Global regulation of alternative RNA splicing by the SR-rich protein RBM39.

BACKGROUND: RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. METHODS: In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RESULTS: RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. CONCLUSIONS: RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. GENERAL SIGNIFICANCE: The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels.

Global Regulation of Differential Gene Expression by c-Abl/Arg Oncogenic Kinases.

BACKGROUND Studies have found that c-Abl oncogenic kinases may regulate gene transcription by RNA polymerase II phosphorylation or by direct regulation of specific transcription factors or coactivators. However, the global regulation of differential gene expression by c-Abl/Arg is largely unknown. In this study, differentially expressed genes (DEGs) regulated by c-Abl/Arg were identified, and related cellular functions and associated pathways were investigated. MATERIAL AND METHODS RNA obtained from wild-type and c-Abl/Arg gene-silenced MCF-7 cells was analyzed by RNA-Seq. DEGs were identified using edgeR software and partially validated by qRT-PCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to explore the potential functions of these DEGs. RESULTS A total of 1,034 DEGs were significantly regulated by c-Abl/Arg (399 were up-regulated and 635 were down-regulated after c-Abl/Arg double knockdown). GO and KEGG analyses showed that the DEGs were primarily involved in cellular metabolic processes, neurodegenerative disease, the metabolic process and signaling pathway of cAMP, angiogenesis, and cell proliferation. CONCLUSIONS Our data collectively support the hypothesis that c-Abl/Arg regulate differential gene expression, providing new insights into the biological functions of c-Abl and Arg.

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-ß. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRß in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

Annexin A2 is a discriminative serological candidate in early hepatocellular carcinoma.

To date, the useful markers of hepatocellular carcinoma (HCC) remains incompletely developed. Here, we show that annexin A2 complement alpha-fetoprotein (AFP), a widely used liver cancer marker, in the serologically surveillance and early detection of HCC. First, differentially expressed proteins in HCC were identified using a subcellular proteomic approach. Annexin A2 was then selected for further verification. It was found to be overexpressed in HCC tissues (60.7%, 136/224). Using a self-established sandwich enzyme-linked immunosorbent assay, we found that annexin A2 significantly increased in the sera of HCC (n = 175, median, 24.75 ng/µl) compared with the healthy (n = 49, median, 16.69 ng/µl), benign tumors (n = 19, median, 19.92 ng/µl), hepatitis (n = 23, median, 6.48 ng/µl) and cirrhosis (n = 51, median, 7.39 ng/µl) controls and other malignant tumors (n = 87). Importantly, raised concentrations of annexin A2 were observed in 83.2% (79/95) of early stage (median, 24.32 ng/µl) and 78.4% (58/74) of AFP-negative (median, 24.09 ng/µl) patients. Annexin A2 alone had a better area under the receiver-operating characteristic curve (AUC = 0.79, 95% confidence interval: 0.73-0.85) in comparison with AFP (AUC = 0.73, 95% confidence interval: 0.66-0.80) in detecting of early stage HCC. Combining both markers notably improved the diagnostic efficiency of early HCC with an achieved sensitivity of 87.4%. Additionally, the expression characteristics of annexin A2 during hepatocarcinogenesis were detected in p21-HBx gene knockin transgenic mice model. The results showed that annexin A2 expression was substantially elevated in HCC-bearing mice, in accordance with the finding in human samples. In conclusion, annexin A2 may be an independent serological candidate for hepatitis B virus-related HCC, especially in the early stage cases with normal serum AFP.

SNP genotyping for the genetic monitoring of laboratory mice by using a microarray-based method with dualcolour fluorescence hybridisation.

