Potential and application of abortive transcripts as a novel molecular marker of cancers

Abortive transcript(AT)is a 2-19 nt long non-coding RNA that is produced in the abortive initiation stage.Abortive initiation was found to be closely related to RNA polymerase through in vitro experiments.Therefore,the distribution of AT length and the scale of abortive initiation are correlated to the promoter,discriminator,and transcription initiation sequence,and can be affected by transcription elongation factors.AT plays an important role in the occurrence and development of various diseases.Here we summarize the discovery of AT,the factors responsible for AT formation,the detection methods and biological functions of AT,to provide new clues for finding potential targets in the early diagnosis and treatment of cancers.

Circulating metastasis associated in colon cancer 1 transcripts in gastric cancer patient plasma as diagnostic and prognostic biomarker

AIM: To evaluate the diagnostic and prognostic value of circulating Metastasis Associated in Colon Cancer 1(MACC1) transcripts in plasma of gastric cancer patients.METHODS: We provide for the first time a blood-based assay for transcript quantification of the metastasis inducer MACC1 in a prospective study of gastric cancer patient plasma.MACC1 is a strong prognostic biomarker for tumor progression and metastasis in a variety of solid cancers.We conducted a study to define the diagnostic and prognostic power of MACC1 transcripts using 76 plasma samples from gastric cancer patients,either newly diagnosed with gastric cancer,newly diagnosed with metachronous metastasis of gastric cancer,as well as follow-up patients.Findings were controlled by using plasma samples from 54 tumor-free volunteers.Plasma was separated,RNA was isolated,and levels of MACC1 as well as S100A4 transcripts were determined by quantitative RT-PCR.RESULTS: Based on the levels of circulating MACC1 transcripts in plasma we significantly discriminated tumorfree volunteers and gastric cancer patients(P < 0.001).Levels of circulating MACC1 transcripts were increased in gastric cancer patients of each disease stage,compared to tumor-free volunteers: patients with tumors without metastasis(P = 0.005),with synchronous metastasis(P = 0.002),with metachronous metastasis(P = 0.005),and patients during follow-up(P = 0.021).Sensitivity was 0.68(95%CI: 0.45-0.85) and specificity was 0.89(95%CI: 0.77-0.95),respectively.Importantly,gastric cancer patients with high circulating MACC1 transcript levels in plasma demonstrated significantly shorter survival whencompared with patients demonstrating low MACC1 levels(P = 0.0015).Furthermore,gastric cancer patients with high circulating transcript levels of MACC1 as well as of S100A4 in plasma demonstrated significantly shorter survival when compared with patients demonstrating low levels of both biomarkers or with only one biomarker elevated(P = 0.001).CONCLUSION: Levels of circulating MACC1 transcripts in plasma of gastric cancer patients are of diagnostic value and are prognostic for patient survival in a prospective study.

Analyzing the Transcripts of Desperate Housewives from the Perspective of Cooperate Principle

The cooperative principle put forward by Grice is an important part of pragmatics.The four maxims included in the cooperative principle are effective in analyzing people's conversation,and sometimes the violations of the four maxims can often generate the implicit meaning of conversation,which called conversational implicature,or achieve certain communicative effects such as humor,irony and so on.This article tries to analyze some transcripts from a popular TV series of America called Desperate Housewives season I from the perspective of cooperative principle to show how to infer the conversational implicature and communicative effects of people's conversation with cooperative principle.

Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts

A single mammalian transcript normally encodes one protein, but the transcript of GNAS （G-protein u-subunit） contains two reading frames and produces two structurally unrelated proteins, XLas and ALEX. No other confirmed GNAS-Iike dual-coding transcripts have been reported to date, even though many such candidate genes have been predicted by bioinformatics analysis. In this study, we constructed a series of vectors to test how two protein products were translated from a single transcript in vitro. The length of the ORF （open reading frame）, position of the first AUG and the Kozak motif were found to be important factors. These factors, as well as 55-bp NMD （nonsense-mediated mRNA decay） rule, were used in a bioinformatics search for candidate dual-coding transcripts. A total of 1307, 750 and 474 two-ORF-containing transcripts were found in human, mouse and rat, respectively, of which 170, 89 and 70, respectively, were found to be potential dual-coding transcripts. Most transcripts showed low conservation among species. Interestingly, dual-coding transcripts were significantly enriched for transcripts from the zinc-finger protein family, which are usually DNA-binding proteins involved in regulation of the transcription process.

