A novel antisense lncRNA NT5E promotes progression by modulating the expression of SYNCRIP and predicts a poor prognosis in pancreatic cancer.

A novel antisense lncRNA NT5E was identified in a previous microarray that was clearly up-regulated in pancreatic cancer (PC) tissues. However, its biological function remains unclear. Thus, we aimed to explore its function and clinical significance in PC. The lncNT5E expression was determined in PC specimens and cell lines. In vitro and in vivo studies detected the impact of lncNT5E depletion on PC cell proliferation, migration and invasion. Western blotting investigated the epithelial-mesenchymal transition (EMT) markers. The interaction between lncNT5E and the promoter region of SYNCRIP was detected by dual-luciferase reporter assay. The role of lncNT5E in modulating SYNCRIP was investigated in vitro. Our results showed that lncNT5E was significantly up-regulated in PC tissues and cell lines and associated with poor prognosis. LncNT5E depletion inhibited PC cell proliferation, migration, invasion and EMT in vitro and caused tumorigenesis arrest in vivo. Furthermore, SYNCRIP knockdown had effects similar to those of lncNT5E depletion. A significant positive relationship was observed between lncNT5E and SYNCRIP. Moreover, the dual-luciferase reporter assays indicated that lncNT5E depletion significantly inhibited SYNCRIP promoter activity. Importantly, the malignant phenotypes of lncNT5E depletion were rescued by overexpressing SYNCRIP. In conclusion, lncNT5E predicts poor prognosis and promotes PC progression by modulating SYNCRIP expression.

Lowly expressed lncRNA PVT1 suppresses proliferation and advances apoptosis of glioma cells through up-regulating microRNA-128-1-5p and inhibiting PTBP1.

BACKGROUND: Glioma is a primary intracranial malignancy with poor prognosis, of which the pathogenesis remains to be elucidated. Therein, the aim of this study is to discuss the impacts of lncRNA plasmacytoma variant translocation 1 (PVT1)/microRNA-128-1-5p (miR-128-1-5p)/polypyrimidine tract-binding protein 1 (PTBP1) axis on the biological characteristics of glioma cells. METHODS: Glioma tissue samples (72 cases) and normal brain tissue samples (35 cases) were harvested. The expression of PVT1, miR-128-1-5p and PTBP1 in glioma tissues and cells was detected. Glioma cells were transfected with sh-PVT1, miR-128-1-5p mimics or miR-128-1-5p inhibitors to verify the impacts of PVT1 and miR-128-1-5p on DNA damage, cell colony formation, invasion, proliferation, migration and apoptosis of glioma U87 and U251 cells. The growth of transplanted tumor was tested by tumor xenograft in nude mice. The combination of PVT1 and miR-128-1-5p and the targeting relationship between miR-128-1-5p and PTBP1 were verified. RESULTS: PVT1 and PTBP1 expression was enhanced and miR-128-1-5p expression was degraded in glioma tissues and cells. Overexpressed miR-128-1-5p and lowly-expressed PVT1 promoted DNA damage, suppressed colony formation, invasion, proliferation and migration as well as boosted apoptosis of U251 and U87 cells. Up-regulating miR-128-1-5p and down-regulating PVT1 reduced transplanted tumor volume and weight of glioma in mice. Low expression miR-128-1-5p reversed the effect of low expression PVT1 on the biological characteristics of glioma cells. PVT1 specifically bound to miR-128-1-5p and PTBP1 was the target gene of miR-128-1-5p. CONCLUSION: This study suggests that down-regulated PVT1 or up-regulated miR-128-1-5p boosts apoptosis and attenuates proliferation of glioma cells by inhibiting PTBP1 expression. This study is essential for finding new therapeutic targets for glioma.

Long noncoding RNA HCG22 suppresses proliferation and metastasis of bladder cancer cells by regulation of PTBP1.

