Overexpression of phosphatidylserine synthase IbPSS1 enhances salt tolerance by stimulating ethylene signaling-dependent lignin synthesis in sweetpotato roots.

Phosphatidylserine (PS) is an important lipid signaling required for plant growth regulation and salt stress adaptation. However, how PS positively regulate plant salt tolerance is still largely unknown. In this study, IbPSS1-overexpressed sweetpotato plants that exhibited overproduction of PS was employed to explore the mechanisms underlying the PS stimulation of plant salt tolerance. The results revealed that the IbPSS1-overexpressed sweetpotato accumulated less Na+ in the stem and leaf tissues compared with the wild type plants. Proteomic profile of roots showed that lignin synthesis-related proteins over-accumulated in IbPSS1-overexpressed sweetpotato. Correspondingly, the lignin content was enhanced but the influx of Na + into the stele was significantly blocked in IbPSS1-overexpressed sweetpotato. The results further revealed that ethylene synthesis and signaling related genes were upregulated in IbPSS1-overexpressed sweetpotato. Ethylene imaging experiment revealed the enhancement of ethylene mainly localized in the root stele. Inhibition of ethylene synthesis completely reversed the PS-overproduction induced lignin synthesis and Na+ influx pattern in stele tissues. Taken together, our findings demonstrate a mechanism by which PS regulates ethylene signaling and lignin synthesis in the root stele, thus helping sweetpotato plants to block the loading of Na+ into the xylem and to minimize the accumulation of Na+ in the shoots.

Chromosome painting reveals the genomic structure of three polyploid species of Ipomoea.

Cultivated sweetpotato [Ipomoea batatas (L.) Lam.] from the family Convolvulaceae is a hexaploid species with 2n = 6x = 90 and has been controversial regarding its nature as an autopolyploid arising within a species or an allopolyploid forming between species. Here, we developed oligonucleotide-based painting probes for two chromosomes of I. nil, a model diploid Ipomoea species. Using these probes, we revealed the pairing behavior of homoeologous chromosomes in I. batatas and its two possible polyploid ancestral species, tetraploid I. tabascana (2n = 4x = 60) and hexaploid I. trifida (2n = 6x = 90). Chromosome painting analysis revealed a high percentage of quadrivalent formation in zygotene-pachytene cells of I. tabascana, which supported that I. tabascana was an autotetraploid likely derived by doubling of structurally similar and homologous genomes rather than a hybrid between I. batatas and I. trifida (2x). A high frequency of hexavalent/bivalent and tetravalent pairing was observed in I. trifida (6x) and I. batatas. However, the percentage of hexavalent pairing in I. trifida (6x) was far higher than that in I. batatas. Thus, the present results tend to support that I. trifida (6x) is an autohexaploid, while I. batatas is more likely to be a segmental allohexaploid.

Karyotypic stability of Fragaria (strawberry) species revealed by cross-species chromosome painting.

Chromosome karyotyping analysis is particularly useful in determining species relationships and the origin of polyploid species. Identification of individual chromosomes is the foundation for karyotype development. For Fragaria (strawberry) species, definitive identification of the individual chromosomes is extremely difficult because of their small size and similar shape. Here, we identified all chromosomes for 11 representative Fragaria species with different ploidy using a set of oligonucleotide-based probes developed in Fragaria vesca. Comprehensive molecular cytogenetic karyotypes were established based on the individually identified chromosomes. In addition, we used oligo probes to assign the 5S and 45S rDNA loci to specific chromosomes in 16 Fragaria species. We found that these Fragaria species maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements at the cytological level were observed in any of the chromosomes among these species. Despite karyotypic stability and similarity, variations in the signal intensity of oligo probes were observed among the homologous chromosomes in several polyploid species. Moreover, most Fragaria species also showed differences in the distribution patterns of 45S and 5S rDNA. These data provide new insights into the origins of several polyploid Fragaria species.

