Transcribed Ultraconserved Regions Expression Research Articles

Construction of a three-component regulatory network of transcribed ultraconserved regions for the identification of prognostic biomarkers in gastric cancer.

Altered expression and functional roles of the transcribed ultraconserved regions (T-UCRs), as genomic sequences with 100% conservation between the genomes of human, mouse, and rat, in the pathophysiology of neoplasms has already been investigated. Nevertheless, the relevance of the functions for T-UCRs in gastric cancer (GC) is still the subject of inquiry. In the current study, we first used a genome-wide profiling approach to analyze the expression of T-UCRs in GC patients. Then, we constructed a three-component regulatory network and investigated potential diagnostic and prognostic values of the T-UCRs. The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) dataset was used as a resource for the RNA-sequencing data. FeatureCounts was utilized to quantify the number of reads mapped to each T-UCR. Differential expression analysis was then conducted using DESeq2. In the following, interactions between T-UCRs, microRNAs (miRNAs), and messenger RNAs (mRNAs) were combined into a three-component network. Enrichment analyses were performed and a protein-protein interaction (PPI) network was constructed. The R Survival package was utilized to identify survival-related significantly differentially expressed T-UCRs (DET-UCRs). Using an in-house cohort of GC tissues, expression of two DET-UCRs was furthermore experimentally verified. Our results showed that several T-UCRs were dysregulated in TCGA-STAD tumoral samples compared to nontumoral counterparts. The three-component network was constructed which composed of DET-UCRs, miRNAs, and mRNAs nodes. Functional enrichment and PPI network analyses revealed important enriched signaling pathways and gene ontologies such as "pathway in cancer" and regulation of cell proliferation and apoptosis. Five T-UCRs were significantly correlated with the overall survival of GC patients. While no expression of uc.232 was observed in our in-house cohort of GC tissues, uc.343 showed an increased expression, although not statistically significant, in gastric tumoral tissues. The constructed three-component regulatory network of T-UCRs in GC presents a comprehensive understanding of the underlying gene expression regulation processes involved in tumor development and can serve as a basis to investigate potential prognostic biomarkers and therapeutic targets.

The dark matter of the human genome and its role in human cancers.

The transcribed ultra-conserved regions (T-UCRs) are a novel family of non-coding RNAs which are absolutely conserved (100%) across orthologous regions of the human, mouse, and rat genomes. T-UCRs represent a small portion of the human genome that is likely to be functional but does not code for proteins and is referred to as the "dark matter" of the human genome. Although T-UCRs are ubiquitously expressed, tissue- and disease-specific expression of T-UCRs have also been observed. Accumulating evidence suggests that T-UCRs are differentially expressed and involved in the malignant transformation of human tumors through various genetic and epigenetic regulatory mechanisms. Therefore, T-UCRs are novel candidate predisposing biomarkers for cancer development. T-UCRs have shown to drive malignant transformation of human cancers through regulating non-coding RNAs and/or protein coding genes. However, the functions and fate of most T-UCRs remain mysterious. Here, we review and highlight the current knowledge on these ultra-conserved elements in the formation and progression of human cancers.

Overexpression of ultraconserved region 83- induces lung cancer tumorigenesis.

The expression of non–coding RNAs (ncRNAs) is dysregulated in human cancers. The transcribed ultraconserved regions (T-UCRs) express long ncRNAs involved in human carcinogenesis. T-UCRs are non-coding genomic sequence that are 100% conserved across humans, rats and mice. Conservation of genomic sequences across species intrinsically implies an essential functional role and so we considered the expression of T-UCRs in lung cancer. Using a custom microarray we analyzed the global expression of T-UCRs. Among these T-UCRs, the greatest variation was observed for antisense ultraconserved element 83 (uc.83-), which was upregulated in human lung cancer tissues compared with adjacent non cancerous tissues. Even though uc.83- is located within the long intergenic non-protein coding RNA 1876 (LINC01876) gene, we found that the transcribed uc.83- is expressed independently of LINC01876 and was cloned as a 1143-bp RNA gene. In this study, functional analysis confirmed important effects of uc.83- on genes involved in cell growth of human cells. siRNA against uc.83- decreased the growth of lung cancer cells while the upregulation through a vector overexpressing the uc.83- RNA increased cell proliferation. We also show the oncogenic function of uc.83- is mediated by the phosphorylation of AKT and ERK 1/2, two important biomarkers of lung cancer cell proliferation. Based on our findings, inhibition against uc.83- could be a future therapeutic treatment for NSCLC to achieve simultaneous blockade of pathways involved in lung carcinogenesis.