Ensuring the genetic homogeneity of the mice used in laboratory experiments contributes to the Reduction aspect of the Three Rs, by maximising the quality of the data obtained from any animals that are used for these purposes, and ultimately reducing the numbers of animals used. Single nucleotide polymorphism (SNP) genotyping is especially suitable for use in the analysis of the genetic purity of model organisms such as the mouse, because bi-allelic markers remain fully informative when used to characterise crosses between inbred strains. Here, we attempted to apply a microarray-based method for a SNP marker to monitor the genetic quality of inbred mouse strains, so as to validate the reliability, stability and applicability of this SNP genotyping panel. The amplified PCR products containing four different SNP loci from four inbred mouse strains were spotted and immobilised onto amino-modified glass slides to generate a microarray. This was then interrogated through hybridisation with dual-colour probes, to determine the SNP genotypes of each sample. The results indicated that this microarray-based method could effectively determine the genotypes of the four selected SNPs with a high degree of accuracy. We have developed a new SNP genotyping technique for effective use in the genetic monitoring of inbred mouse strains.

Prognostic value of circulating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma.

OBJECTIVE: Circulating tumor cells (CTCs) of patients with malignant tumors can be used as a prognostic marker. However, there are few relevant reports to date on esophageal squamous cell carcinoma (ESCC). Our study assesses the clinical significance of CTCs in ESCC patients. PATIENTS AND METHODS: CTCs were detected in 103 peripheral blood (PB) samples from 59 ESCC patients. Correlation between CTCs and clinical parameters was analyzed using the χ2 test or Fisher's exact test. Overall survival (OS) and progression-free survival (PFS) were analyzed using Kaplan-Meier analysis and univariate and multivariate methods. RESULTS: The CTC detection rate was 79.7% (47/59) at baseline. The frequency of CTC-positive patients increased as the disease stage advanced (88.0% in stages III-IV, 58.9% in stages I-II). CTC counts ≥0/7.5 mL of PB were correlated with the degree of tumor differentiation, tumor infiltration, and lymph node and distant metastases. Overall, the OS and PFS of patients with CTC counts ≥3 or ≥5/7.5 mL of PB before surgery were significantly shorter than those of patients with CTC counts <3 or <5/7.5 mL. Multivariate analysis showed CTC counts ≥5/7.5 mL of PB to be a strong prognostic indicator of OS (hazard ratio [HR] 12.478; 95% confidence interval [CI], 8.2-34.3; P<0.05) and PFS (HR 6.524; 95% CI, 1.2-34.3; P<0.05) in ESCC patients. Patients in whom CTCs changed from positive at baseline to a negative value after surgery had an excellent prognosis. CONCLUSION: CTCs might serve as a reference indicator for the prognosis and monitoring of disease progression and treatment effects in ESCC.

Monitoring disease progression and treatment efficacy with circulating tumor cells in esophageal squamous cell carcinoma: A case report.

This study investigated whether changes in circulating tumor cell (CTC) numbers reflect tumor progression and treatment efficacy in esophageal squamous cell carcinoma (ESCC). A 47-year-old male patient with ESCC is presented in this case study. The patient was evaluated for a series of serum tumor markers and subjected to radiological examinations before and after surgery and during follow-up over the course of five years. In addition, the CTCs in 7.5 mL of peripheral blood were enriched by magnetic-activated cell sorting negative selection and identified by immunofluorescence staining. Serum tumor markers remained within normal ranges and were discordant with imaging scans during the follow-up. Initially, one CTC was detected in the peripheral blood sample, and 14 were observed seven days after the operation. After 12 wk, subcutaneous metastases and bone metastases occurred, and the number of CTCs increased to 84. After 48 wk, lung metastases were noted, and the CTC level was 21. At 104 wk, the number of CTCs was 14, and disease recurrence was detected by positron emission tomography-computed tomography. The CTC counts were in accord with the imaging studies at several time points. The additional information provided by CTC enumeration could thus facilitate monitoring of disease status and treatment efficacy and provide support for treatment decisions.

Novel method for extracting exosomes of hepatocellular carcinoma cells.