Mutations of t-complex testis expressed gene 5 transcripts in the testis of sterile t-haplotype mutant mouse

Aim： To determine the possible roles of the t-complex testis expressed gene 5 （Tctex5） on sperm functions, the fulllength sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice. Methods： We applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences. Results： One long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type （Tctex5^long-＋ and Tctex5^short-＋） and t-haplotype （Tctex5^long-＋ and Tctex5^short-＋） mice, respectively. Being enhanced only in the testis, Tctex5^long-＋ had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5^long-＋, whereas the Tctex5^short-＋ was similar to the Tctex5^short-＋. The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5^long-＋ had significant mutations on putative sites of phosphorylation and PP1 binding. Conclusion： We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues. （Asian J Androl 2008 Mar; 10： 219-226）

C-myc antisense transcripts acceleratec differentiation and start apoptosis in human leukemia cells

To demonstrate that low c-myc expression mightexert the effects on differentiation and survival ofleukemic cells, antisense technique was used. Human 2. 7kb c-myc DNA fragment containing exon l, intron 1 and127nt exon 2 was ligated into retroviral vector pDOR-neoin reverse direction. This recombinant plasmid

Abrupt decrease of c-myc expression by antisense transcripts induces terminal differentiation and apoptosis in human promyelocytic leukemia HL-60 cells

This study was designed using c-myc antisense transcripts to evaluate how alteration of c-myc expression in human myeloid leukemic HL-60 cells could influence the myelomonocytic differentiation and induction of apoptosis. The recombinant plasmid pDACx expressing antisense transcripts to c-myc fragment containing a part of intron 1 and 137 nt exon 2 was constructed. pDACx was transfected into HL-60 cell line by lipofectin reagent.Cytochemical stainings including NBT reduction, peroxidase and α -NAE as well as detection of CD13 and CD33 antigens by flow cytometric analysis indicated occurrence of myelomonocytic differentiation in cells expressing antisense transcripts to c-myc. DNA degradation measured by DNA gel electrophoresis and typical morphological changes observed under electron microscope proved the switchon of apoptosis in terminally differentiating HL-60 cells.

Full-length transcripts facilitates Portunus trituberculatus genome structure annotation

Portunus trituberculatus is an ide al model for elucidating crustacean genetic networks.Here we combined single molecule real-time(SMRT)sequencing and Illumina RNA-seq to characterize the coding genes,non-coding RNAs and pseudogenes and further to improve the genome annotation information of P.tritub erculatus.In this study,we assembled 9694 non-redundancy full-length transcripts,and 658737307-bp repetitive sequences were identified in the P.trituberculatus full-length transcriptome.We also predicted the P.tritub erculatus genome structure based on full-length transcripts,including 18602 genes,28686 non-coding RNAs,1407 pseudogenes,740 motif,and 26434 domain.Meanwhile,14460,10211,5412,7314,and 14448 genes had significant matches with sequences in the NR,KOG,GO,KEGG,and TrEMBL database,respectively.Overall,our work firstly provided the long-read transcriptome and we believed that these data are very necessary to improve the annotation information of P.trituberculatus genome structure,and useful information for the future studies on evolution and physiological regulation of P.trituberculatus.

