associations. Gene expression was measured by immunohistochemistry, western blotting, or quantitative PCR (qPCR). RESULTS: Rbm24-knockout mice developed spontaneous colorectal adenomas with lower expression of phosphatase and tensin homolog (PTEN). Immunohistochemical staining for the proliferation markers Ki-67 and pHH3 and BrdU assay showed intestinal hyperplasia in Rbm24-knockout mice compared to wild-type mice. RBM24 expression in colorectal adenoma tissues of Apcmin/+ mouse was downregulated compared with adjacent normal samples and was positively correlated with PTEN expression. In vitro, RBM24 overexpression suppressed cell proliferation, migration, invasion and increased sensitivity to 5-FU or cisplatin in CRC cells. Mechanistically, RBM24 maintained PTEN mRNA stability by directly binding to the GT-rich region at positions 8101-8251 in the 3'-UTR of PTEN mRNA, prolonging the half-life of PTEN mRNA, thereby increasing PTEN expression. Hence, low expression of RBM24 downregulated PTEN mRNA, causing the activation of PI3K-Akt signalling in CRC cells. Furthermore, RBM24 expression in CRC tissues was lower than adjacent normal samples. RBM24 expression was positively correlated with PTEN expression and negatively correlated with Ki-67 level. CRC patients with high RBM24 expression had a favourable outcome. CONCLUSIONS: Taken together, RBM24 expression is markedly lower in colorectal tumours than in para-carcinoma tissues. Rbm24-knockout mice develop spontaneous colorectal adenomas. RBM24 directly binds and stabilises PTEN mRNA, which could cause the suppression of CRC cell proliferation, migration and invasion, thereby repressing colorectal tumourigenesis. These findings support the tumour-suppressive role of RBM24. Targeting RBM24 holds strong promise for the diagnosis and treatment of CRC.

LHPP-Mediated Histidine Dephosphorylation Suppresses the Self-Renewal of Mouse Embryonic Stem Cells.

Self-renewal of embryonic stem cells (ESCs) is orchestrated by a vast number of genes at the transcriptional and translational levels. However, the molecular mechanisms of post-translational regulatory factors in ESC self-renewal remain unclear. Histidine phosphorylation, also known as hidden phosphorylation, cannot be detected by conventional experimental methods. A recent study defined phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) as a histidine phosphatase, which regulates various biological behaviors in cells via histidine dephosphorylation. In this study, the doxycycline (DOX)-induced hLHPP-overexpressing mouse ESCs and mouse LHPP silenced mESCs were constructed. Quantitative polymerase chain reaction (qPCR), western blotting analysis, immunofluorescence, Flow cytometry, colony formation assays, alkaline phosphatase (AP) and bromodeoxyuridine (Brdu) staining were performed. We found that the histidine phosphorylation level was strikingly reduced following LHPP overexpression. Besides, the expression of Oct4 and Lefty1, indispensable genes in the process of ESCs self-renewal, was significantly down-regulated, while markers related to the differentiation were markedly elevated. Moreover, LHPP-mediated histidine dephosphorylation induced G0/G1 phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of Lhpp promoted the self-renewal of mESCs and reversed the RA induced increased expression of genes associated with differentiation. Mechanistically, our findings suggested that the enzymatic active site of LHPP was the cysteine residue at position 226, not 53. LHPP-mediated histidine dephosphorylation lowered the expression levels of ß-catenin and the cell cycle-related genes CDK4 and CyclinD1, while it up-regulated the cell cycle suppressor genes P21 and P27. Taken together, our findings reveal that LHPP-mediated histidine dephosphorylation plays a role in the self-renewal of ESCs. LHPP-mediated histidine dephosphorylation inhibited the self-renewal of ESCs by negatively regulating the Wnt/ß-catenin pathway and downstream cell cycle-related genes, providing a new perspective and regulatory target for ESCs self-renewal.

Rbm24 regulates inner-ear-specific alternative splicing and is essential for maintaining auditory and motor coordination.