Multifarious biological functions of long noncoding RNAs (lncRNAs) have been reported in various cancers including bladder cancer (BCa). This study aims to determine the biological role of a certain lncRNA in BCa. Consistent with the data of The Cancer Genome Atlas database, it was validated that lncRNA HLA complex group 22 (HCG22) was weakly expressed in BCa samples and lowly expressed HCG22 was closely correlated with low overall survival of the BCa patient. To verify the role of HCG22 in BCa progression, functional experiments were carried out in two representative BCa cells (J82 and T24) and the negative effects of HCG22 expression on the cell proliferation, migration, and epithelial-mesenchymal transition were identified. Mechanistically, polypyrimidine tract-binding protein 1 (PTBP1), which was highly expressed in BCa tissues and cell lines, was negatively regulated by HCG22 and the PTBP1-mediated Warburg effect was also obstructed by HCG22. Furthermore, HCG22 modulated the expression of PTBP1 through destabilizing human antigen R (HuR). And functional rescue assays confirmed that HCG22 functioned in bladder cancer through downregulating PTBP1. In conclusion, the present study revealed that HCG22 inhibited BCa progression via the HuR/PTBP1 axis, opening new prospects for potent therapeutic regimens for BCa patients.

Glucose Controls the Expression of Polypyrimidine Tract-Binding Protein 1 via the Insulin Receptor Signaling Pathway in Pancreatic ß Cells.

In pancreatic ß cells, glucose stimulates the biosynthesis of insulin at transcriptional and post-transcriptional levels. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), also named hnRNP I, acts as a critical mediator of insulin biosynthesis through binding to the pyrimidine-rich region in the 3'-untranslated region (UTR) of insulin mRNA. However, the underlying mechanism that regulates its expression in ß cells is unclear. Here, we report that glucose induces the expression of PTBP1 via the insulin receptor (IR) signaling pathway in ß cells. PTBP1 is present in ß cells of both mouse and monkey, where its levels are increased by glucose and insulin, but not by insulin-like growth factor 1. PTBP1 levels in immortalized ß cells established from wild-type (ßIRWT) mice are higher than levels in ß cells established from IR-null (ßIRKO) mice, and ectopic re-expression of IR-WT in ßIRKO cells restored PTBP1 levels. However, PTBP1 levels were not altered in ßIRKO cells transfected with IR-3YA, in which the Tyr1158/1162/1163 residues are substituted with Ala. Consistently, treatment with glucose or insulin elevated PTBP1 levels in ßIRWT cells, but not in ßIRKO cells. In addition, silencing Akt significantly lowered PTBP1 levels. Thus, our results identify insulin as a pivotal mediator of glucose-induced PTBP1 expression in pancreatic ß cells.

Post-Translational Modifications in Polypyrimidine Tract Binding Proteins PTBP1 and PTBP2.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue-specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post-translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N-terminal, linker 1, and linker 2 regions. We find that the two proteins have many overlapping acetate modifications in the RNA recognition motifs (RRMs) with a few distinct sites in PTBP1 RRM2 and RRM3. Our data also reveal that lysine residues in the nuclear localization sequence of PTBP2 are acetylated. Collectively, our results highlight important differences in post-translational modifications between the paralogs and suggest a role for them in the differential splicing activity of PTBP1 and PTBP2.

Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA.

The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56+ TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56+ TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens.

Securinine enhances SMN2 exon 7 inclusion in spinal muscular atrophy cells.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron gene (SMN1) on chromosome 5q13. A second copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is truncated and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. In this study, we screened for the compounds that enhance SMN2 exon 7 inclusion by using SMN2 minigene-luciferase reporter system. We found that securinine can increase luciferase activity, indicating that securinine promoted SMN2 exon 7 inclusion. In addition, securinine increased full-length SMN2 mRNA and SMN protein expression in SMA patient-derived lymphoid cell lines. To investigate the mechanism of securinine effect on SMN2 splicing, we compared the protein levels of relevant splicing factors between securinine-treated and untreated cells. We found that securinine downregulated hnRNP A1 and Sam68 and upregulated Tra2-ß1 expression. However, securinine, unlike HDAC inhibitors, did not enhance tra2-ß1 gene transcription, indicating a post-transcriptional mechanism for Tra2-ß1 upregulation. Furthermore, we treated SMA-like mice with securinine by i.p. injection and found that securinine treatment increased SMN2 exon 7 inclusion and SMN protein expression in the brain and spinal cord. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease.