Investigation of DNA Sequences Related to Latency-Associated Transcripts in the Genome of Canine Herpesvirus Type 1 (CHV-1) by Means of Bioinformatics Tools

A characteristic common to herpesviruses is the ability to establish a latent infection in the hosts, a transcriptionally active region has detected during latency as well as a set of RNA that are known as Latency Associated Transcripts (LATs), their functions have been clarified in recent work. The present work was carried using different bioinformatics method in order to determine if Herpesvirus Canine 1 (CHV-1) has a region associated with latency. Our result was the selection of nine sequences candidate of micro RNA (miRNA) (MIREval 2.0 software), and 26 miRNA (miRNAFold v.1.0 software), of them, were selected 14 with real precursors of miRNA, two were found between the RL2 and RS1 genes, one in the RL2 gene and 11 in the RS1 gene. The results showed that the similarities of these regions are very low among the herpesviruses analyzed, so it was not possible to deduce the presence of the LAT gene in canine herpesvirus type 1 with bioinformatics. On the other hand, the comparison showed that the miRNA predicted: chv1-mir-mirnafold-8 has similarity with the ebv-mir-BART7-3p of Epstein-Barr Virus (EBV), in this way, the microRNAs predicted by means of bioinformatic programs met the theoretical requirements of these molecules, however at not having a degree of preservation in other herpesviruses, the expression by CHV-1 in latency cannot be confirmed and it is necessary to identify through experimental tests.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

Identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling

As part of our efforts to identify novel contraceptive targets in the epididymis we performed transcriptional profiling on each of the 10 and 19 segments of the mouse and rat epididymidis, respectively, using Affymetrix whole genome microarrays. A total of 17 096 and 16 360 probe sets representing transcripts were identified as being expressed in the segmented mouse and rat epididymal transcriptomes, respectively. Comparison of the expressed murine transcripts against a mouse transcriptional profiling database derived from 22 other mouse tissues identified 77 transcripts that were expressed uniquely in the epididymis. The expression of these genes was further evaluated by reverse transcription polymerase chain reaction （RT-PCR） analysis of RNA from 21 mouse tissues. RT-PCR analysis confirmed epididymis-specific expression of Defensin Beta 13 and identified two additional genes with expression restricted only to the epididymis and testis. Comparison of the 16 360 expressed transcripts in the rat epididymis with data of 21 other tissues from a rat transcriptional profiling database identified 110 transcripts specific for the epididymis. Sixty-two of these transcripts were further investigated by qPCR analysis. Only Defensin 22 （E3 epididymal protein） was shown to be completely specific for the epididymis. In addition, 14 transcripts showed more than 100-fold selective expression in the epididymis. The products of these genes might play important roles in epididymal and/or sperm function and further investigation and validation as contraceptive targets are warranted. The results of the studies described in this report are available at the Mammalian Reproductive Genetics （MRG） Database （http：//mrg. genetics.washington.edu/）. （Asian J Androl 2007July; 9： 522-527）

Systematic Identification of the Light-quality Responding Anthocyanin Synthesis-related Transcripts in Petunia Petals

Previous studies have shown that high light intensity can induce anthocyanin synthesis(AS)in petunia plants.To identifywhich kind of light quality plays a role in inducing such metabolic process,and what transcripts participate in controlling it,we carried out whole-transcriptome sequencing and analysis of petunia petals treated with different light-quality conditions.Among the red and white light treatments,a total of 2205 differentially expressed genes and 15,22,and 20 differentially expressed circRNAs,miRNAs,and lncRNAs,were identified respectively.The AS-related genes,including the structural genes CHSj,F3H,F35H,DFR,and ANS,and the regulatory genes AN4,DPL,PHZ and MYBx were found to be downregulated under red light condition compared with their levels under white light condition.Furthermore,the light photoreceptor Cryptochrome 3(CRY3)and a series of light-dependent genes,such as PIF,HY5,andBBXs,were also determined to respond to the light treatments.The anthocyanin contents in early petunia petals under red light were significantly lower than that under white and blue light.The results of qRT-PCR further confirmed the expression pattern of some AS-related and light-response genes in response to different light quality.Yeast two-hybrid results showed that the key elements in the light signal pathway,HY5 can interact with BBX19,BBX24 and BBX25.And PHZ,the important AS regulator can induce anthocyanin synthesis in response to blue light quality fromtransient expression analysis in petunia petals.These findings presented here not only deepen our understanding of how light quality controls anthocyanin synthesis,but also allow us to explore potential target genes for improving pigment production in petunia flower petals.

