A Systematic Analysis on DNA Methylation and the Expression of Both mRNA and microRNA in Bladder Cancer

Background: DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported. Methodology/Principal Findings: The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to "neurogenesis" and "cell differentiation" by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples. Conclusions/Significance: We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research

Synthetic miRNA-Mowers Targeting miR-183-96-182 Cluster or miR-210 Inhibit Growth and Migration and Induce Apoptosis in Bladder Cancer Cells

Background: MicroRNAs (miRNAs) function as endogenous regulators of biological behaviors of human cancers. Several natural non-coding RNAs are reported to inhibit miRNAs by base-pairing interactions. These phenomena raise questions about the ability of artificial device to regulate miRNAs. The purpose of this study is to create synthetic devices that target a single miRNA or a miRNA cluster and to ascertain their therapeutic effects on the phenotypes of bladder cancer cells. Methodology/Principal Findings: Tandem bulged miRNA binding sites were inserted into the 39 untranslated region (UTR) of the SV-40 promoter-driven Renilla luciferase gene to construct two "miRNA-mowers" for suppression of miR-183-96-182 cluster or miR-210. A third device with tandem repeat sequences not complementary to any known miRNA was generated as an untargeted-control. In functional analyses, bladder cancer T24 and UM-UC-3 cells were transfected with each of the three devices, followed by assays for detection of their impacts. Luciferase assays indicated that the activities of the luciferase reporters in the miRNA-mowers were decreased to 30-50% of the untargeted-control. Using Real-Time qPCR, the expression levels of the target miRNAs were shown to be reduced 2-3-fold by the corresponding miRNA-mower. Cell growth, apoptosis, and migration were tested by MTT assay, flow cytometry assay, and in vitro scratch assay, respectively. Cell growth inhibition, increased apoptosis, and decreased motility were observed in miRNA-mowers-transfected bladder cancer cells. Conclusions/Significance: Not only a single target miRNA but also the whole members of a target miRNA cluster can be blocked using this modular design strategy. Anti-cancer effects are induced by the synthetic miRNA-mowers in the bladder cancer cell lines. miR-183/96/182 cluster and miR-210 are shown to play oncogenic roles in bladder cancer. A potentially useful synthetic biology platform for miRNA loss-of-function study and cancer treatment has been established in this work

<b>Table2.</b> The inhibitory effects of miRNA-mowers on the expressions of target miRNAs in bladder cancer cells.

<p>a The values are mean ± SD. Each experiment in the two cell lines was performed in triplicate for three independent times.</p

Effects of the synthetic miRNA-mowers on migration of bladder cancer cells.

<p>T24 and UM-UC-3 cells were transfected with the devices in 12-well plates. Cell migration was measured by the scratch assay. (A) miRM-183/96/182 and miRM-210 inhibited cell migration in T24 cells independently. (B) miRM-183/96/182 and miRM-210 inhibited cell migration in UM-UC-3 cells independently. Each experiment was performed at least three times and a representative picture was shown. Results were shown in mean ± SD. ** P<0.01, compared with the untargeted-control.</p

Effects of the synthetic miRNA-mowers on growth of bladder cancer cells.

<p>T24 and UM-UC-3 cells were transfected with the devices in 96-well plates. Cell growth was measured by MTT assay at different time intervals. ANOVA was used for the comparison of curves of cell growth. (A) miRM-183/96/182 inhibited cell growth in T24 cells (P<0.05). (B) miRM-183/96/182 inhibited cell growth in UM-UC-3 cells (P<0.05). (C) miRM-210 inhibited cell growth in T24 cells (P<0.01). (D) miRM-210 inhibited cell growth in UM-UC-3 cells (P<0.01). Data were the average of three independent experiments; bars, SD.</p

<b>Table1.</b> The inhibitory effects of endogenous miRNAs on the activities of the luciferase reporters in the miRNA-mowers.

<p>a The values are mean ± SD. Each experiment in the two cell lines was performed in triplicate for three independent times.</p

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

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

Genomic structures of our synthetic bacteriophages.

<p>(A) The S13 genome is 5386 nucleotides long and it codes for 11 genes. The four mutation sites are located at the F coding region. (B) S13/G4 chimeric recombinant genome. This new phage genotype consists of the S13 genome with its allele of F replaced by the homologue from G4. Blue areas represent regions of S13 phage and the green area represents the F gene of G4 phage. The label “G:F” represents the untranslated region between genes G and F and the other label “F:J” represents the untranslated region between genes F and J.</p