Prenatal arsenic exposure alters REST/NRSF and microRNA regulators of embryonic neural stem cell fate in a sex-dependent manner.

Exposure to arsenic, a common environmental toxin found in drinking water, leads to a host of neurological pathologies. We have previously demonstrated that developmental exposure to a low level of arsenic (50ppb) alters epigenetic processes that underlie deficits in adult hippocampal neurogenesis leading to aberrant behavior. It is unclear if arsenic impacts the programming and regulation of embryonic neurogenesis during development when exposure occurs. The master negative regulator of neural-lineage, REST/NRSF, controls the precise timing of fate specification and differentiation of neural stem cells (NSCs). Early in development (embryonic day 14), we observed increased expression of Rest, its co-repressor, CoREST, and the inhibitory RNA binding/splicing protein, Ptbp1, and altered expression of mRNA spliced isoforms of Pbx1 that are directly regulated by these factors in the male brain in response to prenatal 50ppb arsenic exposure. These increases were concurrent with decreased expression of microRNA-9 (miR-9), miR-9*, and miR-124, all of which are REST/NRSF targets and inversely regulate Rest expression to allow for maturation of NSCs. Exposure to arsenic decreased the formation of neuroblasts in vitro from NSCs derived from male pup brains. The female response to arsenic was limited to increased expression of CoREST and Ptbp2, an RNA binding protein that allows for appropriate splicing of genes involved in the progression of neurogenesis. These changes were accompanied by increased neuroblast formation in vitro from NSCs derived from female pups. Unexposed male mice express transcriptomic factors to induce differentiation earlier in development compared to unexposed females. Thus, arsenic exposure likely delays differentiation of NSCs in males while potentially inducing precocious differentiation in females early in development. These effects are mitigated by embryonic day 18 of development. Arsenic-induced dysregulation of the regulatory loop formed by REST/NRSF, its target microRNAs, miR-9 and miR-124, and RNA splicing proteins, PTBP1 and 2, leads to aberrant programming of NSC function that is perhaps perpetuated into adulthood inducing deficits in differentiation we have previously observed.

Synaptotagmin 7 and SYNCRIP proteins are ubiquitously expressed in the rat brain and co-localize in Purkinje neurons.

Synaptotagmin 7 (SYT7) is ubiquitously expressed calcium sensor, involved in neuronal membrane trafficking. Immunoprecipitation experiments demonstrated that SYT7 interacts with Synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a component of mRNA granules, which are transported to dendrites and are prerequisite for synaptic plasticity. Given the potential significance of SYT7 regulation in processes of neurodegeneration, which are characterized by high level of synaptic vulnerability, we aimed to analyse and compare the distribution of SYT7 and SYNCRIP proteins in the adult rat striatum, hippocampus, cerebral and cerebellar cortex. We investigated the degree of SYT7-SYNCRIP co-localization in order to examine possible functional interaction of these two proteins. We found that SYT7 is abundantly distributed in neuropil of all examined anatomical areas of the brain, most prominently in axons. On the contrary, SYNCRIP had cytoplasmic somatodendritic pattern of expression, which was most prominent in the hippocampus and cerebellum. In the striatum, hippocampus and cerebral cortex SYT7 and SYNCRIP immunofluorescent signals were mutually excluded, thus diminishing the probability for their physiological interaction. In somata of Purkinje neurons in the cerebellar cortex, both SYT7 and SYNCRIP were expressed and partially co-localized suggesting possible functional connection between SYT7 and SYNCRIP proteins in Purkinje neurons.

Transcriptome sequencing reveals aberrant alternative splicing in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene-encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component, however, has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of the BA4 (Brodmann area 4) motor cortex from seven human HD brains and seven controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using two expanded panels with a total of 108 BA4 tissues from patients and controls, we identified four splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD.

