Serum long non­coding RNA NNT­AS1 protected by exosome is a potential biomarker and functions as an oncogene via the miR­496/RAP2C axis in colorectal cancer.

Increasing evidence has indicated that long non­coding RNAs (lncRNAs) serve an essential role in carcinogenesis and cancer development. It has been reported that lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT­AS1) serves a crucial role in several types of cancer. However, the clinical significance of circulating NNT­AS1 expression in colorectal cancer (CRC) remains to be elucidated. The current study aimed to investigate the potential role of NNT­AS1 and the clinical significance of its serum expression levels in patients with CRC. The expression of NNT­AS1 was measured in 40 pairs of tumor and adjacent normal tissues from patients with CRC via reverse transcription­quantitative PCR. The serum expression levels of NNT­AS1 were assayed in an independent cohort of healthy controls and patients with CRC. The levels of NNT­AS1 were also compared between paired preoperative and postoperative serum samples. In addition, the presence of exosomal NNT­AS1 in serum was explored. Furthermore, the biological roles of NNT­AS1 were investigated in CRC cells in vitro. The expression of NNT­AS1 was significantly upregulated in tumor tissues compared with adjacent normal tissues (P<0.05). A higher level of NNT­AS1 was associated with an advanced CRC stage. The serum levels of NNT­AS1 were significantly upregulated in patients with CRC compared with healthy subjects (P<0.05). Furthermore, the NNT­AS1 levels were significantly decreased in postoperative samples compared with preoperative samples (P<0.01). In addition, it was also identified that NNT­AS1 was upregulated in CRC exosomes (P<0.01), whereas no significant difference was observed in NNT­AS1 levels between serum and exosomes. Silencing of NNT­AS1 inhibited the proliferation, migration and invasion of CRC cells. It was also identified that NNT­AS1 exerted its effects via regulation of the microRNA­496/Ras­related protein Rap­2c axis. The present study demonstrated that circulating NNT­AS1, which may be protected by exosomes, could be a novel potential biomarker and therapeutic target in CRC.

Long Non-Coding RNA MACC1-AS1 Is Involved in Distant Recurrence of Hepatocellular Carcinoma After Surgical Resection.

BACKGROUND We explored the role of MACC1-AS1 in hepatocellular carcinoma (HCC). MATERIAL AND METHODS Measurement of preoperative plasma levels of MACC1-AS1 was performed by qPCR, and the comparison between the HCC and Control group was performed by unpaired t test. The overexpression of TGF-ß1 in SNU-182 and SNU-398 cells was confirmed by qPCR. RESULTS MACC1-AS1 was overexpressed in HCC patients. In comparison to pretreatment level, distant recurrence (DR) was accompanied by increased levels of MACC1-AS1 in plasma, but this phenomenon was not observed in cases of local recurrence (LR) or non-recurrence (NR). In HCC cells, MACC1-AS1 positively regulated the expression of TGF-ß1. MACC1-AS1 overexpression resulted in increased invasion and migration rates of HCC cells, while siRNA silencing resulted in reduced rates. Moreover, TGF-ß1 overexpression reduced the effects of MACC1-AS1 siRNA silencing. CONCLUSIONS MACC1-AS1 is involved in the distant recurrence of HCC, and its actions are possibly mediated by TGF-ß1.

Expression Analysis of BDNF, BACE1, and Their Natural Occurring Antisenses in Autistic Patients.

Autism spectrum disorder (ASD) as a multifaceted neurological syndrome affects many aspects of neuropsychologic functions. Dysregulated expressions of several genes have been documented in ASD patients. The current project aimed at comparison of transcript levels of brain derived neurotrophic factor (BDNF), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), and their natural occurring antisenses in the peripheral blood of ASD individuals (n = 50, male/female = 38/12, age (mean ± standard deviation (SD)): 6 ± 1.4, age range: 3-8) and matched healthy persons (n = 50, male/female = 37/13, age (mean ± SD): 6 ± 1.74, age range: 3-8). We demonstrated remarkable higher levels of these genes in ASD patients. BACE1 transcript levels were correlated with transcript levels of BACE1-AS in all study participants. However, BACE1 transcript levels were not correlated with participants' age. BACE1-AS and BDNF transcript levels were correlated with age in female participants. Significant correlations were detected between transcript levels of BDNF and those of other genes in all study groups. The current results render further indications for contribution of BDNF, BACE1, and their antisenses in the course of ASD and suggested expression levels of these transcripts as putative markers for this neurobehavioral disorder. Such results might be applied in clinical setting for diagnosis of complicated ASD cases.