Tissue-specific alternative splicing (AS) is emerging as one of the most exciting types of mechanisms associated with organ development and disease. In the auditory system, many hearing-related genes undergo AS, and errors in this process result in syndromic or non-syndromic hearing loss. However, little is known about the factors and mechanisms directing AS in the inner ear. In the present study, we identified a novel RNA-binding protein, Rbm24, which was critically involved in regulating inner-ear-specific AS. Rbm24 deletion resulted in hearing loss and defects in motor coordination. Global splicing analysis showed Rbm24 was required for correct splicing of a subset of pre-mRNA transcripts with essential roles in stereocilia integrity and survival of hair cells. Furthermore, we identified that Rbm24 directly regulated the splicing of Cdh23, a known disease gene responsible for human Usher syndrome 1D and non-syndromic autosomal recessive deafness DFNB12. In conclusion, our findings demonstrated that Rbm24 was a critical factor in regulating inner-ear-specific splicing and maintaining the hearing and motor coordination function of the inner ear. Our data not only offer mechanistic insights but also provide functional annotation of Rbm24 splicing targets that contribute to hearing loss.

Expression of two non-mutated genetic elements is sufficient to stimulate oncogenic transformation of human mammary epithelial cells.

Trefoil factor 3 (TFF3) expression is positively associated with advanced clinicopathological features of mammary carcinoma (MC). Herein, we provide evidence for a functional role of TFF3 in oncogenic transformation of immortalized, but otherwise normal human mammary epithelial cells (HMECs), namely, HMEC-hTERT, MCF10A, and MCF12A. Forced expression of TFF3 in immortalized-HMECs enhanced cell proliferation, cell survival, anchorage-independent growth, produced highly disorganised three-dimensional (3D) acinar structures and generated tumours in immunocompromised mice. Forced expression of TFF3 in immortalized-HMECs stimulated STAT3 activity that was required for TFF3-stimulated cell proliferation, survival, and anchorage-independent growth. TFF3 specifically utilised STAT3 activity to govern a transcriptional program, which was required for TFF3-stimulated oncogenic transformation of immortalized-HMECs, including transcriptional upregulation of CCND1 and BCL2. siRNA-mediated depletion or functional inhibition of STAT3 significantly inhibited the TFF3-stimulated transcription of CCND1 and BCL2 and oncogenicity in immortalized-HMECs. Furthermore, DOX-inducible expression of TFF3 in HMEC-hTERT cells also permitted anchorage-independent growth and produced disorganized acinar structures in 3D Matrigel culture. Removal of DOX-induced expression of TFF3 in HMEC-hTERT cells, previously grown with DOX, resulted in efficient normalisation of the disorganized acinar architecture and attenuated cell viability in Matrigel culture. Cumulatively, these findings suggest that TFF3 is a potent oncogene and its increased expression along with hTERT in HMECs is sufficient to produce oncogenic transformation.

STAT3-Inducible Mouse ESCs: A Model to Study the Role of STAT3 in ESC Maintenance and Lineage Differentiation.

Studies have demonstrated that STAT3 is essential in maintaining self-renewal of embryonic stem cells (ESCs) and modulates ESC differentiation. However, there is still lack of direct evidence on STAT3 functions in ESCs and embryogenesis because constitutive STAT3 knockout (KO) mouse is embryonic lethal at E6.5-E7.5, prior to potential functional role in early development can be assessed. Therefore, in this study, two inducible STAT3 ESC lines were established, including the STAT3 knockout (InSTAT3 KO) and pSTAT3 overexpressed (InSTAT3 CA) using Tet-on inducible system in which STAT3 expression can be strictly controlled by doxycycline (Dox) stimulation. Through genotyping, deletion of STAT3 alleles was detected in InSTAT3 KO ESCs following 24 hours Dox stimulation. Western blot also showed that pSTAT3 and STAT3 protein levels were significantly reduced in InSTAT3 KO ESCs while dominantly elevated in InSTAT3 CA ECSs upon Dox stimulation. Likewise, it was found that STAT3-null ESCs would affect the differentiation of ESCs into mesoderm and cardiac lineage. Taken together, the findings of this study indicated that InSTAT3 KO and InSTAT3 CA ESCs could provide a new tool to clarify the direct targets of STAT3 and its role in ESC maintenance, which will facilitate the elaboration of the mechanisms whereby STAT3 maintains ESC pluripotency and regulates ESC differentiation during mammalian embryogenesis.

