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Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish. [ABSTRACT FROM AUTHOR]

The GAS5 gene encodes a long non-coding RNA (lncRNA) and intron-located small nucleolar RNAs (snoRNAs). Its structure, splice variants, and diverse functions in mammalian cells have been thoroughly investigated. However, there are still no data on a successful knockout of GAS5 in human cells, with most of the loss-of-function experiments utilizing standard techniques to produce knockdowns. By using CRISPR/Cas9 to introduce double-strand breaks in the terminal intronic box C/D snoRNA genes (SNORDs), we created monoclonal cell lines carrying continuous deletions in one of the GAS5 alleles. The levels of GAS5-encoded box C/D snoRNAs and lncRNA GAS5 were assessed, and the formation of the novel splice variants was analyzed. To comprehensively evaluate the influence of specific SNORD mutations, human cell lines with individual mutations in SNORD74 and SNORD81 were obtained. Specific mutations in SNORD74 led to the downregulation of all GAS5-encoded SNORDs and GAS5 lncRNA. Further analysis revealed that SNORD74 contains a specific regulatory element modulating the maturation of the GAS5 precursor transcript. The results demonstrate that the maturation of GAS5 occurs through the m6A-associated pathway in a SNORD-dependent manner, which is a quite intriguing epitranscriptomic mechanism. [ABSTRACT FROM AUTHOR]

Previous studies indicated long non-coding RNAs (lncRNAs) participated in the pathogenesis of atrial fibrillation (AF). However, little is known about the role of lncRNAs in AF-induced electrical remodeling. This study aimed to investigate the regulatory effect of lncRNA GAS5 (GAS5) on the electrical remodeling of neonatal rat cardiomyocytes (NRCMs) induced by rapid pacing (RP). RNA microarray analysis yielded reduced GAS5 level in NRCMs after RP. RT-qPCR, western blot, and immunofluorescence yielded downregulated levels of Nav1.5, Kv4.2, and Cav1.2 after RP, and whole-cell patch-clamp yielded decreased sodium, potassium, and calcium current. Overexpression of GAS5 attenuated electrical remodeling. Bioinformatics tool prediction analysis and dual luciferase reporter assay confirmed a direct negative regulatory effect for miR-27a-3p on lncRNA-GAS5 and HOXa10. Further analysis demonstrated that either miR-27a-3p overexpression or the knockdown of HOXa10 further downregulated Nav1.5, Kv4.2, and Cav1.2 expression. GAS5 overexpression antagonized such effects in Nav1.5 and Kv4.2 but not in Cav1.2. These results indicate that, in RP-treated NRCMs, GAS5 could restore Nav1.5 and Kv4.2 expression via the miR-27a-3p/HOXa10 pathway. However, the mechanism of GAS5 restoring Cav1.2 level remains unclear. Our study suggested that GAS5 regulated cardiac ion channels via the GAS5/miR-27a-3p/HOXa10 pathway and might be a potential therapeutic target for AF. [ABSTRACT FROM AUTHOR]

Asthma is a heterogeneous entity encompassing distinct endotypes and varying phenotypes, characterized by common clinical manifestations, such as shortness of breath, wheezing, and variable airflow obstruction. Two major asthma endotypes based on molecular patterns are described: type 2 endotype (allergic-asthma) and T2 low endotype (obesity-related asthma). Long noncoding RNAs (lncRNAs) are transcripts of more than 200 nucleotides in length, currently involved in many diverse biological functions, such as chromatin remodeling, gene transcription, protein transport, and microRNA processing. Despite the efforts to accurately classify and discriminate all the asthma endotypes and phenotypes, if long noncoding RNAs could play a role as biomarkers in allergic asthmatic and adolescent obesity-related asthma, adolescents remain unknown. To compare expression levels of lncRNAs: HOTAIRM1, OIP5-AS1, MZF1-AS1, and GAS5 from whole blood of Healthy Adolescents (HA), Obese adolescents (O), allergic asthmatic adolescents (AA) and Obesity-related asthma adolescents (OA). We measured and compared expression levels from the whole blood of the groups mentioned above through RT-q-PCR. We found differentially expressed levels of these lncRNAs between the groups of interest. In addition, we found a discriminative value of previously mentioned lncRNAs between studied groups. Finally, we generated an interaction network through bioinformatics. Expression levels of OIP5-AS1, MZF1-AS1, HOTAIRM1, and GAS5 in whole blood from the healthy adolescent population, obese adolescents, allergic asthma adolescents, and obesity-related asthma adolescents are differently expressed. Moreover, these lncRNAs could act as molecular biomarkers that help to discriminate between all studied groups, probably through molecular mechanisms with several genes and miRNAs implicated. [ABSTRACT FROM AUTHOR]