Overexpression of lncRNA TINCR is associated with high-grade, invasive, and recurring tumors, and facilitates proliferation in vitro and in vivo of urothelial carcinoma of the bladder.

OBJECTIVES: The aberrant expression of long noncoding RNAs (lncRNAs) plays roles in cancer development. In this work, we measured the expression of lncRNA terminal differentiation-induced non-coding RNA (TINCR) in urothelial carcinoma of the bladder (UCB), determined its impact on the proliferation of UCB in vitro and in vivo and identified its possible targets. METHODS: The expression levels of genes were measured by Real-Time quantitative Polymerase Chain Reaction (qPCR). Cell proliferation, cell motility, and cell apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, wound healing assay, and ELISA, respectively. Tumor growth in vivo was determined by xenograft formation assay in nude mice. The predicted binding site between TINCR and hsa-miR-125b was confirmed by dual luciferase reporter assay. RESULTS: The expression levels of TINCR were higher in cancerous tissues than that in paired noncancerous tissues of UCB. Higher expression levels of TINCR were positively associated with high-grade, invasive, and recurring tumors. Depletion of TINCR retarded proliferation, decreased motility, increased apoptosis in UCB cells, and markedly reduced tumor growth in xenograft nude mice. The predicted binding site between TINCR and hsa-miR-125b was functional. TINCR downregulated hsa-miR-125b in UCB cells. Hsa-miR-125b mimic reversed the proliferation caused by TINCR up-expression in UCB cells. CONCLUSIONS: Up-regulation of TINCR may act as an unfavorable indicator for the diseasing status of UCB. TINCR facilitates bladder cancer proliferation in vitro and in vivo. Hsa-miR-125b is a target for TINCR in UCB.

Amplification and adaptation of centromeric repeats in polyploid switchgrass species.

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats from a single satellite repeat family. Why centromeres are dominated by a single satellite repeat and how the satellite repeats originate and evolve are among the most intriguing and long-standing questions in centromere biology. We identified eight satellite repeats in the centromeres of tetraploid switchgrass (Panicum virgatum). Seven repeats showed characteristics associated with classical centromeric repeats with monomeric lengths ranging from 166 to 187 bp. Interestingly, these repeats share an 80-bp DNA motif. We demonstrate that this 80-bp motif may dictate translational and rotational phasing of the centromeric repeats with the cenH3 nucleosomes. The sequence of the last centromeric repeat, Pv156, is identical to the 5S ribosomal RNA genes. We demonstrate that a 5S ribosomal RNA gene array was recruited to be the functional centromere for one of the switchgrass chromosomes. Our findings reveal that certain types of satellite repeats, which are associated with unique sequence features and are composed of monomers in mono-nucleosomal length, are favorable for centromeres. Centromeric repeats may undergo dynamic amplification and adaptation before the centromeres in the same species become dominated by the best adapted satellite repeat.

Synthesizing oncogenic signal-processing systems that function as both "signal counters" and "signal blockers" in cancer cells.

RNA-protein interaction plays a significant role in regulating eukaryotic translation. This phenomenon raises questions about the ability of artificial biological systems to take the advantage of protein-RNA interaction. Here, we designed an oncogenic signal-processing system expressing both a Renilla luciferase reporter gene controlled by RNA-protein interaction in its 5'-untranslated region (5'-UTR) and a Firefly luciferase normalization gene. To test the ability of the designed system, we then constructed vectors targeting the nuclear factor-κB (NF-κB) or the ß-catenin signal. We found that the inhibition (%) of luciferase expression was correlated to the targeted protein content, allowing quantitative measurement of oncogenic signal intensity in cancer cells. The systems inhibited the expression of oncogenic signal downstream genes and induced bladder cancer cell proliferation inhibition and apoptosis without affecting normal urothelial cells. Compared to traditional methods (ELISA and quantitative immunoblotting), the bio-systems provided highly accurate, consistent, and reproducible quantification of protein signals and were able to discriminate between cancerous and non-cancerous cells. In conclusion, the synthetic systems function as both "signal counters" and "signal blockers" in cancer cells. This approach provides a synthetic biology platform for oncogenic signal measurement and cancer treatment.