Global profiling of Rbm24 bound RNAs uncovers a multi-tasking RNA binding protein.

RNA binding proteins serve as critical molecular switches in a multitude of post-transcriptional regulatory processes. In the heart and muscles, the tissue specific RNA binding protein, Rbm24, is known to play important developmental roles via driving different post-transcriptional processes. Nonetheless, the currently identified molecular targets and regulatory pathways seem inadequate to completely explain the observed developmental effects upon Rbm24 knockdown/knockout. Here, by performing RNA Immunoprecipitation and coupling it to microarrays (RIP-Chip), we have generated an atlas of the mRNA binding repertoire of Rbm24. Further functional evaluation of its targets led to the elucidation of novel roles for Rbm24 in post-transcriptional processing, besides its already known roles in regulation of mRNA stability and alternative splicing. Interestingly, Rbm24 is found to cause the destabilization of Chrm2 via binding to an element in the coding region. In addition, Rbm24 is also found to have an uncharacterized role in driving the generation of isoforms with alternative transcriptional start sites. We have, for the first time, demonstrated that Rbm24 is a multi-tasking RNA binding protein capable of regulating its bound targets via a range of mechanisms.

Corrigendum: Stk38 Modulates Rbm24 Protein Stability to Regulate Sarcomere Assembly in Cardiomyocytes.

This corrects the article DOI: 10.1038/srep44870.

Stk38 Modulates Rbm24 Protein Stability to Regulate Sarcomere Assembly in Cardiomyocytes.

RNA-binding protein Rbm24 is a key regulator of heart development and required for sarcomere assembly and heart contractility. Yet, its underlying mechanism remains unclear. Here, we link serine/threonine kinase 38 (Stk38) signaling to the regulation of Rbm24 by showing that Rbm24 phosphorylation and its function could be modulated by Stk38. Using co-immunoprecipitation coupled with mass spectrometry technique, we identified Stk38 as an endogenous binding partner of Rbm24. Stk38 knockdown resulted in decreased Rbm24 protein level in cardiomyocytes. Further studies using Stk38 kinase inhibitor or activator showed that Rbm24 protein stability was regulated in a kinase activity-dependent manner. Deficiency of Stk38 caused reduction of sarcomere proteins and disarrangement of sarcomere, suggesting that Stk38 is essential for Rbm24 to regulate sarcomere assembly. Our results revealed that Stk38 kinase catalyzes the phosphorylation of Rbm24 during sarcomerogensis and this orchestrates accurate sarcomere alignment. This furthers our understanding of the regulatory mechanism of cardiac sarcomere assembly in both physiologic and pathologic contexts, and uncovers a potential novel pathway to cardiomyopathy through modulating the Stk38/Rbm24 protein activity.

Rbm24 Regulates Alternative Splicing Switch in Embryonic Stem Cell Cardiac Lineage Differentiation.