Differentially Methylated Ultra-Conserved Regions Uc160 and Uc283 in Adenomas and Adenocarcinomas Are Associated with Overall Survival of Colorectal Cancer Patients.

Deregulation of the transcribed ultra-conserved regions (T-UCRs) Uc160, Uc283, and Uc346 has been reported in colorectal cancer (CRC) recently. Here, we investigated promoter methylation of these T-UCRs during the adenoma–carcinoma sequence and their clinical significance in CRC patients. Methylation levels were assessed in CRC, adenomas, infiltrated lymph nodes, and metastatic tissue specimens. In situ hybridization was performed in representative tissue specimens. T-UCRs expression levels were also evaluated in HT-29 colon cancer cells before and after the acquired resistance to 5-fluorouracil (5-FU) and oxaliplatin. A gradual increase in T-UCRs methylation levels from hyperplastic polyps to adenomas and to in situ carcinomas (ISC) and a gradual decrease from ISC to infiltrative and metastatic carcinomas was observed (p < 0.001 for Uc160 and Uc283, p = 0.018 for Uc346). Uc160 and Uc283 methylation was associated with the grade of dysplasia in adenoma specimens (p = 0.034 and p = 0.019, respectively). Furthermore, higher Uc160 methylation, mainly in stage III and IV patients, was related to improved overall survival (OS) in univariate (p = 0.009; HR, 0.366) and multivariate analysis (p = 0.005; HR, 0.240). Similarly, higher methylation of Uc283 was associated with longer OS (p = 0.030). Finally, T-UCRs expression was significantly reduced in HT-29 cells after resistance to chemotherapy. This study suggests that promoter methylation of Uc160, Uc283, and Uc346 is altered during CRC development and that Uc160 and Uc283 methylation may have prognostic significance for CRC patients.

Abstract 2011: Expression analysis of transcribed-ultraconserved regions in cancer stem cell population using colorectal cancer organoids

Abstract Background: The transcribed-ultraconserved regions (T-UCRs) are a novel class of long noncoding RNAs transcribed from ultraconserved regions (UCRs) that are located in both intra- and intergenic regions and absolutely conserved among the orthologous regions in most of the vertebrates. There are at least 481 UCRs, and these regions can produce 962 T-UCRs. T-UCRs have distinct signatures in human cancers. However, little has been known about the correlation between T-UCRs and tissue/cancer stem cells so far. Organoid is a novel 3D in vitro stem cell culture technology, which sustains stem cell-driven formation of near-physiologic, self-renewing tissues through using the specific niche factors in a dish. As it can be established from patient-derived tissue samples and recapitulates the genetic diversity of the original tissues, patient-derived tumor organoids could be a reliable model of disease. Because of its biologic features, organoid is assumed to harbor many stem and progenitor cells, so that it is suitable for stem cell analysis. In this study, we aimed to identify T-UCRs that are specifically expressed in cancer stem cell of colorectal cancer (CRC) and validate their functional role in CRC stem cells. Methods: We focused on 61 T-UCRs that were reported as the dysregulated T-UCRs in CRC by microarray analysis. We examined them using RNA samples isolated from 3 pairs of CRC and non-neoplastic organoids by qRT-PCR. Biologic roles of the T-UCRs upregulated in CRC organoids were analyzed using a vector containing the whole sequences of the T-UCRs. Results: Through the validation, we found that Uc.91+, Uc.182+, Uc.249+A and Uc.266+A were specifically upregulated in CRC organoids compared to those in normal organoids. We also validated the T-UCRs expression in several CRC cases using both primary tumors and organoids, which further supported the validity of CRC stem cell specific signature. Each of the expression of the 4 T-UCRs was highly upregulated in 2 CRC cell lines, HCT-116 and SW480, while we did not see any similarities in representative genetic abnormalities between the 2 CRC cell lines. Further in-depth in vitro studies are ongoing to clarify the solid evidences, especially the direct effect of T-UCRs onto the stem cells features such as dormancy and sphere-forming ability. Conclusion: We identified that Uc.91+, Uc.182+, Uc.249+A and Uc.266+A were specifically upregulated in CRC organoids, and the 4 T-UCRs showed similar trend of expression in several CRC cell lines. These 4 T-UCRs are promising candidates that could potentially have an important role in cancer stem cell biology. Citation Format: Ririno Honma, Naoya Sakamoto, Akira Ishikawa, Daiki Taniyama, Kaho Fukada, Yohei Sekino, Shoichiro Mukai, Kazuhiro Sentani, Naohide Oue, Takao Hinoi, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. Expression analysis of transcribed-ultraconserved regions in cancer stem cell population using colorectal cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2011.

