Expression Profile Of Isoforms Research Articles

High expression of the p53 isoform \u03b3 is associated with reduced progression-free survival in uterine serous carcinoma

BackgroundUterine serous carcinoma (USC) is a rare but aggressive subtype of endometrial carcinoma. Large-scale comprehensive efforts have resulted in an improved molecular understanding of its pathogenesis, and the p53 pathway has been proposed as a key player and is potentially targetable. Here we attempt to further portray the p53 pathway in USC by assessing p53 isoform expression.MethodsWe applied quantitative Real-Time PCRs (RT-qPCR) for expression analyses of total p53 mRNA as well as quantitative distinction of p53β, p53γ, and the total mRNA of amino-terminal truncated Δ40p53 and Δ133p53 in a retrospective cohort of 37 patients with USC. TP53 mutation status was assessed by targeted massive parallel sequencing. Findings were correlated with clinical data.ResultsThe p53 isoform expression landscape in USCs was heterogeneous and dominated by total Δ133p53, while the distinct p53β and p53γ variants were found at much lower levels. The isoform expression profiles varied between samples, while their expression was independent of TP53 mutation status. We found high relative p53γ expression to be associated with reduced progression-free survival (PFS).ConclusionsThis is the first indication that elevated p53γ expression is associated with reduced PFS in USC. This single-center study may offer some insight in the landscape of p53 isoform expression in USC, but further validation studies are crucial to understand the context-dependent and tissue-specific role of the p53 isoform network in gynecological cancer.

Acute and long-term NCX activation reduces brain injury and restores behavioral functions in mice subjected to neonatal brain ischemia

Hypoxic-ischemic encephalopathy (HI) accounts for the majority of developmental, motor and cognitive deficits in children, leading to life-long neurological impairments. Since the plasmamembrane sodium/calcium exchanger (NCX) plays a fundamental role in maintaining ionic homeostasis during adult brain ischemia, in the present work we aimed to demonstrate (1)the involvement of NCX in the pathophysiology of neonatal HI and (2)a possible NCX-based pharmacological intervention.HI was induced in neonatal mice at postnatal day 7(P7) by unilateral cut of the right common carotid artery, followed by 60 min exposure to 8%O2. Expression profiles of NCX isoforms from embryos stage to adulthood was evaluated in the hippocampus of hypoxic-ischemic and control mice. To assess the effect of NCX pharmacological stimulation, brain infarct volume was evaluated in brain sections, obtained at several time intervals after systemic administration of the newly synthesized NCX activator neurounina. Moreover, the long term effect of NCX activation was evaluated in adult mice (P60) subjected to neonatal HI and daily treated with neurounina for three weeks.Hypoxic-ischemic insult induced a reduction of NCX1 and NCX3 expression starting from day 7 until day 60. Notably, 8 weeks after HI induction in P7 mice, NCX pharmacological stimulation not only reduced infarct volume but improved also motor behaviour, spatial and visual memory.The present study highlights the significant role of NCX in the evolution of neonatal brain injury and in the learning and memory processes that are impaired in mice injured in the neonatal period.

Abstract 5418: Deciphering the dynamics of alternative pre-mRNA processing of glutaminase in metastatic ovarian cancer