The transition of embryonic stem cell (ESC) pluripotency to differentiation is accompanied by an expansion of mRNA and proteomic diversity. Post-transcriptional regulation of ESCs is critically governed by cell type-specific splicing. However, little is known about the splicing factors and the molecular mechanisms directing ESC early lineage differentiation. Our study identifies RNA binding motif protein 24 (Rbm24) as a key splicing regulator that plays an essential role in controlling post-transcriptional networks during ESC transition into cardiac differentiation. Using an inducible mouse ESC line in which gene expression could be temporally regulated, we demonstrated that forced expression of Rbm24 in ESCs dramatically induced a switch to cardiac specification. Genome-wide RNA sequencing analysis identified more than 200 Rbm24-regulated alternative splicing events (AS) which occurred in genes essential for the ESC pluripotency or differentiation. Remarkably, AS genes regulated by Rbm24 composed of transcriptional factors, cytoskeleton proteins, and ATPase gene family members which are critical components required for cardiac development and functionality. Furthermore, we show that Rbm24 regulates ESC differentiation by promoting alternative splicing of pluripotency genes. Among the Rbm24-regulated events, Tpm1, an actin filament family gene, was identified to possess ESC/tissue specific isoforms. We demonstrated that these isoforms were functionally distinct and that their exon AS switch was essential for ESC differentiation. Our results suggest that ESC's switching into the differentiation state can be initiated by a tissue-specific splicing regulator, Rbm24. This finding offers a global view on how an RNA binding protein influences ESC lineage differentiation by a splicing-mediated regulatory mechanism. Stem Cells 2016;34:1776-1789.

Evaluation of two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods for the analysis of baclofen and gabapentin in feeds by liquid chromatography tandem mass spectrometry.

This paper presents a new analytical method for the simultaneous determination of baclofen and gabapentin in feeds based on two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods and liquid chromatography tandem mass spectrometry (LC-MS/MS). For the two modified QuEChERS methods, samples were first extracted with acidified acetonitrile (5.0% acetic acid, v/v) without using acetonitrile salting-out extraction. Then, the first modified QuEChERS method was established according to the original QuEChERS cleanup procedure. For the second modified QuEChERS method, the extract was evaporated to dryness and reconstituted in acetonitrile. Subsequently, the analytes in the reconstituted solution were retained by primary secondary amine (PSA) and released from PSA with 1.0% formic acid in methanol. Finally, the eluate was evaporated and dissolved in 0.1% formic acid solution/methanol (v/v, 80:20). All of the samples were analyzed by LC-MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as the mobile phase with gradient elution. The matrix effect, recovery, and repeatability, within laboratory reproducibility, and the LODs and LOQs of the two modified QuEChERS sample preparation methods were investigated and compared. Comparative results showed that the second method was obviously superior to the first method.

Simultaneous determination of six resorcylic acid lactones in feed using liquid chromatography-tandem mass spectrometry and multi-walled carbon nanotubes as a dispersive solid phase extraction sorbent.

A simple and cost-effective pre-treatment procedure was developed for six resorcylic acid lactones (RALs) in feed using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra-high performance liquid chromatography-negative electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). After extraction with acetonitrile/water (80:20, v/v) and dilution with water, a dSPE procedure was carried out with MWCNTs. The pH value of the extract, the extraction time for MWCNTs, the type and amount of MWCNTs and the type of eluent were optimised to increase the sample throughput and the sensitivity. The samples were quantified using the internal standard zearalenone-D6. The absolute recoveries of the target compounds from feed samples were most efficient when using 100mg of MWCNTs with an outer diameter of less than 8nm and a length of 10-30µm, and ethyl acetate was shown to be the most suitable solvent for desorbing the target compounds from the MWCNTs. The mean recoveries from fortified swine mixed feed samples ranged from 95.3% to 107.2% and had relative standard deviations lower than 10%; the limits of detection and quantification for RALs were in the ranges of 0.20-0.29µg/kg and 0.54-0.78µg/kg, respectively.

Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for determination of 10 cephalosporins and desacetylcefapirin in milk.