Ischemic Stroke Alters the Expression of the Transcribed Ultraconserved Regions of the Genome in Rat Brain.

Human and rodent genomes diverged ≈75 million years ago. However, 481 regions of their genomes (200-779 nucleotide each) remained absolutely conserved and form noncoding RNAs known as transcribed ultraconserved regions (T-UCRs). The functional significance of T-UCRs is not apparent, but their altered expression is associated with many diseases, and thus thought to be critical for life. We presently investigated the poststroke temporal changes in the expression of T-UCRs with potential functional significance. Male, spontaneously hypertensive rats were subjected to transient middle cerebral artery occlusion. Expression profile of T-UCRs was determined at 3, 6, and 12 hours of reperfusion using microarrays and real-time polymerase chain reaction in the peri-infarct cortex. The putative functional significance of stroke-responsive T-UCRs was identified by bioinformatics. Ischemia altered expression of 69 T-UCRs at ≥1 time points of reperfusion compared with sham. Poststroke expression of the intragenic T-UCRs is independent of the expression of their parent gene mRNAs. Bioinformatics showed that the upstream/downstream and the parent genes of the T-UCRs modulate several biological and molecular functions, including metabolism, response to stimuli, cell communication, protein and nucleic acid binding. This first report shows that ischemic stroke temporally alters the noncoding ultraconserved RNAs in spontaneously hypertensive rats, but their functional significance is yet to be evaluated.

Deregulation of methylation of transcribed-ultra conserved regions in colorectal cancer and their diagnostic and prognostic value.

e15130 Background: Expression of Transcribed Ultra Conserved Regions (T-UCRs) is often deregulated in cancer. We investigated the role of three T-UCRs (Uc160, Uc283 and Uc346) in colorectal adenocarcinomas and their prognostic and diagnostic value. Methods: Expression and methylation levels of the T-UCRs were assessed in neoplastic and paired non-malignant fresh frozen (FF) tissue specimens from 64 colorectal cancer (CRC) patients, as well as in 6 FF adenoma tissue specimens. In addition, T-UCR methylation levels were assessed in FFPE tumor tissues from 80 CRC patients and in plasma from 161 patients (50 CRC, 59 adenoma patients, 40 healthy subjects and 12 patients with colon inflammation or diverticulosis). Results: Expression levels of all three T-UCRs were lower in neoplastic, compared to non-malignant tissues, although at a statistically significant level only for Uc160 ( p&lt; 0.001). Also, methylation levels of Uc160, Uc283 and Uc346 were higher in tumors compared to non-malignant tissues ( p&lt; 0.001, p= 0.001 and p= 0.004 respectively). Tissue methylation levels of Uc160 were associated with TTP ( p= 0.017). The combination of Uc283 and Uc346 methylation levels was related to OS, however without reaching statistical significance ( p= 0.066). Methylation status of Uc160 and Uc346 in plasma differed significantly among the four patient groups with CRC patients exhibiting the higher levels. Moreover, a strong correlation was found between Uc160 plasma methylation levels and adenoma or adenocarcinoma size and lymph node infiltration ( p&lt; 0.001 and p= 0.024 respectively). When methylation status was used to predict if a subject has CRC, sensitivity and specificity were 35% and 89% respectively, while the values changed to 45% and 74.3% respectively when we combined the sum of the three T-UCR plasma methylation levels. For adenomas, the combination of Uc160 and Uc346 plasma methylation displayed 30.2% (sensitivity) and 80.7% (specificity). Conclusions: T-UCR expression and methylation is deregulated in CRC while their methylation has prognostic value and appears a promising non-invasive screening test for CRC and adenomas, provided that the sensitivity of the assay is improved.

Expression and function of Uc.160+, a transcribed ultraconserved region, in gastric cancer.