LncRNA GATA6-AS Promotes Cancer Cell Proliferation and Inhibits Apoptosis in Glioma by Downregulating lncRNA TUG1.

Background: Long noncoding RNA (lncRNA) GATA6-AS regulates the growth of endothelial cells, indicating its involvement in human diseases. The present study aimed to investigate the roles of GATA6-AS in glioma. Results: GATA6-AS was significantly upregulated in glioma. GATA6-AS levels in plasma was positively and significantly correlated with its expression levels in glioma tissues. TUG1 was significantly downregulated in glioma and was inversely correlated with GATA6-AS. GATA6-AS overexpression led to TUG1 downregulation, whereas TUG1 overexpression failed to significantly affect GATA6-AS. GATA6-AS overexpression promoted glioma cell proliferation and inhibited apoptosis, TUG1 overexpression inhibited cell proliferation and attenuated the effects of GATA6-AS overexpression. Conclusions: Therefore, lncRNA GATA6-AS affects cell proliferation and apoptosis and regulates TUG1 expression in glioma.

Sex-based dimorphisms in expression of BDNF and BACE1 in bipolar patients.

Bipolar disorder (BD) is a chronic, serious mental disorder distinguished by repeated episodes of mania and depression. Previous studies have demonstrated dysregulation of a number of transcripts in brain tissue or peripheral blood of BD patients. In the present study, we compared expression of two protein coding genes (brain-derived neurotrophic factor (BDNF) and beta-secretase 1 (BACE1)) and their natural occurring anti-sense (AS) RNAs (BDNF-AS and BACE1-AS) in peripheral blood of 50 BD patients (mean age ±â€¯standard deviation (SD) = 36.5 ±â€¯9.32) and 50 healthy subjects (mean age ±â€¯SD = 33.62 ±â€¯8.59). BDNF and BACE1 were significantly up-regulated in peripheral blood of total BD patients compared with total healthy subjects (Expression ratio = 2.2, P value = 0.003; Expression ratio = 2.2, P value = 0.002 respectively). However, comparison of their levels in sex-based subgroups showed their up-regulations only in male patients compared with male health subjects (Expression ratio = 2.48, P value = 0.006; Expression ratio = 2.1, P value = 0.01). No significant differences were found in expressions of BDNF-AS and BACE1-AS between BD and health subjects. We detected a significant correlation between BDNF expression and age at disease onset in BD group after adjustment of the effects of sex (R = 0.26, P value = 0.03). Moreover, there were trends toward correlations between BDNF expression and disease duration in BD group and between BDNF expression and age in health subjects (P values = 0.05). Combination of BDNF, BDNF-AS and BACE1 expression levels could differentiate BD patients from healthy subjects with 68% sensitivity and 82% specificity (area under curve = 0.72, P value = 0.0001). The current study suggests a sex-based dimorphic pattern in expression of BDNF and BACE1. Moreover, our results imply that expression pattern of these genes could be diagnostic markers in BD. Future studies are needed to assess this speculation in larger patient samples.

Expression analysis of beta-secretase 1 (BACE1) and its naturally occurring antisense (BACE1-AS) in blood of epileptic patients.

Beta-secretase 1 (BACE1) gene encodes a transmembrane protease from the peptidase A1 family of aspartic proteases whose role in the pathogenesis of Alzheimer's disease has been assessed. The enzymatic activity of BACE1 on several proteins implicated in epileptogenesis implies its role in the pathogenesis of epilepsy. In the present study, we assessed expression of BACE1 and its naturally occurring antisense (BACE1-AS) in peripheral blood of 40 epileptic patients and 40 age- and sex-matched healthy subjects. We did not detect either any difference in the expression of these genes between cases and controls or significant correlation between their expressions and participants' age. However, we demonstrated a significant correlation between expression levels of BACE1 and BACE1-AS which supports the previously suggested feed-forward mechanism of regulation between these two transcripts. Future studies in larger sample sizes are needed to elaborate the function of BACE1 in epilepsy.

Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria.

Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in understanding the in vivo pathological adaptations of these parasites.

Analysis of remnant reticulocyte mRNA reveals new genes and antisense transcripts expressed in the human erythroid lineage.