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 10 cephalosporins and desacetylcefapirin in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Samples were directly purified through HLB cartridge after dilution with 50mM phosphate buffer solution (pH 8.5). Then the eluate was dried under nitrogen and the residue was redissolved in mobile phase. Samples were analyzed by LC-MS/MS on an Acquity UPLC BEH Shield RP18 column with gradient elution. The samples were quantified using ceftiofur-D3 as internal standard. The proposed method was validated according to the European Commission Decision 2002/657/EC. The CCα values were 111, 0.04, 140, 55, 55, 67, 23, 23, 68, 0.10 and 113µg/kg for cefalexin, cefradine, cefacetrile, cefazolin, cefoperazone, cefapirin, cefalonium, cefquinome, desacetylcefapirin, cefotaxime and ceftiofur, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CVr), and reproducibility (CVR) varied from 94.6% to 117.1%, from 5.6% to 13.6% (CVr), and from 5.9% to 27.9% (CVR), respectively. The method is demonstrated to be suitable for the determination of 10 cephalosporins and desacetylcefapirin in bovine milk. The total time required for the analysis of one sample, including sample preparation, was about 40min.

Perspective from the heart: the potential of human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cells (hPSC) are self-renewing cells with the potential to differentiate into a variety of human cells. They hold great promise for regenerative medicine and serve as useful in vitro models for studying human biology. For the past few years, there is vast interest in applying these cells to advance cardiovascular medicine. Human cardiomyocytes can be readily generated from hPSC and they have been characterized extensively with regards to molecular and functional properties. They have been transplanted into animal models of cardiovascular diseases and also shown to be potentially useful reagents for drug discovery. Yet, despite great progress in this field, significant technical hurdles remain before these cells could be used clinically or for pharmaceutical research and development. Further research using novel approaches will be required to overcome these bottlenecks.

RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility.

AIMS: The factors responsible for cardiomyopathy are not fully understood. Our studies of the transcriptome of human embryonic stem cell-derived cardiomyocytes identified novel genes up-regulated during cardiac differentiation, including RBM24. We therefore studied how its deficiency affected heart development. METHODS AND RESULTS: The expression of Rbm24 was detected in mouse cardiomyocytes and embryonic myocardium of zebrafish at the RNA and protein level. The Rbm24 loss-of-function showed that Rbm24 deficiency resulted in a reduction in sarcomeric proteins, Z-disc abnormality, and diminished heart contractility, resulting in the absence of circulation in zebrafish embryos. Gene expression profiling revealed down-regulation of multiple pathways associated with sarcomere assembly and vasculature development in Rbm24 deficiency. CONCLUSION: We identified a novel role of the tissue-specific RNA-binding protein (RBP) Rbm24 involving in the regulation of cardiac gene expression, sarcomeric assembly, and cardiac contractility. This study uncovers a potential novel pathway to cardiomyopathy through down-regulation of the RBP Rbm24.

Global expression profile of highly enriched cardiomyocytes derived from human embryonic stem cells.

Human embryonic stem cells (hESC), with their ability to differentiate into cardiomyocytes in culture, hold great potential for cell replacement therapies and provide an in vitro model of human heart development. A genomewide characterization of the molecular phenotype of hESC-derived cardiomyocytes is important for their envisioned applications. We have employed a lineage selection strategy to generate a pure population of cardiomyocytes (>99%) from transgenic hESC lines. Global gene expression profiling showed that these cardiomyocytes are distinct from pluripotent and differentiated hESC cultures. Pure cardiomyocytes displayed similarities with heart tissue, but in many aspects presented an individual transcriptome pattern. A subset of 1,311 cardiac-enriched transcripts was identified, which were significantly overpresented (p < .01) in the Gene Ontology (GO) categories of heart function and heart development. Focused analysis of the GO categories ion transport, sarcomere, and heart development uncovered a unique molecular signature of hESC cardiomyocytes. Pathway analysis revealed an extensive cardiac transcription factor network and novel peroxisome proliferator-activated receptor signaling components within the cardiac-enriched genes. Notably, approximately 80% of these genes were previously uncharacterized. We have evaluated the biological relevance of four candidates-Rbm24, Tcea3, Fhod3, and C15orf52-by in situ hybridization during early mouse development and report that all were prominently expressed in cardiac structures. Our results provide the fundamental basis for a comprehensive understanding of gene expression patterns of hESC cardiomyocytes and will greatly help define biological processes and signaling pathways involved in hESC cardiomyogenic differentiation and in human heart development.