PCBP1/HNRNP E1 Protects Chromosomal Integrity by Translational Regulation of CDC27.

UNLABELLED: CDC27 is a core component of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, whose oscillatory activity is responsible for the metaphase-to-anaphase transition and mitotic exit. Here, in normal murine mammary gland epithelial cells (NMuMG), CDC27 expression is controlled posttranscriptionally through the RNA binding protein poly(rC) binding protein 1 (PCBP1)/heterogeneous nuclear ribonucleoprotein E1 (HNRNP E1). shRNA-mediated knockdown of HNRNP E1 abrogates translational silencing of the Cdc27 transcript, resulting in constitutive expression of CDC27. Dysregulated expression of CDC27 leads to premature activation of the G2-M-APC/C-CDC20 complex, resulting in the aberrant degradation of FZR1/CDH1, a cofactor of the G1 and late G2-M-APC/C and a substrate normally reserved for the SCF-ßTRCP ligase. Loss of CDH1 expression and of APC/C-CDH1 activity, upon constitutive expression of CDC27, results in mitotic aberrations and aneuploidy in NMuMG cells. Furthermore, tissue microarray of breast cancer patient tumor samples reveals high CDC27 levels compared with nonneoplastic breast tissue and a significant correlation between disease recurrence and CDC27 expression. These results suggest that dysregulation of HNRNP E1-mediated translational regulation of Cdc27 leads to chromosomal instability and aneuploidy and that CDC27 expression represents a significant predictor of breast cancer recurrence. IMPLICATIONS: The RNA-binding protein HNRNP E1 mediates translational regulation of the cell-cycle regulator CDC27 and that dysregulation of CDC27 leads to aneuploidy. In addition, high CDC27 expression in breast cancer patient tumor specimens significantly predicts disease recurrence, suggesting a novel role for CDC27 as a predictor of relapse. Mol Cancer Res; 14(7); 634-46. ©2016 AACR.

RBM4a-regulated splicing cascade modulates the differentiation and metabolic activities of brown adipocytes.

RNA-binding motif protein 4a (RBM4a) reportedly reprograms splicing profiles of the insulin receptor (IR) and myocyte enhancer factor 2C (MEF2C) genes, facilitating the differentiation of brown adipocytes. Using an RNA-sequencing analysis, we first compared the gene expressing profiles between wild-type and RBM4a(-/-) brown adipocytes. The ablation of RBM4a led to increases in the PTBP1, PTBP2 (nPTB), and Nova1 proteins, whereas elevated RBM4a reduced the expression of PTBP1 and PTBP2 proteins in brown adipocytes through an alternative splicing-coupled nonsense-mediated decay mechanism. Subsequently, RBM4a indirectly shortened the half-life of the Nova1 transcript which was comparatively stable in the presence of PTBP2. RBM4a diminished the influence of PTBP2 in adipogenic development by reprogramming the splicing profiles of the FGFR2 and PKM genes. These results constitute a mechanistic understanding of the RBM4a-modulated splicing cascade during the brown adipogenesis.

Downregulated Poly-C binding protein-1 is a novel predictor associated with poor prognosis in Acute Myeloid Leukemia.

BACKGROUND: Depletion of Poly-C binding protein-1(PCBP1) is implicated in various human malignancies. However, the underlying biological effect of PCBP1 in cancers, including acute myeloid leukemia (AML), still remains elusive. The purpose of this study was to examine the expression and clinical outcome of PCBP1in acute myeloid leukemia. METHODS: Bone marrow fluids of 88 newly diagnosed AML patients were sampled, and the PCBP1 mRNA expression level was evaluated using quantitative RT-PCR. The association between PCBP1 expression and clinicopathological features or the survival status of the patients was assessed by Chi-square test and Kaplan-Meier method. RESULTS: Comparing newly diagnosed AML patients to normal healthy donors, PCBP1 expression was significantly decreased in AML patients (P < 0.001). Conversely, PCBP1 expression had accordingly recovered back to normal in patients with complete remission (P < 0.001). Clinical feature analyses showed that PCBP1 expression was negatively correlated with white blood cell count (P = 0.024). In addition, patients with low PCBP1 expression had poor disease-free survival (11.8% vs. 45.3%; P = 0.01) and overall survival (18.2% vs. 42.4%; P = 0.032), respectively. CONCLUSIONS: Taken together, our results showed for the first time that expression of PCBP1 was down-regulated in newly diagnosed AML patients and might be an independent prognostic marker in AML and should to be further investigated.