Genome-wide mapping of conserved microRNAs and their host transcripts in Tribolium castaneum

MicroRNAs （miRNAs） are endogenous 22-nt RNAs, which play important regulatory roles by post-transcriptional gene silencing. A computational strategy has been developed for the identification of conserved miRNAs based on features of known metazoan miRNAs in red flour beetle （Tribolium castaneum）, which is regarded as one of the major laboratory models of arthropods. Among 118 putative miRNAs, 47% and 53% of the predicted miRNAs from the red flour beetle are harbored by known protein-coding genes （intronic） and genes located outside （intergenic miRNA）, respectively. There are 31 intronic miRNAs in the same transcriptional orientation as the host genes, which may share RNA polymerase II and spliceosomal machinery with their host genes for their biogenesis. A hypothetical feed-back model has been proposed based on the analysis of the relationship between intronic miRNAs and their host genes in the development of red flour beetle.

INHIBITION OF C-MYC PROTEIN EXPRESSION AND CELL PROLIFERATION IN HL-60 CELLS BY ANTISENSE TRANSCRIPTS TO c-myc

The human promyelocytic cell line HL-60 overexpresses the c-myc protooncogene. Plasmid pDACx carrying antisense human c-myc DNA and neo gene was introduced into HL-60 cells with lipofectin reagent. Upon DNA entering the tar-geted celis and expression of antisense transcripts to c-myc, C-MYC protein level, cell proliferation and colony-forming potentiality were all definitely inhibited.

MicroRNA Primary Transcripts and Promoter Elements Analysis in Soybean (Glycine max L. Merril.)

The importance of microRNA （miRNA） at the post-transcriptional regulation level has recently been recognized in both animals and plants. In recent years, many studies focused on miRNA target identification and functional analysis. However, little is known about the transcription and regulation of miRNAs themselves. In this study, the transcription start sites （TSSs） for 11 miRNA primary transcripts of soybean from 11 miRNA loci （of 50 loci tested） were cloned by a 5＂ rapid amplification of cDNA ends （5＂ RACE） procedure using total RNA from 30-d-old seedlings. The features consistent with a RNA polymerase II mechanism of transcription were found among these miRNA loci. A position weight matrix algorithm was used to identify conserved motifs in miRNA core promoter regions. A canonical TATA box motif was identified upstream of the major start site at 8 （76%） of the mapped miRNA loci. Several cis-acting elements were predicted in the 2 kb 5＂ to the TSSs. Potential spatial and temporal expression patterns of the miRNAs were found. The target genes for these miRNAs were also predicted and further elucidated for the potential function of the miRNAs. This research provides a molecular basis to explore regulatory mechanisms of miRNA expression, and a way to understand miRNA-mediated regulatory pathways and networks in soybean.

Expression of estrogen receptor (ER) -α and -βtranscripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb

In the present study expression of estrogen receptor subtype -alpha (ERalpha) and -beta (ERbeta) in the cerebral cortex, cerebellum, and olfactory bulb was investigated and compared between neonatal (1 to approximately 3-days-old) and adult (250 to approximately 350 g) rats, using reverse transcription-polymerase chain reaction (RT-PCR). No ERalpha transcripts were detectable in the adult cerebellum and olfactory bulb, whereas very weak expression of ERalpha was present in the adult cerebral cortex. No significant difference in ERbeta transcripts was detectable between the neonatal and adult rats. While transcripts for both ER subtypes were co-expressed in these brain areas of neonatal rats, although ERalpha expression was significantly weaker than ERbeta. Even in the cerebral cortex known to contain both ER subtypes in adult rats, ERalpha transcripts in neonatal rats were much higher than in adult. These observations provide evidence for the existence of different expression patterns of ERalpha/ERbeta transcripts in these three brain areas between the neonatal and adult rats, suggesting that each ER subtype may play a distinct role in the regulation of differentiation, development, and functions of the brain by estrogen.