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.

Recursive SVM feature selection and sample classification for mass-spectrometry and microarray data.

BACKGROUND: Like microarray-based investigations, high-throughput proteomics techniques require machine learning algorithms to identify biomarkers that are informative for biological classification problems. Feature selection and classification algorithms need to be robust to noise and outliers in the data. RESULTS: We developed a recursive support vector machine (R-SVM) algorithm to select important genes/biomarkers for the classification of noisy data. We compared its performance to a similar, state-of-the-art method (SVM recursive feature elimination or SVM-RFE), paying special attention to the ability of recovering the true informative genes/biomarkers and the robustness to outliers in the data. Simulation experiments show that a 5%- approximately 20% improvement over SVM-RFE can be achieved regard to these properties. The SVM-based methods are also compared with a conventional univariate method and their respective strengths and weaknesses are discussed. R-SVM was applied to two sets of SELDI-TOF-MS proteomics data, one from a human breast cancer study and the other from a study on rat liver cirrhosis. Important biomarkers found by the algorithm were validated by follow-up biological experiments. CONCLUSION: The proposed R-SVM method is suitable for analyzing noisy high-throughput proteomics and microarray data and it outperforms SVM-RFE in the robustness to noise and in the ability to recover informative features. The multivariate SVM-based method outperforms the univariate method in the classification performance, but univariate methods can reveal more of the differentially expressed features especially when there are correlations between the features.

Gene expression profiling to identify oncogenic determinants of autocrine human growth hormone in human mammary carcinoma.

We have exploited a discrepancy in the oncogenic potential of autocrine and exogenous human growth hormone (hGH) in an attempt to identify molecules that could potentially be involved in oncogenic transformation of the human mammary epithelial cell. Microarray analysis of 19,000 human genes identified a subset of 305 genes in a human mammary carcinoma cell line that were remarkably different in their response to autocrine and exogenous hGH. Autocrine and exogenous hGH also regulated 167 common genes. Semiquantitative reverse transcription-PCR confirmed differential regulation of genes by either autocrine or exogenous hGH. Functional analysis of one of the identified autocrine hGH-regulated genes, TFF3, determined that its expression is sufficient to support anchorage-independent growth of human mammary carcinoma cells. Small interfering RNA-mediated knockdown of TFF3 concordantly abrogated anchorage-independent growth of mammary carcinoma cells and abrogated the ability of autocrine hGH to stimulate oncogenic transformation of immortalized human mammary epithelial cells. Further functional characterization of the identified subset of specifically autocrine hGH regulated genes will delineate additional novel oncogenes for the human mammary epithelial cell.

Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state.

Human embryonic stem cells (hESCs) are an important source of stem cells in regenerative medicine, and much remains unknown about their molecular characteristics. To develop a detailed genomic profile of ESC lines in two different species, we compared transcriptomes of one murine and two different hESC lines by massively parallel signature sequencing (MPSS). Over 2 million signature tags from each line and their differentiating embryoid bodies were sequenced. Major differences and conserved similarities between species identified by MPSS were validated by reverse transcription polymerase chain reaction (RT-PCR) and microarray. The two hESC lines were similar overall, with differences that are attributable to alleles and propagation. Human-mouse comparisons, however, identified only a small (core) set of conserved genes that included genes known to be important in ESC biology, as well as additional novel genes. Identified were major differences in leukemia inhibitory factor, transforming growth factor-beta, and Wnt and fibroblast growth factor signaling pathways, as well as the expression of genes encoding metabolic, cytoskeletal, and matrix proteins, many of which were verified by RT-PCR or by comparing them with published databases. The study reported here underscores the importance of cross-species comparisons and the versatility and sensitivity of MPSS as a powerful complement to current array technology.