Abstract The chronically proliferative cancer phenotype requires metabolic reprogramming to meet the increased energy and biosynthetic demands of rapid cell division. This results in increased glucose uptake and usage (the Warburg effect). Cancer cells that undergo the Warburg effect may also become reliant on glutamine, a term called ‘glutamine addiction’. Recent studies in ovarian cancer suggest that highly invasive ovarian cancer cells show a remarkable dependence on glutamine, hence implicating glutamine metabolism in metastasis. Glutaminase is the first enzyme that is involved in glutaminolysis and it catalyzes the rate-limiting conversion of glutamine to glutamate and ammonia. There are two genes that code for glutaminase in the human genome, glutaminase 1 (GLS1), and glutaminase 2 (GLS2). GLS2 is associated with cell differentiation whereas GLS1 expression is up-regulated in cancer. Hence, GLS1 had been proposed to be better adapted to meet the altered metabolic needs of the tumor phenotype. GLS1 can be alternatively spliced into two isoforms, KGA and GAC. Although both KGA and GAC have been implicated in cancer cell metabolism, there is still controversy over the actual isoform that is most important for tumorigenesis. Whereas the KGA isoform is repressed by miR-23, there is no documented miRNA repression of GAC suggesting that GAC is more tumorigenic. Using next-generation sequencing, we recently identified an unannotated novel KGA transcript with a truncated 3′ untranslated region (3′UTR) which is more stable than the traditionally known KGA transcript. Our goal is to determine the expression profile of different glutaminase isoforms in the highly lethal gynecological malignancy, ovarian cancer, and their role in tumorigenesis and metastasis. We have developed amplicons to measure total GLS1 and GLS2 transcripts using quantitative real time PCR (qRT-PCR). Although we unexpectedly detected both GLS2 and GLS1 transcripts, the mRNA levels of GLS2 were significantly lower than those of GLS1. To determine the specific GLS1 isoforms expressed we used isoform specific amplicons and detected both GAC and KGA using qRT-PCR. Western blot analysis was able to detect both GAC and KGA protein in one cell line only suggesting that the KGA mRNA we detected was subject to miR-23 repression. Inhibition of total GLS1 activity with a glutaminase inhibitor resulted in decreased colony formation as shown by soft agar assays. In conclusion, the expression profile of different GLS1 isoforms in highly invasive ovarian cancer supports the role of glutamine metabolism in maintaining the metastatic phenotype. This makes glutamine metabolism a viable therapeutic target for metastatic ovarian cancer. Citation Format: Chioniso P. Masamha, Patrick LaFontaine, Bettine E. Gibbs. Deciphering the dynamics of alternative pre-mRNA processing of glutaminase in metastatic ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2017-5418

Abstract 4482: Osteopontin, p53 and PTEN isoforms expression patterns in endometrium carcinoma cell lines

Abstract Total osteopontin (OPN) is overexpressed in endometrium carcinoma (EC) and modifications at TP53 and PTEN genes correspond to major genetic alterations in these tumors. Althought total OPN expression has been correlated to p53 and PTEN expression, no data is available regarding OPN splice variants and their association to p53 and PTEN isoforms expression. OPN has three splicing isoforms (OPN-SI), named OPNa, OPNb and OPNc, while p53 has at least 12 variants, such as p53 (full lenght), p53β, p53γ, Δ40p53, Δ133p53 and Δ160p53. Moreover, PTEN splicing isoforms (PTEN-SI) are named vs-fl (full lenght variant), vs-3a-3c, vs-5a-5d, and vs-D6. We aimed to evaluate the expression profile of OPN, p53 and PTEN isoforms as a first approach to establish their presumptive associations and functional interactions in endometrial tumoral and non tumoral cells. Total RNA has been extracted from endometrial non-tumoral (E6/E7 and EM42) and tumoral (Ishikawa, RL95-2, AN3CA and KLE) cell lines, followed by cDNA synthesis. Transcriptional and protein expression patterns have been evaluated for OPN, p53 or PTEN isoforms, using quantitative real time PCR, immunoblot or immunofluorescence. OPNa is overexpressed in tumoral and non-tumoral cell lines in relation to OPNb and OPNc. Regarding p53, the full lengh isoform is overexpressed, followed by Δ40p53, in relation to other variants in EC cells. Conversely, Δ40p53 it is the major expressed transcript in both EM42 and E6/E7 non-tumoral cells. Otherwise, in EC and endometrial non-tumoral cells, PTEN vs-fl isoform is overexpressed in relation to the other splice variants. Specifically in E6/E7 cells, all PTEN-SI, except vs-3b e vs-D6, are overexpressed in relation to EC cell lines. At the protein level, both p53 and PTEN isoforms are overexpressed in E6/E7 cells, when compared to EC cells. Of note, distinct isoforms for most of these variant transcripts have not been detected at the protein level. Our data demonstrate that full lenght OPN, p53 and PTEN are the major expressed variants in EC tumor cell lines. However, most of these transcript variants display differential expression between endometrial tumor and non-tumoral cells and also among distinct EC cell lines. These data provide early evidence that these variants can differently modulate the expression and functional roles of their full length couterparts. Further work should investigate the impact of OPNa overerexpression on p53 and PTEN isoforms and their putative cellular and molecular roles on modulating p53 and PTEN tumor supressive roles. Citation Format: Vanessa F. Franco, Nataly dos Santos Melo, Iaci N. Soares, Ya-Ting Hsu, Ya-Ting, Tim H. Huang, Wallace M. Araujo, José A. Morgado-Diaz, Jerson L. Silva, Etel Rodrigues Pereira Gimba. Osteopontin, p53 and PTEN isoforms expression patterns in endometrium carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2017-4482