AIM: To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells. METHODS: Unlike the traditional extraction method, the supernatants of cell cultures were concentrated, and the exosomes were isolated promptly and effectively using a novel nanomaterial called ExoQuick. Coomassie brilliant blue staining was used for protein quantification, and the morphology of the exosomes extracted by both methods was visualized by transmission electron microscopy. Exosome marker proteins were detected by Western blot analysis. Two potential hepatoma-associated proteins, tissue transglutaminase 2 (TGM2) and annexin A2, were analyzed. RESULTS: The exosomes separated by the new extraction assay based on the nanomaterial were disc-shaped, intact vesicles with lipid bilayer membranes. They were approximately 30-100 nm in diameter, which is similar to the diameter of exosomes isolated by the traditional method. The protein concentration of exosomes extracted by the new method was approximately 780 µg/10(8) cells, and therefore, it was 19 times higher than that of exosomes extracted in the traditional manner. There were differences between the total proteins of Huh-7 cells and the exosomal proteins. Typical exosome proteins, such as the transmembrane protein CD63 and heat shock protein 70, were confirmed. Two potential hepatoma-associated proteins were also identified. TGM2 was first found to exist in the exosomes of human liver cancer cells, but annexin A2 was not secreted into exosomes. CONCLUSION: The new extraction method based on the nanomaterial is quick and efficient. The cancer-associated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway, and it may be a valuable tumor marker.

Chronotype and a PERIOD3 variable number tandem repeat polymorphism in Han Chinese pilots.

An association has been determined between variable number tandem-repeat (VNTR) polymorphisms in the PERIOD3 gene (PER3, rs57875989) and chronotype. An association has been found in which the longer PER3(5) allele is correlated with diurnal preference and shorter PER3(4) allele is linked with preference for evening, respectively. In this study, we explored the genotype frequency and relationship to the chronotype of a PER3 VNTR polymorphism in Han Chinese pilots compared to other populations to further develop aviation safety research. DNA samples were genotyped with respect to the 4-repeat and 5-repeat alleles of the PER3 VNTR polymorphism. We compared and analyzed PER3 VNTR genotype frequencies of a general Han Chinese population and Han Chinese pilots. The chronotypes of our subjects were evaluated by the morningness-eveningness questionnaire (MEQ). The distribution of PER3 VNTR genotype frequencies from 240 Han Chinese was determined (PER3(4/4), 78.3%; PER3(4/5), 20.0%; PER3(5/5), 1.7%) and compared to the genotype frequencies of 126 Han Chinese pilots (PER3(4/4), 71.4%; PER3(4/5), 26.1%; PER3(5/5), 2.4%). Statistical analysis revealed no significant difference between the general Han Chinese population and Han Chinese pilots regarding the PER3 VNTR genotype and allele frequencies (x(2) = 2.170, p > 0.05). Furthermore, MEQ results showed no association between the PER3 VTNR polymorphism and chronotype. However, PER3 VNTR genotype frequencies differed significantly between Han Chinese and other ethnic groups previously reported, such as Caucasians, African Americans and Italians. These data indicate that the proposed role of the PER3 VNTR needs further clarification and the role of PER3(5) allele in sleep regulation needs to be investigated in more detail. In particular, a study of PER3 polymorphisms with a larger sample size of Han Chinese individuals and Han Chinese pilots may be required.

Non-receptor tyrosine kinases c-Abl and Arg regulate the activity of C/EBPbeta.

The CCAAT/enhancer-binding protein beta (C/EBPbeta) is a critical transcription factor that regulates gene expression during numerous biological processes, including differentiation, metabolism, homeostasis, proliferation, tumorigenesis, inflammation, and apoptosis. In this study, interactions between C/EBPbeta and either the Abelson murine leukemia viral oncogene homolog 1 (c-Abl) or the Abl-related gene (Arg) were demonstrated in vitro and in vivo with a direct binding assay and by co-immunoprecipitation, respectively. The Y79 amino acid residue of C/EBPbeta was phosphorylated by c-Abl or Arg. The phosphorylation of C/EBPbeta resulted in an increased C/EBPbeta stability and a potentiation of C/EBPbeta transcription activation activity in cells. Expression of the C/EBPbeta(Y79F) mutant in HEK293, and K562, and in other cell lines, resulted in less of a delay in the cell cycle compared to the wild type C/EBPbeta; furthermore, the C/EBPbeta (Y79F) mutant induced an increased apoptosis compared to the wild type C/EBPbeta. These findings suggest that the c-Abl family non-receptor tyrosine kinases have a role in the regulation of the C/EBPbeta transcription factor.