Inducing cell proliferation inhibition and apoptosis via silencing Dicer, Drosha, and Exportin 5 in urothelial carcinoma of the bladder.

BACKGROUND AND OBJECTIVES: MicroRNAs (miRNAs) are aberrantly expressed in cancers. Dicer, Drosha, and Exportin 5 are essential for miRNA processing. In this study, the expression patterns of Dicer, Drosha, and Exportin 5 and the cell proliferation inhibition and apoptosis induced by silencing these genes in urothelial carcinoma of the bladder were determined. METHODS: The expression levels of Dicer, Drosha, and Exportin 5 were determined using Real-Time qPCR in 40 patients with urothelial carcinoma of the bladder. Bladder urothelial carcinoma T24 and 5637 cells were transfected with Dicer, Drosha, or Exportin 5 siRNA or negative control siRNA. Cell proliferation was determined using MTT assay. Apoptosis was evaluated using ELISA assay. RESULTS: All the three genes were up-regulated in bladder urothelial carcinoma compared to matched normal urothelium. Dicer, Drosha, and Exportin 5 expression levels were higher in high grade carcinomas than that in low grade carcinomas. Invasive carcinomas had higher expression levels than non-invasive carcinomas. Silencing Dicer, Drosha, or Exportin 5 induced cell proliferation inhibition and apoptosis in bladder urothelial carcinoma T24 and 5637 cells. CONCLUSIONS: Dicer, Drosha, and Exportin 5 are promising biomarkers and therapeutic targets for urothelial carcinoma of the bladder.

Inducing cell proliferation inhibition, apoptosis, and motility reduction by silencing long noncoding ribonucleic acid metastasis-associated lung adenocarcinoma transcript 1 in urothelial carcinoma of the bladder.

OBJECTIVE: To study the expression patterns of long noncoding ribonucleic acid (RNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the cell proliferation inhibition, apoptosis, and motility changes induced by silencing MALAT1 in urothelial carcinoma of the bladder. MATERIALS AND METHODS: The expression levels of MALAT1 were determined using real-time quantitative polymerase chain reaction in cancerous tissues and paired normal tissues in a total of 36 patients with urothelial carcinoma of the bladder. Expression differences were analyzed according to the grade and stage. Bladder urothelial carcinoma T24 and 5637 cells were transfected with MALAT1 small interfering RNA or negative control small interfering RNA. The cell proliferation changes of the transfected bladder urothelial carcinoma cells were determined using the MTT assay. Apoptosis caused by silencing MALAT1 was evaluated using the flow cytometry assay and enzyme-linked immunosorbent assay. The motility changes induced by silencing MALAT1 were measured using the wound healing assay. RESULTS: MALAT1 was upregulated in bladder urothelial carcinoma compared with matched normal urothelium (P=.008). The MALAT1 expression levels were greater in high-grade carcinomas than in low-grade carcinoma (P=.001). The MALAT1 expression levels were greater in invasive carcinoma than in noninvasive carcinoma (P=.018). Cell proliferation inhibition, increased apoptosis, and decreased motility were observed in MALAT1 small interfering RNA-transfected bladder urothelial carcinoma T24 and 5637 cells. CONCLUSION: MALAT1 plays an oncogenic role in urothelial carcinoma of the bladder. Silencing MALAT1 is a potential novel therapeutic approach for this cancer.

Long intergenic non-coding RNA TUG1 is overexpressed in urothelial carcinoma of the bladder.