Poly r(C) binding protein-1 is central to maintenance of cancer stem cells in prostate cancer cells.

AIMS: To investigate global proteomic changes induced in CD44+CD24- stem cells isolated from the prostate cancer cell lines, LNCaP and DU145, post prolonged TGF-ß treatment in order to understand underlying mechanisms that promote stemness in prostate cancer cells. METHODS: CD44+CD133+α2ß1Integrin+CD24- population was isolated from mock or TGF-ß treated (7 days) prostate cancer cell line, LNCaP, through fluorescent activated cell sorting. Cell lysates were obtained from the ±TGF-ß cell population and proteomics profiling (MS/MS) was performed by mass spectrometry. Relative enrichment or depletion in the CD44+CD24-population post-TGF-ß treatment was determined relative to mock-treated CD44+CD24- cells post normalization to GAPDH expression levels. RESULTS obtained from MS/MS were validated using immunoblotting. Functional validation of one putative regulator was performed using gain-of-function strategy to investigate its role in rendering stemness in LNCaP and DU145 cells in vitro and in promoting tumorigenicity in vivo. RESULTS: TGF-ß treatment caused significant enrichment of CD44+CD24- population in LNCaP cells (22.35 ± 0.94% in mock treated vs 95.23 ± 2.34% in TGF-ß treated cells; P < 0.01), which were also positive for CD133 and α2ß1Integrin. Mass spectrometry analysis of the enriched cell population revealed that sixty-three proteins were either up- or down-regulated greater than five folds, out of which the poly r(C) binding protein (PCBP)-1 was the most down-regulated (9.31 ± 0.05 folds). Ectopic overexpression of PCBP1 in LNCaP and DU145 cells not only attenuated enrichment of CD44+CD133+CD24- population in these cells following TGF-ß treatment, but also significantly decreased tumorigenicity of the stem cell subset, as assessed by in vitro soft agar colony formation and in vivo xenograft assays. CONCLUSION: Our proteomic profiling and subsequent validation indicate that PCBP1 is central to CSCs enrichment and functionality in prostate cancer.

Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.

Heterogeneous nuclear ribonucleoprotein U-like 1 and Poly (ADP-ribose) polymerase 1 are downregulated in renal cell carcinoma and connected with the prognosis.

OBJECTIVE: To examine the expression of heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1) and poly (ADP-ribose) polymerase 1 (PARP-1) in renal cell carcinoma (RCC) tissues and the connection between the expressions and prognosis of RCC. MATERIAL AND METHODS: Total RNAs were extracted from 36 pairs of RCC and their adjacent non-tumor tissues and real-time qRT-PCR was performed. RESULTS: The expression of hnRPUL1 was remarkably downregulation in RCC tissues (14/36, 38.9%), compared with matched adjacent non-tumor tissues. And the expression of PARP1 was also remarkably downregulation in RCC tissues (12/36, 30.0%). In the stratification of clinical stage, downregulation in hnRPUL1 and PARP1 were both connected with the advanced clinical stage (P=0.013 and P=0.009). In addition, significantly increased risk of developing with a moderately and poorly differentiated tumor nuclear grade was found in the downregulation of hnRPUL1 patients (P=0.027). CONCLUSIONS: Despite the limitations, hnRPUL1 and PARP1 were downregulated in renal cell carcinoma and connected with the prognosis.

Amiloride modulates alternative splicing in leukemic cells and resensitizes Bcr-AblT315I mutant cells to imatinib.

