Associations between low serum levels of ANRIL and some common gene SNPs in Iranian patients with premature coronary artery disease.

Coronary artery disease (CAD) is the major cause of mortality in the world. Premature development of CAD can be attributed to women under 55 and men under 45. Many genetic factors play a part in premature CAD. Among them, ANRIL, a long noncoding RNA is located at the 9p21 risk locus, and its expression seems to be correlated with CAD. In the current study, premature CAD and control blood samples, with and without Type 2 Diabetes (T2D), were genotyped for six SNPs at the 9p21 locus. Additionally, ANRIL serum expression was assessed in both groups using real-time PCR. It was performed using different primers targeting exons 1, 5-6, and 19. The χ2 test for association, along with t-tests and ANOVA, was employed for statistical analysis. In this study, we did not find any significant correlation between premature coronary artery disease and rs10757274, rs2383206, rs2383207, rs496892, rs10757278 and rs10738605. However, a lower ANRIL expression was correlated with each SNP risk genotype. Despite the correlation between lower ANRIL expression and CAD, Type 2 diabetes was associated with higher ANRIL expression. Altogether, the correlation between ANRIL expression and the genotypes of the studied SNPs indicated that genetic variants, even those in intronic regions, affect long noncoding RNA expression levels. In conclusion, we recommend combining genetic variants with expression analysis when developing screening strategies for families with premature CAD. To prevent the devastating outcomes of CAD in young adults, it is crucial to discover noninvasive genetic-based screening tests.

Differential Expression Pattern of linc-ROR Spliced Variants in Pluripotent and Non-Pluripotent Cell Lines.

OBJECTIVE: The human large intergenic non-coding RNA-regulator of reprogramming program (linc-ROR) is known as a stem cell specific linc-RNA. linc-ROR counteracts differentiation via sequestering microRNA-145 (miR-145) that targets OCT4 transcript. Despite the research on the expression and function, the exact structure of linc-ROR transcripts is not clear. Considering the contribution of alternative splicing in transcripts structures and function, identifying different spliced variants of linc-ROR is necessary for further functional analyses. We aimed to find the alternatively spliced transcripts of linc-ROR and investigate their expression pattern in stem and cancer cell lines and during neural differentiation of NT2 cells as a model for understanding linc-ROR role in stem cell and differentiation. MATERIALS AND METHODS: In this experimental study, linc-ROR locus was scanned for identifying novel exons. Different primer sets were used to detect new spliced variants by reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing. Quantitative PCR (qPCR) and RT-PCR were employed to profile expression of linc-ROR transcripts in different cell lines and during neural differentiation of stem cells. RESULTS: We could discover 13 novel spliced variants of linc-ROR harboring unique array of exons. Our work uncovered six novel exons, some of which were the product of exonized transposable elements. Monitoring expression profile of the linc-ROR spliced variants in a panel of pluripotent and non-pluripotent cells exhibited that all transcripts were primarily expressed in pluripotent cells. Moreover, the examined linc-ROR spliced variants showed a similar downregulation during neural differentiation of NT2 cells. CONCLUSION: Altogether, our data showed despite the difference in the structure and composition of exons, various spliced variants of linc-ROR showed similar expression pattern in stem cells and through differentiation.

Endothelial nitric oxide synthase Asp298Glu (894G/T) gene polymorphism as a possible risk factor for the coronary slow flow phenomenon among Iranians.

BACKGROUND: Mounting evidence indicates an association between endothelial dysfunction and the coronary slow flow phenomenon (CSFP). In the present study, we aimed to evaluate the possible role of endothelial nitric oxide synthase (eNOS) 894G/T and interleukin-1ß (IL-1ß) 315C/T polymorphisms as possible risk factors for CSFP. METHODS: This prospective study enrolled patients with CSFP and individuals with normal coronary arteries. Genotypes were assessed using regular polymerase chain reaction and direct Sanger-sequencing techniques. RESULTS: The study population consisted of 267 individuals: 180 patients with CSFP (49 women [27.2%]) at a median age of 55 (48-62) years and 87 controls with normal coronary arteries (56 women [64.4%]) at a median age of 47 (41-58) years. The allelic distribution of eNOS 894G/T was significantly associated with CSFP (odds ratio [OR], 1.58; 95% confidence interval (CI), 1.04-2.42; P = 0.03). This polymorphism increased the risk of CSFP under the dominant model (OR 1.73; 95% CI I.02-2.95; P = 0.04). However, the allelic frequencies (1.05; 95% CI 0.68-1.59; P = 0.83) and genotypic frequencies (0.88; 95% CI 0.52-1.49; P = 0.63) of the IL-1ß 315C/T polymorphism were not associated with the incidence of CSFP in the Iranian population. CONCLUSIONS: The CSFP and control groups were statistically different regarding the eNOS 894G/T polymorphism. Our findings also demonstrated that the IL-1ß 315C/T polymorphism was not a risk factor for CSFP.

