Aifa Tang

MicroRNA Expression Signatures of Bladder Cancer Revealed by Deep Sequencing

Background MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They are aberrantly expressed in many types of cancers. In this study, we determined the genome-wide miRNA profiles in bladder urothelial carcinoma by deep sequencing. Methodology/Principal Findings We detected 656 differentially expressed known human miRNAs and miRNA antisense sequences (miRNA*s) in nine bladder urothelial carcinoma patients by deep sequencing. Many miRNAs and miRNA*s were significantly upregulated or downregulated in bladder urothelial carcinoma compared to matched histologically normal urothelium. hsa-miR-96 was the most significantly upregulated miRNA and hsa-miR-490-5p was the most significantly downregulated one. Upregulated miRNAs were more common than downregulated ones. The hsa-miR-183, hsa-miR-200b∼429, hsa-miR-200c∼141 and hsa-miR-17∼92 clusters were significantly upregulated. The hsa-miR-143∼145 cluster was significantly downregulated. hsa-miR-182, hsa-miR-183, hsa-miR-200a, hsa-miR-143 and hsa-miR-195 were evaluated by Real-Time qPCR in a total of fifty-one bladder urothelial carcinoma patients. They were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium (p<0.001 for each miRNA). Conclusions/Significance To date, this is the first study to determine genome-wide miRNA expression patterns in human bladder urothelial carcinoma by deep sequencing. We found that a collection of miRNAs were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium, suggesting that they might play roles as oncogenes or tumor suppressors in the development and/or progression of this cancer. Our data provide novel insights into cancer biology.

Epigenetic inactivation of PCDH10 in human prostate cancer cell lines

PCDH10 (protocadherin‐10), a novel tumour suppressor gene, is down‐regulated in several human cancers due to hypermethylation of promoter CGIs (CpG islands). Here, we investigated the expression of PCDH10 in different normal adult tissues and in a panel of prostate cancer cell lines. PCDH10 was widely expressed in normal tissues with higher levels in the prostate. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell lines compared with normal adult prostate tissue. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Furthermore, the DNA demethylating agent 5′‐azacytidin restored PCDH10 expression by suppressing PCDH10 promoter methylation in prostate cancer cell lines. Treatment with Trichostatin A alone had no significant effect on the expression of PCDH10 but enhanced the effect of 5′‐azacytidin. In conclusion, we found that the decreased PCDH10 expression in prostate cancer cells was associated with the aberrant methylation of PCDH10 promoter CGI. Our results may contribute to the understanding of the role of PCDH10 inactivation in the progression of prostate cancers.

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1-7 days of differentiation), mid-stage EBs groups (9-15 days of differentiation), maturing EBs groups (17-45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.

Whole-Genome Synthesis and Characterization of Viable S13-Like Bacteriophages

Background Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template. Methodology/Principal Findings The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl2, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition. Conclusions/Significance The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

Developmental expression pattern of a novel gene, TSG23/Tsg23, suggests a role in spermatogenesis.

A novel gene, TSG23/Tsg23, was identified by comparing the expression profiles of human adult and fetal testis using Affymetrix Genechips. RT-PCR analysis from multiple human and mouse tissues indicated TSG23/Tsg23 mRNA was mainly expressed in the testis. In situ hybridization revealed that TSG23/Tsg23 mRNA was located in spermatocytes and round spermatids of the seminiferous tubules in human and mouse testis. To further confirm the result from RT-PCR, the antibody for human TSG23 was generated against the protein encoded by the gene. Western blot analysis demonstrated that TSG23 was mainly expressed in human testis, with a molecular weight of about 23 kDa. Immunohistochemistry showed that TSG23 was predominantly located in spermatocytes and round spermatids, consistent with the results from in situ hybridization. In order to explore the function of TSG23 in spermatogenesis, the study compared the expression of TSG23 in the testis from fertile persons and from patients with azoospermia. The results showed that there was less expression in patients with obstructive azoospermia compared with fertile persons, and no detectable TSG23 at mRNA and protein levels in patients with non-obstructive azoospermia. The expression of Tsg23 mRNA was considerably decreased in a time-dependent manner in the testis of an azoospermic mouse model induced by Busulfan. These data suggest that TSG23/Tsg23 is involved in human and mouse spermatogenesis.