Transcribed ultraconserved regions (T-UCRs) are a novel class of noncoding RNAs that are highly conserved among the orthologous regions in most vertebrates. It has been reported that T-UCRs have distinct signatures in human cancers. We previously discovered the downregulation of T-UCR expression in gastric cancer (GC), indicating that T-UCRs could play an important role in GC biology. Uc.160+, a T-UCR reported to be downregulated in human cancer, has not been examined in GC. We analyzed the expression pattern of Uc.160+ in nonneoplastic and tumor tissues of the stomach by using uantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH), specifically focusing on the mechanism of transcriptional regulation and target genes that are regulated by T-UCRs. We also attempted to determine the effect of Uc.160+ expression on biological features of GC cell lines by Western blotting. On the basis of the qRT-PCR and ISH results, Uc.160+ expression in adenoma and GC tissues was clearly downregulated compared with that in nonneoplastic mucosa tissues of the stomach. Cancer-specific DNA methylation in the promoter region of Uc.160 was observed by bisulfite genomic DNA sequencing analysis. The effect of DNA methylation on Uc.160+ expression was further confirmed by reporter gene assay. We also revealed that Uc.160+ inhibited the phosphorylation of Akt by regulating phosphatase and tensin homolog (PTEN) expression. These results indicate that Uc.160+ could possibly have a tumor suppressive role in GC.

New Cross-Talk Layer between Ultraconserved Non-Coding RNAs, MicroRNAs and Polycomb Protein YY1 in Bladder Cancer.

MicroRNAs (miRNAs) are highly conserved elements in mammals, and exert key regulatory functions. Growing evidence shows that miRNAs can interact with another class of non-coding RNAs, so-called transcribed ultraconserved regions (T-UCRs), which take part in transcriptional, post-transcriptional and epigenetic regulation processes. We report here the interaction of miRNAs and T-UCRs as a network modulating the availability of these non-coding RNAs in bladder cancer cells. In our cell system, antagomiR-596 increased the expression of T-UCR 201+. Moreover, T-UCR 8+ silencing increased miR-596 expression, which in turn reduced total T-UCR 283+, showing that the perturbation of one element in this network changes the expression of other interactors. In addition, we identify the polycomb protein Yin Yang 1 (YY1) as mediator of binding between miR-596 and T-UCR 8+. These new findings describe for the first time a network between T-UCRs, miRNAs and YY1 protein, highlighting the existence of an additional layer of gene expression regulation.

570P - Diagnostic value of methylation status of T-UCRs for colorectal cancer

Expression of Transcribed Ultra Conserved Regions (T-UCRs) is often dysregulated in various types of cancer. Regulation of T-UCR expression includes epigenetic mechanisms, and in particular CpG island methylation. Three T-UCRs (160, 283 and 346) have been found to be methylated in colon adenocarcinomas. The present study assesses the use of the T-UCR methylation status in circulating DNA as a diagnostic marker for colorectal cancer (CRC). Expression and methylation levels of T-UCRs 160, 283 and 346 were assessed in neoplastic and paired normal colonic fresh frozen tissue specimens from 75 CRC patients, as well as in 5 fresh frozen adenoma tissue specimens. Methylation status of the three T-UCRs was also determined in plasma from 161 patients (56 CRC patients, 55 adenoma patients, 40 healthy subjects and 10 patients with colon inflammation or diverticulosis). Expression levels of all three T-UCRs were lower in neoplastic tissues, compared to normal adjacent colonic tissues, but only in T-UCR 160 the difference was statistically significant (p < 0.001). Methylation levels of 160, 283 and 346 were higher in colorectal cancer tissues compared to normal adjacent tissues (p < 0.001, p = 0.029 and p = 0.005 respectively). Notably, methylation levels of 160 and 346 in adenomas were higher than those in normal tissues but lower than those in cancer tissues. Methylation status of 160 in plasma differed significantly among the four different groups of patients (p = 0.024) and the difference increased when we compared methylation status in colorectal adenoma or adenocarcinoma patients with healthy subjects or patients with inflammation or diverticulosis (p = 0.007). When methylation status was used to predict if a subject has colorectal adenocarcinoma, specificity and sensitivity were 85% and 30% respectively. Methylation of T-UCR 160 in plasma has great specificity for CRC but low sensitivity. Alteration of the methodological approaches to improve the sensitivity could result in a promising non-invasive screening method for CRC.