De novo assembly of Zea nicaraguensis root transcriptome identified 5261 full-length transcripts

Zea nicaraguensis, a wild relative of cultivated maize （Zea mays subsp, mays）, is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verifi- cation to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L-1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-re- dundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology （GO） annotation, 2 354 received cluster of orthologous groups （COG） terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes （KEGG） Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.

EFFECTS OF CALM /AF10 ANTISENSES ON PRIMARY LEUKEMIC CELLS WITH CALM /AF10 FUSION TRANSCRIPTS IN VITRO

Objectives: To define the involvement of CALM and AF10 fusion transcripts in primary leukaemias with t(10; 11). Methods: The AF10 and CALM fusion in five t(10; 11) leukemia samples were checked by reverse transcriptase-polymerase chain reaction (RT-PCR), and effects of CALM/AF10 antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs) on chemotherapy sensitivity and apoptosis of leukemia cells in vitro were observed. Results: Five different-sized AF10-CALM products and four different-sized CALM/AF10 products were detected by RT-PCR. The chemotherapy sensitivity of leukemic cells with t(10; 11) to drugs in vitro was lower than that of leukemic cells without t(10; 11). AS PS-ODNs increased the chemotherapy sensitivity and apoptotic rate. There were 4 cases positive at 5 μmol/L concentration, all cases positive at 10 μmol/L and 20 μmol/L concentration, P<0.01 vs only chemotherapeutic drugs (3 cases positive), and chemotherapeutic drugs + S-PS-ODNs (10 μmol/L) (3 cases positive). After cells were treated with 10 μmol/L AS-PS-ODNs + chemotherapeutic drugs for 48 h, 72 h, 96 h, the apoptotic indexes were 14.22±2.86, 29.39±3.57, and 41.26±4.52, respectively. These were significantly higher than those of only chemotherapeutic drugs-treated cells and chemotherapeutic drugs + S-PS-ODNs-treated cells at corresponding time (P<0.01). There was no difference between only drugs group and S-PS-ODNs group at corresponding time (P>0.05). Conclusion: The CALM and AF10 fusion transcripts are involved in the pathogenesis of haematological malignancies with t(10, 11), and is associated with a poor prognosis. AS-PS-ODNs might be useful in therapy of t(10, 11) leukemia. Key words AF10 - CALM - Fusion transcript - Primary leukemia cell - In vitro sensitivity - Antisense oligodeoxynucleotide CLC number R733.7 Biography: LIU Ge-xiu (1968–), male, associate professor, Institute of Hematology, Medical College, Jinan University, majors in hematology.

Large-scale long terminal repeat insertions produced a significant set of novel transcripts in cotton

Genomic analysis has revealed that the 1,637-Mb Gossypium arboreum genome contains approximately 81%transposable elements(TEs),while only 57%of the 735-Mb G.raimondii genome is occupied by TEs.In this study,we investigated whether there were unknown transcripts associated with TE or TE fragments and,if so,how these new transcripts were evolved and regulated.As sequence depths increased from 4 to 100 G,a total of 10,284 novel intergenic transcripts(intergenic genes)were discovered.On average,approximately 84%of these intergenic transcripts possibly overlapped with the long terminal repeat(LTR)insertions in the otherwise untranscribed intergenic regions and were expressed at relatively low levels.Most of these intergenic transcripts possessed no transcription activation markers,while the majority of the regular genic genes possessed at least one such marker.Genes without transcription activation markers formed their+1 and-1 nucleosomes more closely(only(117±1.4)bp apart),while twice as big spaces(approximately(403.5±46.0)bp apart)were detected for genes with the activation markers.The analysis of 183 previously assembled genomes across three different kingdoms demonstrated systematically that intergenic transcript numbers in a given genome correlated positively with its LTR content.Evolutionary analysis revealed that genic genes originated during one of the whole-genome duplication events around 137.7million years ago(MYA)for all eudicot genomes or 13.7 MYA for the Gossypium family,respectively,while the intergenic transcripts evolved around 1.6 MYA,resultant of the last LTR insertion.The characterization of these low-transcribed intergenic transcripts can facilitate our understanding of the potential biological roles played by LTRs during speciation and diversifications.

Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.