Expression profiles of different glutathione S-transferase isoforms in scallop Chlamys farreri exposed to benzo[a]pyrene and chrysene in combination and alone

Aquatic organisms are increasingly exposed to polycyclic aromatic hydrocarbons (PAHs) due to anthropogenic pressure. This study aimed at evaluating the response of Glutathione S-transferases (GSTs) in scallop Chlamys farreri against benzo[a]pyrene (BaP) and chrysene (CHR) exposure under laboratory conditions. Nine published GST genes were classified into six subfamilies and a new member of rho family was identified for the first time. Twelve GSTs (including nine published GST genes and three in transcriptome established by our laboratory) mRNA transcript levels in the gills, digestive glands, adductor muscle, mantle, testis, ovaries, blood cells of scallops were measured by real-time PCR. The results showed that the mRNA transcript levels of twelve GSTs, except GST-zeta, GST-mu and GST-microsomal, were highest in digestive gland. Accordingly, the mRNA expression levels of GSTs were measured in digestive glands of scallops exposed to BaP (0.1μg/L and 1μg/L), CHR (0.1μg/L and 1μg/L) and their mixtures (0.1μg/L BaP +0.1μg/L CHR and 1μg/L BaP +1μg/L CHR). The results indicated that different GST had specific response to different pollution exposure. In BaP exposure experiment, the mRNA expression level of GST-theta was a potential suitable biomarker. GST-sigma-2 and GST-3, which belonged to sigma class, were sensitive to CHR exposure while GST-microsomal was considered a potential ideal bioindicator to joint exposure of BaP and CHR. In summary, this study investigated the classification of GSTs and provided information about the expression profiles of different class GSTs after PAHs exposure.

Genome-wide mRNA analysis reveals a TUBD1 isoform profile as a potential biomarker for diabetic retinopathy development

Diabetic retinopathy (DR) affects approximately one third of all diabetic subjects and is the leading cause of blindness in young to middle-aged adults in the developed world. While early diagnosis is crucial for preventing DR-associated visual loss, the identification of accessible biomarkers that could lead to presymptomatic recognition of the disease is of great clinical importance. The aim of this work was to investigate the possible involvement of alternative splicing events in DR development by performing a genome-wide transcriptional profiling comparing blood-derived RNA from DR subjects and from diabetic-non DR controls. A total of 95 RNA samples, 67 from patients with bilateral DR and 28 from diabetic patients without DR after a period of at least 10 years with type 2 DM, were compared in a genome-wide transcriptome analysis using the GeneChip® Human Gene 2.0 ST Array which contains probe sets covering all exons of ∼33,500 coding transcripts of annotated genes. Microarray data analysis followed by RT-PCR and cDNA sequencing identified important differential splicing events in TUBD1 (Tubulin, Delta-1) isoforms between DR and DM samples. Specifically, the co-expression of particular TUBD1 isoforms was significantly associated with NPDR risk (p = 0.039 by Pearson's chi-squared test; OR (CI 95%): 8.1 (1.0–72.7)). Analysis of TUBD1 signal pathways and regulating networks using a MetaCore platform showed that HIF-1, a molecule playing an important role in the pathogenesis of DR, is a direct regulator of TUBD1 expression. In conjunction, our data suggest that TUBD1 mRNA isoform expression profile in peripheral blood could be an accessible biomarker for predicting the risk for diabetic retinopathy development.

Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression.

Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab®. New treatments will aim to target the bronchial epithelium–immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air–liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC (p < 0.05); in vitro HBEC re-differentiation from SA displays a particular isoform expression profile compared to MA and HC (p = 0.03); HLA-G*01:06 frequency in MA and SA was significantly higher than in the healthy population (p = 0.03 and p < 0.001, respectively); and IL-13 exposure had no impact on HLA-G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs.

The Role of Insulin Receptor Isoforms in Diabetes and Its Metabolic and Vascular Complications.

The insulin receptor (IR) presents by alternative splicing two isoforms: IRA and IRB. The differential physiological and pathological role of both isoforms is not completely known, and it is determinant the different binding affinity for insulin-like growth factor. IRB is more abundant in adult tissues and it exerts mainly the metabolic actions of insulin, whereas IRA is mainly expressed in fetal and prenatal period and exerts mitogenic actions. However, the change in the expression profile of both IR isoforms and its dysregulation are associated with the development of different pathologies, such as cancer, insulin resistance, diabetes, obesity, and atherosclerosis. In some of them, there is a significant increase of IRA/IRB ratio conferring a proliferative and migratory advantage to different cell types and favouring IGF-II actions with a sustained detriment in the metabolic effects of insulin. This review discussed specifically the role of IR isoforms as well as IGF-IR in diabetes and its associated complications as obesity and atherosclerosis. Future research with new IR modulators might be considered as possible targets to improve the treatment of diabetes and its associated complications.

Knock-Down of the NFAT2 Long and Intermediate Isoforms Leads to CLL Acceleration

NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation.We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice.While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells.In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration DisclosuresNo relevant conflicts of interest to declare.

Development of a Specific Substrate—Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Proteomic Analysis of Parkin Isoforms Expression in Different Rat Brain Areas

PARK2 gene's mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.

Lack of age-related clinical progression in PGC-1α-deficient mice – implications for mitochondrial encephalopathies

Impaired peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) function has been demonstrated in several neurodegenerative diseases, and murine whole-body knockouts of PGC-1α have been considered as models for Huntington’s disease. Recent neuropathological studies, however, rather propose these animals to be morphological models of mitochondrial encephalopathies, with special reminiscence of Kearns-Sayre syndrome. PGC-1α-deficient animals have already been subjected to behavioral assessments; however, the contradictory findings and the paucity of data assessing long-term progression necessitated further examinations. This study provides a comprehensive neurological phenotypic profiling of full-length-(FL-)PGC-1α-deficient mice in a broad age spectrum, with special focus on previously controversial findings, the issue of long-term phenotypic progression, the histopathological assessment of previously non-characterized tissues of potential clinicopathological relevance, and the gene expression profile of novel brain-specific isoforms of PGC-1α. Our findings demonstrate moderate hypomotility with signs of gait and trunk ataxia in addition to severe impairments in coordination and muscle strength in FL-PGC-1α-deficient mice, phenotypic features consistent of a mitochondrial disease. Intriguingly, however, these early alterations did not progress with age, the understanding of which may unveil mechanisms of potential therapeutic relevance, as discussed. The observed phenotype did not associate with retinal or spinal cord alterations, and was accompanied by mild myopathic changes. Based on these, FL-PGC-1α-deficient mice can be regarded not only as morphological but behavioral models of mitochondrial encephalopathies, with an important temporal limitation that has now been clarified. The mechanisms capable of halting a potentially lethal phenotype are to be unveiled, as they may hold therapeutic value for mitochondrial diseases.

Transforming growth factor β-related genes in human retinal pigment epithelial cells after tacrolimus treatment.