Altered T-UCRs expression profile in the spinal cord of mice with neuropathic pain

Spinal cord plays an important role in the transmission and modulation of nociceptive information. Global changes in gene expression in the spinal cord contribute to the induction and maintenance of neuropathic pain. Transcribed Ultraconserved Regions (T-UCRs), a novel class of long noncoding RNAs, can regulate gene expression at both transcriptional and post-transcriptional levels and are related to many human diseases such as cancer, Alzheimer's disease, and heart diseases. In this study, we screened abnormal T-UCRs expression in the spinal cord under spinal nerve ligation (SNL)-induced neuropathic pain condition. Microarray data showed the alternation of T-UCRs at the transcriptional level in the spinal cord 10 days after SNL. Among 78 altered T-UCRs, 23 T-UCRs were upregulated by more than 1.5-fold and 55 ones downregulated by less than 0.5-fold after SNL. Hierarchical cluster analysis of T-UCRs expression profiles showed the opposite expression pattern between SNL and sham-operated mice. The quantitative real-time reverse transcription polymerase chain reaction analysis further confirmed the expression patterns of uc.305, uc.189, uc.46, and uc.217 after SNL. The gene ontology annotation and signaling pathway analysis for the T-UCRs host genes indicated that differentially expressed T-UCRs were involved in several intracellular activities and signaling pathways, including Ephrin receptor activity, soluble NSF attachment protein receptor (SNARE) interactions in vesicular transport pathway, and WNT signaling pathway. Collectively, the current data suggest the possible role of T-UCR in the pathogenesis of neuropathic pain. T-UCRs may serve as a new kind of target for the treatment of neuropathic pain.

Globally increased ultraconserved noncoding RNA expression in pancreatic adenocarcinoma.

Transcribed ultraconserved regions (T-UCRs) are a class of non-coding RNAs with 100% sequence conservation among human, rat and mouse genomes. T-UCRs are differentially expressed in several cancers, however their expression in pancreatic adenocarcinoma (PDAC) has not been studied. We used a qPCR array to profile all 481 T-UCRs in pancreatic cancer specimens, pancreatic cancer cell lines, during experimental pancreatic desmoplasia and in the pancreases of P48Cre/wt; KrasLSL-G12D/wt mice. Fourteen, 57 and 29% of the detectable T-UCRs were differentially expressed in the cell lines, human tumors and transgenic mouse pancreases, respectively. The vast majority of the differentially expressed T-UCRs had increased expression in the cancer. T-UCRs were monitored using an in vitro model of the desmoplastic reaction. Twenty-five % of the expressed T-UCRs were increased in the HPDE cells cultured on PANC-1 cellular matrix. UC.190, UC.233 and UC.270 were increased in all three human data sets. siRNA knockdown of each of these three T-UCRs reduced the proliferation of MIA PaCa-2 cells up to 60%. The expression pattern among many T-UCRs in the human and mouse pancreases closely correlated with one another, suggesting that groups of T-UCRs are co-activated in PDAC. Successful knockout of the transcription factor EGR1 in PANC-1 cells caused a reduction in the expression of a subset of T-UCRs suggesting that EGR1 may control T-UCR expression in PDAC. We report a global increase in expression of T-UCRs in both human and mouse PDAC. Commonalties in their expression pattern suggest a similar mechanism of transcriptional upregulation for T-UCRs in PDAC.

Expression Levels of Transcribed Ultraconserved Regions uc.73 and uc.388 Are Altered in Colorectal Cancer

Objectives: The development of colorectal cancer (CRC) is characterized by multiple genetic alterations. Transcribed ultraconserved regions (T-UCRs) are a subset of 481 sequenc es longer than 200 bp, which are absolutely conserved between orthologous regions of human, rat and mouse genomes, and are actively transcribed. It has recently been proven in cancer systems that differentially expressed T-UCRs could alter the functional characteristics of malignant cells. Genome-wide profiling revealed that T-UCRs have distinct signatures in human leukemia and carcinoma. Methods: In our study, we examined the expression levels of uc.43, uc.73, uc.134, uc.230, uc.339, uc.388 and uc.399 in 54 samples of primary colorectal carcinomas and 15 samples of non-tumoral adjacent tissues by real-time PCR. T-UCR expression levels were also correlated with commonly used clinicopathological features of CRC. Results: Expression levels of uc.73 (p = 0.0139) and uc.388 (p = 0.0325) were significantly decreased in CRC tissue, and uc.73 indicated a positive correlation with overall survival (p = 0.0315). The lower expression of uc.388 was associated with the distal location of CRC (p = 0.0183), but no correlation of any evaluated T-UCR with clinical stage, grade and tumor diameter was observed. Conclusion: Our preliminary results suggest that uc.73 and uc.388 could be potential diagnostic and prognostic biomarkers in CRC patients.

An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.