Although the imbalance of circulating levels of Thyroid Hormones (THs) affects female fertility in vertebrates, its involvement in the promotion of Premature Ovarian Aging (POA) is debated. Therefore, altered synthesis of THs in both thyroid and ovary can be a trait of POA. We investigated the relationship between abnormal TH signaling, dysthyroidism, and POA in evolutionary distant vertebrates: from zebrafish to humans. Ovarian T3 signaling/metabolism was evaluated by measuring T3 levels, T3 responsive transcript, and protein levels along with transcripts governing T3 availability (deiodinases) and signaling (TH receptors) in distinct models of POA depending on genetic background and environmental exposures (e.g., diets, pesticides). Expression levels of well-known (Amh, Gdf9, and Inhibins) and novel (miR143/145 and Gas5) biomarkers of POA were assessed. Ovarian dysthyroidism was slightly influenced by genetics since very few differences were found between C57BL/6J and FVB/NJ females. However, diets exacerbated it in a strain-dependent manner. Similar findings were observed in zebrafish and mouse models of POA induced by developmental and long-life exposure to low-dose chlorpyrifos (CPF). Lastly, the T3 decrease in follicular fluids from women affected by diminished ovarian reserve, as well as of the transcripts modulating T3 signaling/availability in the cumulus cells, confirmed ovarian dysthyroidism as a common and evolutionary conserved trait of POA. [ABSTRACT FROM AUTHOR]

Lately, the interest in long non-coding RNAs (lncRNAs) as potential drug targets and predictive markers in the context of HIV-1 has peaked, but their in vivo expression and regulation remains largely unexplored. Therefore, the present study examined lncRNA expression patterns during a clinical antiretroviral treatment interruption (ATI) trial. Peripheral blood mononuclear cells were isolated from ten patients at four timepoints: prior to ATI, 7–15 days after stop, at viral rebound and 3 months post antiretroviral therapy re-initiation. RNA was extracted and RT-qPCR on five known HIV-1-related lncRNAs (HEAL, MALAT1, NEAT1, GAS5 and NRON) was performed and correlated with HIV-1 and host marker expression. All lncRNAs correlated stronger with interferon stimulated genes (ISGs) than with HIV-1 reservoir and replication markers. However, one lncRNA, HEAL, showed significant upregulation at viral rebound during ATI compared to baseline and re-initiation of therapy (p = 0.0010 and p = 0.0094, respectively), following a similar viral-load-driven expression pattern to ISGs. In vitro knockdown of HEAL caused a significant reduction in HIV-1 infection levels, validating HEAL's importance for HIV-1 replication. We conclude that the HIV-1-promoting lncRNA HEAL is upregulated at viral rebound during ATI, most likely induced by viral cues. [ABSTRACT FROM AUTHOR]

Insulin resistance (IR), designated as the blunted response of insulin target tissues to physiological level of insulin, plays crucial roles in the development and progression of diabetes, nonalcoholic fatty liver disease (NAFLD) and other diseases. So far, the distinct mechanism(s) of IR still needs further exploration. Long non-coding RNA (lncRNA) is a class of non-protein coding RNA molecules with a length greater than 200 nucleotides. LncRNAs are widely involved in many biological processes including cell differentiation, proliferation, apoptosis and metabolism. More recently, there has been increasing evidence that lncRNAs participated in the pathogenesis of IR, and the dysregulated lncRNA profile played important roles in the pathogenesis of metabolic diseases including obesity, diabetes and NAFLD. For example, the lncRNAs MEG3, H19, MALAT1, GAS5, lncSHGL and several other lncRNAs have been shown to regulate insulin signaling and glucose/lipid metabolism in various tissues. In this review, we briefly introduced the general features of lncRNA and the methods for lncRNA research, and then summarized and discussed the recent advances on the roles and mechanisms of lncRNAs in IR, particularly focused on liver, skeletal muscle and adipose tissues. [ABSTRACT FROM AUTHOR]

Glucocorticoids are essential for stress coping, stress resilience, and homeostasis. In this Special Issue, two studies report the importance of elucidating stress mechanisms and stress-related translational research. 34072036 7 Nemoto T., Kakinuma Y. Prenatal and postnatal methyl-modulator intervention corrects the stress-induced glucocorticoid response in low-birthweight rats. Sex differences regulate ghrelin responsiveness, and aging triggers a persistent stress response. [Extracted from the article]

Long noncoding RNAs (lncRNAs) have been identified as contributors to the development and progression of cancer through various functions and mechanisms. LncRNA GAS5 is downregulated in multiple cancers and acts as a tumor suppressor in breast cancer. GAS5 interacts with various proteins (e.g., E2F1, EZH2, and YAP), DNA (e.g., the insulin receptor promoter), and various microRNAs (miRNAs). In breast cancer, GAS5 binds with miR-21, miR-222, miR-221-3p, miR-196a-5p, and miR-378a-5p that indicates the presence of several elements for miRNA binding (MREs) in GAS5. Mediated by the listed miRNAs, GAS5 is involved in the upregulation of a number of mRNAs of suppressor proteins such as PTEN, PDCD4, DKK2, FOXO1, and SUFU. Furthermore, the aberrant promoter methylation is involved in the regulation of GAS5 gene expression in triple-negative breast cancer and some other carcinomas. GAS5 can stimulate apoptosis in breast cancer via diverse pathways, including cell death receptors and mitochondrial signaling pathways. GAS5 is also a key player in the regulation of some crucial signal pathways in breast cancer, such as PI3K/AKT/mTOR, Wnt/β-catenin, and NF-κB signaling. Through epigenetic and other mechanisms, GAS5 can increase sensitivity to multiple drugs and improve prognosis. GAS5 is thus a promising target in the treatment of breast cancer patients. [ABSTRACT FROM AUTHOR]