The transforming growth factor β (TGFβ) family plays an important role in the pathogenesis of many diseases, including fibrotic pathologies of the eyes. The difficulties of surgical procedures contribute to the search for new treatment strategies for proliferative vitreoretinopathy. Therefore, the aim of this study was to investigate the expression profile of TGFβ isoforms, their receptors, and TGFβ-related genes in human retinal pigment epithelial cells (RPE) after tacrolimus (FK-506) treatment in the presence or absence of lipopolysaccharide (LPS)-induced inflammation. The expression profile was analyzed using oligonucleotide microarrays and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) techniques. Analysis using oligonucleotide microarrays revealed 20 statistically significant differentially expressed TGFβ-related genes after LPS treatment in relation to control cells, and after tacrolimus and LPS treatment in relation to LPS-treated cells. Moreover, our results showed that mRNA levels for TGFβ2 and TGFβR3 after tacrolimus treatment, and for TGFβR3 after tacrolimus and LPS treatment in RPE cells were decreased. In turn, in the presence of LPS-induced inflammation, TGFβ2 mRNA level was increased. These results can be important in regard to the treatment of proliferative vitreoretinopathy, pathogenesis of which is associated with processes regulated by TGFβ, such as inflammation, proliferation, epithelial-mesenchymal transition (EMT), and fibrosis.

Tomato Jasmonic Acid-Deficient Mutant spr2 Seedling Response to Cadmium Stress

The involvement of jasmonic acid (JA) in the plant response to cadmium (Cd) stress has been addressed in some publications by application experiments or analysis of endogenous contents of JA. In this study, we comparatively investigated the response of tomato wild type (WT) and its JA-deficient mutant spr2 to Cd stress aimed at clarifying the role of JA. One-month-old potted plants were exposed to CdCl2 at final concentrations of 5, 25, and 50 mg kg−1 in soil, respectively, for 15 days. The root and leaf Cd contents were dramatically increased, especially in the spr2 plants, in a CdCl2 dose-effect manner. In the Cd dose-dependent inhibitory effect on plant growth, spr2 plants were more obvious than WT plants. This was also reflected by certain physiological and biochemical metabolisms. We analyzed photosynthesis-related parameters including total chlorophyll, actual efficiency of PS2, ratio of variable to maximum chlorophyll fluorescence, and net photosynthetic rate; relative water content, soluble sugar and proline contents, and starch accumulation; oxidative stress and antioxidative defense including malondialdehyde production, electrolyte leakage, H2O2 levels, activities of superoxide dismutase, peroxidase, and catalase, and their isoform expression profiles. The Cd-induced changes in all these parameters supported the conclusion that endogenous JA deficiency enhanced tomato seedling sensitivity to Cd. This implies that JA positively regulates the tomato plant response to Cd stress.

Analysis of the expression of SDF-1 splicing variants in human colorectal cancer and normal mucosa tissues.

C-X-C motif chemokine ligand 12 (CXCL12), also termed stromal cell-derived factor-1 (SDF-1) is a small protein 8-14 kDa in length that is expressed as six isoforms, consisting of SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1θ. All six isoforms are encoded by the single CXCL12 gene on chromosome 10. This gene regulates leukocyte trafficking and is variably expressed in a number of normal and cancer tissues. The potential role of the novel CXCL12 splice variants as components of the CXCR4 axis in cancer development is not fully understood. The present study aimed to analyze the expression profile of the various SDF-1 isoforms and SDF-1 polymorphisms, and the association with the clinicopathological features and overall survival of patients with colorectal cancer (CRC). SDF-1 polymorphism analysis was performed using restriction fragment length polymorphism (RFLP) analysis in 73 histologically confirmed human CRC tissue samples at various stages of disease. The expression pattern of the SDF-1 isoforms was analyzed by reverse transcription-polymerase chain reaction in 40 histologically confirmed human CRC tissue samples obtained at various stages of disease, as well as in matched adjacent normal mucosa samples. The presence of the CXCL12 gene polymorphism rs1801157 demonstrated an association with local progression of the primary tumor, as indicated by the T stage. The frequency of the GG genotype was slightly increased in patients with stage 3 and 4 tumors (78.0%) compared with the incidence of the GA/AA genotype (69.5%; P=0.067). The expression of SDF-1β was associated with the presence of metastases (P=0.0656) and the expression of SDF-1γ was significantly associated with tumor size (P=0.0423). The present study is the first to analyze the association between the expression profile of the chemokine CXCL12 splice variants in human CRC tissues and their clinical relevance. The present results reveal that the CXCL12 G801A polymorphism is a low-penetrance risk factor for the development of CRC, and was associated with the T stage. All six isoforms of SDF-1 were expressed in CRC tissues. The expression of SDF-1β was found to be associated with metastases and SDF-1γ appears to be a possible tumor marker for local tumor progression.