BACKGROUND AND OBJECTIVES: Long intergenic non-coding RNAs (lincRNAs) are a class of non-coding RNAs that regulate gene expression via chromatin reprogramming. Taurine Up-regulated Gene 1 (TUG1) is a lincRNA that is associated with chromatin-modifying complexes and plays roles in gene regulation. In this study, we determined the expression patterns of TUG1 and the cell proliferation inhibition and apoptosis induced by silencing TUG1 in urothelial carcinoma of the bladder. METHODS: The expression levels of TUG1 were determined using Real-Time qPCR in a total of 44 patients with bladder urothelial carcinomas. Bladder urothelial carcinoma T24 and 5637 cells were transfected with TUG1 siRNA or negative control siRNA. Cell proliferation was evaluated using MTT assay. Apoptosis was determined using ELISA assay. RESULTS: TUG1 was up-regulated in bladder urothelial carcinoma compared to paired normal urothelium. High TUG1 expression levels were associated with high grade and stage carcinomas. Cell proliferation inhibition and apoptosis induction were observed in TUG1 siRNA-transfected bladder urothelial carcinoma T24 and 5637 cells. CONCLUSIONS: Our data suggest that lincRNA TUG1 is emerging as a novel player in the disease state of bladder urothelial carcinoma. TUG1 may have potential roles as a biomarker and/or a therapeutic target in bladder urothelial carcinoma.

Hsa-miR-125b suppresses bladder cancer development by down-regulating oncogene SIRT7 and oncogenic long noncoding RNA MALAT1.

MicroRNAs mainly inhibit coding genes and long non-coding RNA expression. Here, we report that hsa-miR-125b and oncogene SIRT7/ oncogenic long noncoding RNA MALAT1 were inversely expressed in bladder cancer. Hsa-miR-125b mimic downregulated, whereas hsa-miR-125b inhibitor upregulated the expression of SIRT7 and MALAT1. Binding sites were confirmed between hsa-miR-125b and SIRT7/MALAT1. Upregulation of hsa-miR-125b or downregulation of SIRT7 inhibited proliferation, motility and increased apoptosis. The effects of upregulation of hsa-miR-125b were similar to that of silencing MALAT1 in bladder cancer as we had previously described. These data suggest that hsa-miR-125b suppresses bladder cancer development via inhibiting SIRT7 and MALAT1.

Hsa-miR-125b suppresses bladder cancer development by down-regulating oncogene SIRT7 and oncogenic long non-coding RNA MALAT1.

MicroRNAs mainly inhibit coding genes and long non-coding RNA expression. Here, we report that hsa-miR-125b and oncogene SIRT7/oncogenic long non-coding RNA MALAT1 were inversely expressed in bladder cancer. Hsa-miR-125b mimic down-regulated, whereas hsa-miR-125b inhibitor up-regulated the expression of SIRT7 and MALAT1. Binding sites were confirmed between hsa-miR-125b and SIRT7/MALAT1. Up-regulation of hsa-miR-125b or down-regulation of SIRT7 inhibited proliferation, motility and increased apoptosis. The effects of up-regulation of hsa-miR-125b were similar to that of silencing MALAT1 in bladder cancer as we had previously described. These data suggest that hsa-miR-125b suppresses bladder cancer development via inhibiting SIRT7 and MALAT1.

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 3' 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.

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.

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.

Identification and characterization of human PCDH10 gene promoter.

Recent studies have suggested roles for PCDH10 as a novel tumor suppressor gene. In our previous work, we located the core promoter of PCDH10 to a 462-bp segment of 5'-flanking region characterized by a high GC content. Here we further identified and characterized the promoter for PCDH10. Transient transfection of PC3 and LNCaP cells with a series of deleted promoter constructs indicated that the minimal promoter region was between nucleotides -144 and -99. This segment contained a CAAT box, a GT box, and a putative transcription factor binding site for AP-4. Mutational analysis identified that the CAAT box and GT box are necessary for promoter activity. Ectopic expression of NF-Ys increased reporter gene activity, whereas expression of a dominant-negative NF-YA decreased reporter gene activity. Co-transfection of Sp1/Sp3 expression plasmids enhanced reporter gene activity in a dose-dependent manner. Mithramycin A, an inhibitor of Sp-DNA interaction, reduced PCDH10 promoter activity. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp1/Sp3 to the promoter region in vitro and in vivo. Our data show that Sp1/Sp3 and CBF/NF-Y transcription factors play a crucial role in the basal expression of the human PCDH10 gene.