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression.

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

Expression profile of a novel germ cell-specific gene, TSCPA, in mice and human.

In order to identify novel genes involved in spermatogenesis, testis cDNA samples from Balb/C mice of different postnatal days were hybridized with the whole mouse genome Affymetrix chip to screen the testis-specific genes. The characteristics of the selected genes were analyzed by RT-PCR as well as other bioinformatic tools. A novel differentially expressed testis-specific gene (GenBank Accession No: NM_029042) in the developmental stages of testes was identified, and named TSCPA. Cellular mapping prediction of TSCPA indicated that its protein was probably expressed in nuclei, and one putative domain (aa 332–377) was anchoring domain of cAMP-dependent type II PK. The result of subcellular localization of GFP-TSCPA fusion protein in Cos-7 cells showed that TSCPA protein was expressed in nuclei. RT-PCR analysis revealed that TSCPA was expressed specifically in mouse and human testis. TSCPA gene was expressed weakly in 21-day-old mouse testis and the expression was increased gradually from 38th day to 6th month of mouse testes. No expression of hTSCPA was found in cryptorchidism and Sertoli-cell-only syndrome patients. It was concluded that the expression profile of TSCPA in human and mice indicated that TSCPA might play an important role in spermatogenesis.SummaryIn order to identify novel genes involved in spermatogenesis, testis cDNA samples from Balb/C mice of different postnatal days were hybridized with the whole mouse genome Affymetrix chip to screen the testis-specific genes. The characteristics of the selected genes were analyzed by RT-PCR as well as other bioinformatic tools. A novel differentially expressed testis-specific gene (GenBank Accession No: NM_029042) in the developmental stages of testes was identified, and named TSCPA. Cellular mapping prediction of TSCPA indicated that its protein was probably expressed in nuclei, and one putative domain (aa 332–377) was anchoring domain of cAMP-dependent type II PK. The result of subcellular localization of GFP-TSCPA fusion protein in Cos-7 cells showed that TSCPA protein was expressed in nuclei. RT-PCR analysis revealed that TSCPA was expressed specifically in mouse and human testis. TSCPA gene was expressed weakly in 21-day-old mouse testis and the expression was increased gradually from 38th day to 6th month of mouse testes. No expression of hTSCPA was found in cryptorchidism and Sertoli-cell-only syndrome patients. It was concluded that the expression profile of TSCPA in human and mice indicated that TSCPA might play an important role in spermatogenesis.

Expression and Bioinformatics Analysis of SPACA4 in Human and Mice

Objective To analyze the expression of SPACA4 in human and mice. Methods Testes cRNA samples from Balb/c mice of different postnatal days were performed with mouse affymetrix chip to screen the expression of SPACA4 in mice. Sub-quantitative RT-PCR and bioinformatic tools were used here to describe the expression profile of SPACA4 in mice and human. Results The results of gene chip analysis indicated that the expression of mSPACA4 began after d 35 of postnatal testis in mice. Sub-quantitative RT-PCR assay showed that SPACA4 gene expressed exclusively in mouse and human testis, and mouse mSPACA4 gene expressed after d 35 of postnatal testis that was consistency with the results of gene chip analysis. By bioinformatics analysis, mSPACA4 is located in cell membrane (34.8%) or plasma membrane (34.8%), the signal peptide cleavage site between position 19 and 20 amino acids, transmembrane region between 2–20 and 101–126 amino acids, respectively, on mSPACA4 protein. Conclusion mSPACA4 and hSPACA4 were testis-specific genes, and the expression of mSPACA4 begins after d 35 of postnatal testis in mice. SPACA4 is a candidate for targeting in a sperm-based contraceptive vaccine.