Expression Profile of Human PARK2 Splicing Isoforms in Peripheral Blood Cells and Isolated Lymphomonocytes

Expression Profile of Human PARK2 Splicing Isoforms in Peripheral Blood Cells and Isolated Lymphomonocytes

Investigation of Histone Lysine-Specific Demethylase 5D KDM5D) Isoform Expression in Prostate Cancer Cell Lines: a System Approach.

Background:It is now well-demonstrated that histone demethylases play an important role in developmental controls, cell-fate decisions, and a variety of diseases such as cancer. Lysine-specific demethylase 5D (KDM5D) is a male-specific histone demethylase that specifically demethylates di- and tri-methyl H3K4 at the start site of active gene. In this light, the aim of this study was to investigate isoform/transcript-specific expression profiles of KDM5D in three prostate cancer cell lines, Du-145, LNCaP, and PC3. Methods:Real-time PCR analysis was performed to determine the expression levels of different KDM5D transcripts in the prostate cell lines. A gene regulatory network was established to analyze the gene expression profile. Results:Significantly different expression levels of both isoforms were found among the three cell lines. Interestingly, isoform I was expressed in three cell lines while isoform III did only in DU-145. The expression levels of both isoforms were higher in DU-145 when compared to other cell lines (P<0.0001). The observed expression profile was determined by using regulatory network analyses. Conclusion: The present study, for the first time, not only showed the expression profiles of KDM5D isoforms in prostate cancer cell lines but also evaluated the effects of the gene regulatory network on the expression profile of this gene.

Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

Altered α-synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains.

Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform-specific primers and exon-specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α-synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphilin-1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over-expressed in the striatum and cerebellar cortex, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal control brains. We have focused on brain regions that are severely affected by α-synuclein pathology and neurodegeneration in MSA. The reported changes of isoform expression profiles suggest alterations in the regulation of transcription that may be important for aggregation of α-synuclein in the brain.

CircNet: a database of circular RNAs derived from transcriptome sequencing data.

Circular RNAs (circRNAs) represent a new type of regulatory noncoding RNA that only recently has been identified and cataloged. Emerging evidence indicates that circRNAs exert a new layer of post-transcriptional regulation of gene expression. In this study, we utilized transcriptome sequencing datasets to systematically identify the expression of circRNAs (including known and newly identified ones by our pipeline) in 464 RNA-seq samples, and then constructed the CircNet database (http://circnet.mbc.nctu.edu.tw/) that provides the following resources: (i) novel circRNAs, (ii) integrated miRNA-target networks, (iii) expression profiles of circRNA isoforms, (iv) genomic annotations of circRNA isoforms (e.g. 282 948 exon positions), and (v) sequences of circRNA isoforms. The CircNet database is to our knowledge the first public database that provides tissue-specific circRNA expression profiles and circRNA–miRNA-gene regulatory networks. It not only extends the most up to date catalog of circRNAs but also provides a thorough expression analysis of both previously reported and novel circRNAs. Furthermore, it generates an integrated regulatory network that illustrates the regulation between circRNAs, miRNAs and genes.