Divergence in centromere structure distinguishes related genomes in Coix lacryma-jobi and its wild relative.

Knowledge about the composition and structure of centromeres is critical for understanding how centromeres perform their functional roles. Here, we report the sequences of one centromere-associated bacterial artificial chromosome clone from a Coix lacryma-jobi library. Two Ty3/gypsy-class retrotransposons, centromeric retrotransposon of C. lacryma-jobi (CRC) and peri-centromeric retrotransposon of C. lacryma-jobi, and a (peri)centromere-specific tandem repeat with a unit length of 153 bp were identified. The CRC is highly homologous to centromere-specific retrotransposons reported in grass species. An 80-bp DNA region in the 153-bp satellite repeat was found to be conserved to centromeric satellite repeats from maize, rice, and pearl millet. Fluorescence in situ hybridization showed that the three repetitive sequences were located in (peri-)centromeric regions of both C. lacryma-jobi and Coix aquatica. However, the 153-bp satellite repeat was only detected on 20 out of the 30 chromosomes in C. aquatica. Immunostaining with an antibody against rice CENH3 indicates that the 153-bp satellite repeat and CRC might be both the major components for functional centromeres, but not all the 153-bp satellite repeats or CRC sequences are associated with CENH3. The evolution of centromeric repeats of C. lacryma-jobi during the polyploidization was discussed.

Comparative physical localization of maize mir1 gene in Zea mays L. and Coix lacryma-jobi L.

The maize gene mir1 encoded a cystein proteinase which is resistant to fall armyworm. Previously, RFLP map indicated that the mir1 was mapped on chromosome 6. However, physical location of the mir1 gene on chromosome 6 has not been reported. In this study, the mir1 gene was physically located on the short arm of metaphase and pachytene chromosome 6 by dual-color fluorescence in situ hybridization (FISH) with 45S rDNA as a reference marker. The results of Southern blotting suggested that there were sequences homologous to mir1 in Coix lacryma-jobi L. genome. Then, the sequences were mapped on the distal region of long arm of chromosome 7 in C. lacryma-jobi by FISH. The percent distance from the signal site to centromere was 73.33 +/- 0.15.

Family-based association study of the NOTCH4 gene in schizophrenia using Japanese and Chinese samples.

BACKGROUND: A family based association study in a British sample found the NOTCH4 gene to be associated with schizophrenia; however, all six replication studies failed to confirm the finding. METHODS: We performed a family based association study of NOTCH4 and schizophrenia in 123 trios (16 Japanese and 107 Chinese). In addition to the original study's polymorphisms, we examined four new single nucleotide polymorphisms (SNPs)--SNPs_A, B, C and D--around SNP1 of the original study. We genotyped all samples for SNPs_A-D and for SNP1 and (CTG)n of the original study. RESULTS: We found no significant associations between NOTCH4 and schizophrenia or its subtypes for all polymorphisms, regardless of gender. The finding remained negative when the Chinese sample was analyzed separately. Exploratory analyses suggested that SNP_A may be associated with early-onset schizophrenia and that SNP1 may be associated with schizophrenia characterized by numerous negative symptoms. CONCLUSIONS: NOTCH4 is not a significant susceptibility gene for schizophrenia when clinical heterogeneity is ignored; however, NOTCH4 may be associated with early-onset schizophrenia or schizophrenia with many negative symptoms, but these findings should be interpreted cautiously.