Linc-ROR has a Potential ceRNA Activity for OCT4A by Sequestering miR-335-5p in the HEK293T Cell Line.

Linc-ROR has a regulatory role in reprogramming, and the core stem cell transcription factors, OCT4, SOX2, and NANOG, regulate its expression. MicroRNAs (miRNAs) are also a critical constituent of pivotal posttranscriptional regulatory pathways. One of such interactions is a competing endogenous RNA interaction that connects small and long non-coding RNAs with coding transcripts. Here, we aimed to investigate the existence of such associations between OCT4A, Linc-ROR, hsa-miR-335-5p, and hsa-miR-544. Bioinformatic analysis was performed to evaluate the expression status of OCT4A, Linc-ROR, miR-335, and miR-544 throughout differentiation as well as in various differentiated cells. The complete lengths of OCT4A and Linc-ROR, and OCT4A 3'-UTR were cloned in the luciferase reporter vector, and the precursors of miR-335 and miR-544 were cloned in expression vectors. Following the overexpression of miR-335 and miR-544 in the 5637 cell line, the endogenous expression of OCT4A and Linc-ROR was evaluated. Afterward, the expression vectors of miRNAs and the reporter vectors of OCT4A/Linc-ROR were co-transfected in the HEK293T cell line. Via the Dual-Luciferase assay, the effect of the overexpression of miRNAs on their two possible targets (Linc-ROR and OCT4A) was investigated. The bioinformatic analysis demonstrated a relatively similar expression pattern for OCT4A and Linc-ROR, while miR-335 showed a different expression status. Both miR-335 and miR-544 inhibited the endogenous expression of OCT4A. The Dual-Luciferase assay likewise confirmed the inhibitory effect of miR-335 and miR-544 on OCT4A expression. In contrast, the miR-335 inhibitory effect was reversed in the presence of Linc-ROR, resulting in the upregulation of OCT4A. Such evidence suggests that Linc-ROR may compete with OCT4A to interact with miR-335.

Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain tumors.

Long non-coding RNAs (lncRNAs) are important modulators of various cellular and molecular events, including cancer-associated pathways. The Anti-differentiation ncRNA (ANCR) is a key regulator of keratinocyte differentiation, where its expression is necessary to maintain epidermal progenitor's cells. Herein, we investigated the expression pattern of ANCR in the course of neural differentiation. Moreover, we used published RNAseq data and clinical samples to evaluate the alteration of ANCR expression in different cell types and brain tumors. Furthermore, we manipulated ANCR expression in glioma cell lines to clarify a potential functional role for ANCR in tumorigenesis. Our qRT-PCR results revealed a significant upregulation of ANCR in more malignant and less differentiated types of brain tumors (P = 0.03). This data was in accordance with down regulation of ANCR during neural differentiation. ANCR suppression caused an elevation in apoptosis rate, as well as a G1 cell cycle arrest in glioblastoma cell line. Altogether, our data demonstrated that ANCR may play a role in glioma genesis and that it could be considered as a potential diagnostic and therapeutic target to combat brain cancers.

Novel spliced variants of OCT4, OCT4C and OCT4C1, with distinct expression patterns and functions in pluripotent and tumor cell lines.

OCT4 is a major regulator of pluripotency which has several spliced variants and expressed pseudogenes. Here, we are reporting the existence of two additional novel spliced variants of OCT4, OCT4C and OCT4C1, which lack Exon1 (E1) but start at a novel exon (E0) located ∼14kb upstream of E2. OCT4C/C1 is highly expressed in ES and iPS cells, and their expression was sharply turned off, upon the induction of neural differentiation. The long non-coding RNA (lncRNA) PSORS1C3, is located ∼9kb downstream of the E0 of OCT4C/C1. PSORS1C3 is vigorously spliced to generate nine novel variants, however, none of its exons incorporated in alternatively spliced variants of OCT4. Interestingly, the exons of OCT4 and PSORS1C3 are intertwined, with a novel exon (E0) of PSORS1C3 located ∼4kb upstream of OCT4 E0. This exon participates in generating some more variants of PSORS1C3 (variants 10-24). OCT4C/C1 knock-down in ES and iPS cell lines caused a slight downregulation of PSORS1C3 and OCT4A, a slight upregulation of OCT4B1, and a dramatic upregulation of OCT4B. Altogether, our data revisited the current view of OCT4 gene structure and regulation, and revealed its complex genomic features and expression regulation in stem and tumor cells.