BACKGROUND AND OBJECTIVES: We studied the gene expression profile of human purified reticulocytes to provide a transcriptional basis for the study of erythroid biology, differentiation and hematologic disorders. DESIGN AND METHODS: We screened highly purified blood reticulocytes from ten healthy adult volunteers. We chose a modified protocol of serial analysis of gene expression (SAGE), the serial analysis of downsized extracts (SADE). RESULTS: Data analysis revealed that 64% of gene signatures (tags) matched with known genes; mainly hemoglobin. In addition to the abundant globin mRNA, SAGE analysis identified previously described genes and new transcripts. In reticulocytes, which are poor in mRNA, we also identified 9% of EST and 27% of tags that did not match with any known genes. Mining our data, 70% of the unknown tags and 39% of tags identifying EST were found to be specific to the reticulocyte. We demonstrated the presence of a mRNA that matched with the reverse sequence of the hemoglobin b (HBB) transcript. INTERPRETATION AND CONCLUSIONS: This is the first description of an antisense transcript of the human HBB gene suggesting regulation by way of sense-antisense pairing. The well-characterized genes found in the SAGE library were genes specific to the blood cell lineage, housekeeping genes and, interestingly, genes not previously described in the reticulocyte. Furthermore the study provides markers of the erythroid lineage regulated during the differentiation process as observed in in vitro experiments.

Tissue disposition of 2'-O-(2-methoxy) ethyl modified antisense oligonucleotides in monkeys.

This study examined the plasma pharmacokinetics, tissue distribution, and metabolism of three second generation antisense oligonucleotides in monkeys. Three groups of monkeys were treated with 10 mg/kg of each test compound by a single 2-h intravenous infusion. Oligonucleotide concentrations were measured in plasma, tissues, and urine using capillary gel electrophoresis (CGE). HPLC-MS was used to identify the metabolite(s) of the study compounds. Plasma-concentration-time profiles after infusion for the two phosphorothioate oligonucleotides were mono-exponential, but was bi- exponential for the phosphodiester oligonucleotide. Plasma clearance for the phosphodiester oligonucleotide was four- to sevenfold higher than the two phosphorothioate oligonucleotides, which was attributed to the plasma protein binding and reduced nuclease resistance. 2'-O-(2-methoxy) ethyl (MOE) modification at both 3' and 5' ends of a phosphorothioate oligonucleotide greatly enhanced the resistance to nucleases in plasma and tissue. MOE modification only at the 3' end enhanced the resistance to nucleases in plasma, but only moderately enhanced the resistance to nucleases in tissues. Urinary excretion was a minor elimination pathway for the phosphorothioate oligonucleotide, but was a major elimination pathway for the phosphodiester oligonucleotide. The results characterize the relationships between structure and disposition and will direct future modifications for therapeutic use.

Detection of minus-strand hepatitis C virus RNA in tumor tissues of hepatocellular carcinoma.

BACKGROUND: Recently, the detection of hepatitis C virus (HCV) antibody has been widely performed for clinical serum testing of HCV infection and can be identified in most hepatocellular carcinoma (HCC) cases in Japan. In the current study, the authors detected not only plus-strand but also minus-strand HCV RNA as a template for RNA replication in hepatocytes in the resected tumors of HCC, and they investigated those significant for HCC. METHODS: The plus-strand and minus-strand HCV RNA were detected using reverse transcription-polymerase chain reaction after the extraction of RNA from the tumor and nontumor tissues of hepatectomized liver, respectively. RESULTS: The detection of HCV RNA in liver tissues from hepatectomized specimens of 20 cases was as follows: in nontumor regions, both of the plus- and minus-strand HCV RNA were detected in 14 cases. Among those cases, plus- and minus-strand HCV RNA were detected in 11 cases, and in another 2 cases, only plus-strand HCV RNA was found in the tumor regions. CONCLUSIONS: These results suggest that HCV RNA is able to replicate in tumor tissues of HCC and may be involved in the development of HCC and also that HCV in remnant hepatocytes may cause the recurrence of HCC as secondary carcinogenesis after hepatectomy.

Regulation of class II MHC gene expression by the inducible anti-sense RNA in transgenic mice.

We have established a gene regulatory system in mice by the inducible anti-sense RNA. We have generated transgenic mice carrying the anti-sense DNA composed of the class II MHC gene under the control of the human metallothionein IIa gene promoter. The detectable amount of anti-sense RNA was constitutively produced in spleen and bone marrow from transgenic mice and the amount in spleen was increased about fivefold by the stimulation of mice with heavy metal ions. We have previously reported that the reduction of class II MHC molecules on early B lineage cells by the anti-sense RNA results in delay of their development in the bone marrow culture. The early B cell development was slightly delayed in the culture from the transgenic mice. This delay was augmented in the culture by the addition of heavy metal ions in proportion to its concentration. These results suggest that the inducible anti-sense RNA reduces the expression of class II MHC molecules on B lineage cells.