The antihypertensive drug amiloride is being considered as a tactic to improve cancer therapy including that for chronic myelogenous leukemia. In this study, we show that amiloride modulates the alternative splicing of various cancer genes, including Bcl-x, HIPK3, and BCR/ABL, and that this effect is not mainly related to pH alteration, which is a known effect of the drug. Splice modulation involved various splicing factors, with the phosphorylation state of serine-arginine-rich (SR) proteins also altered during the splicing process. Pretreatment with okadaic acid to inhibit protein phosphatase PP1 reversed partially the phosphorylation levels of SR proteins and also the amiloride-modulated yields of Bcl-xs and HIPK3 U(-) isoforms. Genome-wide detection of alternative splicing further revealed that many other apoptotic genes were regulated by amiloride, including APAF-1, CRK, and SURVIVIN. Various proteins of the Bcl-2 family and MAPK kinases were found to be involved in amiloride-induced apoptosis. Moreover, the effect of amiloride on mRNA levels of Bcl-x was demonstrated to translate to the protein levels. Cotreatment of K562 and BaF3/Bcr-AblT315I cells with amiloride and imatinib induced more loss of cell viability than either agent alone. Our findings suggest that amiloride may offer a potential treatment option for chronic myelogenous leukemia either alone or in combination with imatinib.

Expression levels of hnRNP G and hTra2-beta1 correlate with opposite outcomes in endometrial cancer biology.

HnRNP G is a member of heterogeneous nuclear ribonucleoprotein (hnRNP) family with potent tumor suppressive activities. Human transformer-2-beta1 (hTra2-beta1) belongs to the arginine-serine rich like proteins and is found over-expressed in various human cancers. It was recently shown that hnRNP G and hTra2-beta1 exert antagonistic effects on alternative splicing. In our study we explored the impact of these two factors in tumor biology of endometrial cancer (EC). EC tissues (n = 139) were tested for hnRNP G and hTra2-beta1 expression on mRNA level by real time PCR and on protein level by immunohistochemistry. HTra2-beta1 mRNA level was found being induced in advanced International Federation of Gynecology and Obstetrics (FIGO) stages (p = 0.016). HnRNP G protein nuclear expression was found more prominent in patients without distant organ metastases (p = 0.033) and in FIGO Stages I/II group (p < 0.001). HTra2-beta1 protein nuclear levels were elevated in poorly differentiated (p = 0.044) and lymph node metastases (p = 0.003) cancers. Kaplan-Meier survival curves revealed that elevated hnRNP G mRNA (p = 0.029) and protein (p = 0.022) levels were associated with a favorable patient outcome. Multivariate Cox-regression analyses identified nuclear hnRNP G level [hazard ratio (HR) 0.468, p = 0.026) as well as hTra2-beta1 level (hazard ratio 5.760, p = 0.004) as independent prognostic factors for EC progression-free survival. Our results indicate that the antagonistic functional effects of hnRNP G and hTra2-beta1 on alternative splicing correlate directly to their opposite clinical effects on EC patient outcome.

The expression pattern of heterogeneous nuclear ribonucleoprotein R in rat retina.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) play important roles in DNA repairing, cell signaling, telomere biogenesis, and in regulating gene expression at both transcriptional and translational levels. In the present study, we demonstrated that the expression of hnRNP-R1 and hnRNP-R2 is developmentally regulated in rat retina. The neural specific isoform hnRNP-R2 is expressed in 7-day postnatal rat retina, but not in the adult retina. The positive immunohistochemistry signal of hnRNP-R1 is extensively distributed in the outer plexiform layer, inner nuclear layer and ganglion cell layer of rat retina. Double staining experiments showed that the positive signal of hnRNP-R1 is distributed in ON-type bipolar cells and localized in the cytoplasm, dendrites and axon terminals. In addition, the hnRNP-R1 distribution is regulated in rat retina during circadian. The present investigation suggests that hnRNP-R may play roles in retinal development and light-elicited cellular activities.