Effects of long non-coding RNA myocardial infarction-associated transcript on retinal neovascularization in a newborn mouse model of oxygen-induced retinopathy

Whether long non-coding RNA myocardial infarction-associated transcript is involved in oxygen-induced retinopathy remains poorly understood. To validate this hypothesis, we established a newborn mouse model of oxygen-induced retinopathy by feeding in an oxygen concentration of 75 ± 2% from postnatal day 8 to postnatal day 12, followed by in normal air. On postnatal day 11, the mice were injected with the myocardial infarction-associated transcript siRNA plasmid via the vitreous cavity to knockdown long non-coding RNA myocardial infarction-associated transcript. Myocardial infarction-associated transcript siRNA transcription significantly inhibited myocardial infarctionassociated transcript mRNA expression, reduced the phosphatidylinosital-3-kinase, phosphorylated Akt and vascular endothelial growth factor immunopositivities, protein and mRNA expression, and alleviated the pathological damage to the retina of oxygen-induced retinopathy mouse models. These findings suggest that myocardial infarction-associated transcript is likely involved in the retinal neovascularization in retinopathy of prematurity and that inhibition of myocardial infarction-associated transcript can downregulate phosphatidylinosital-3-kinase, phosphorylated Akt and vascular endothelial growth factor expression levels and inhibit neovascularization. This study was approved by the Animal Ethics Committee of Shengjing Hospital of China Medical University, China(approval No. 2016 PS074 K) on February 25, 2016.

Regulatory mechanisms of long non-coding RNAs

Long non-coding RNAs(lncRNAs) belong to a large and complex family of RNAs, which play many important roles in regulating gene expression. However, the mechanism underlying the dynamic expression of lncRNAs is still not very clear. In order to identify lncRNAs and clarify the mechanisms involved, we collected basic information and highlighted the mechanisms underlying lncRNA expression and regulation. Overall, lncRNAs are regulated by several similar transcription factors and protein-coding genes. Epigenetic modification(DNA methylation and histone modification) can also downregulate lncRNA levels in tissues and cells. Moreover, lncRNAs may be degraded or cleaved via interaction with miRNAs and miRNAassociated protein complexes. Furthermore, alternative RNA splicing(AS) may play a significant role in the post-transcriptional regulation of lncRNAs.

Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation

Objective:This study explores the mechanism of action of Danhongqing formula(DHQ),a compoundbased Chinese medicine formula,in the treatment of cholestatic liver fibrosis.Methods:In vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout(Mdr2-/-)mice as an animal model of cholestatic liver fibrosis.DHQ was administered orally for 8 weeks,and its impact on cholestatic liver fibrosis was evaluated by assessing liver function,liver histopathology,and the expression of liver fibrosis-related proteins.Real-time polymerase chain reaction,Western blot,immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19(H19)and signal transducer and activator of transcription 3(STAT3)phosphorylation in the liver tissue of Mdr2-/-mice.In addition,cholangiocytes and hepatic stellate cells(HSCs)were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression.Cholangiocytes overexpressing H19 were constructed,and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation.The intervention effect of DHQ on these processes was also investigated.HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ.Results:DHQ alleviated liver injury,ductular reaction,and fibrosis in Mdr2-/-mice,and inhibited H19expression,STAT3 expression and STAT3 phosphorylation.This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19,inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium,and decreased the expression of activation markers in HSCs.The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation,and DHQ was able to successfully inhibit these effects.Conclusion:DHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/-mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC,thereby suppressing cell activation.

The long non-coding RNA DANA2 positively regulates drought tolerance by recruitingERF84 to promote JMJ29-mediated histone demethylation

Tens of thousands of long non-coding RNAs have been uncovered in plants,but few of them have been comprehensively studied for their biological function and molecular mechanism of their mode of action.Here,we show that the Arabidopsis long non-coding RNA DANA2 interacts with an AP2/ERF transcription factor ERF84 in the cell nucleus and then affects the transcription of JMJ29 that encodes a Jumonji C domain-containing histone H3K9 demethylase.Both RNA sequencing(RNA-seq)and genetic analyses demonstrate that DANA2 positively regulates drought stress responses through JMJ29.JMJ29 positively regulates the expression of ERF15 and GOLS2 by modulation of H3K9me2 demethylation.Accordingly,mutation of JMJ29 causes decreased ERF15 and GOLS2 expression,resulting in impaired drought tolerance,in agreement with drought-sensitive phenotypes of dana2 and erf84 mutants.Taken together,these results demonstrate that DANA2 is a positive regulator of drought response and works jointly with the transcriptional activator ERF84 to modulate JMJ29 expression in plant response to drought.

Transcriptional and Post-transcriptional Gene Regulation by Long Non-coding RNA

Advances in genomics technology over recent years have led to the surprising discovery that the genome is far more pervasively transcribed than was previously appreciated. Much of the newly-discovered transcriptome appears to represent long non-coding RNA （lncRNA）, a heteroge- neous group of largely uncharacterised transcripts. Understanding the biological function of these molecules represents a major challenge and in this review we discuss some of the progress made to date. One major theme of lncRNA biology seems to be the existence of a network of interactions with microRNA （miRNA） pathways, lncRNA has been shown to act as both a source and an inhi- bitory regulator of miRNA. At the transcriptional level, a model is emerging whereby lncRNA bridges DNA and protein by binding to chromatin and serving as a scaffold for modifying protein complexes. Such a mechanism can bridge promoters to enhancers or enhancer-like non-coding genes by regulating chromatin looping, as well as conferring specificity on histone modifying com- plexes by directing them to specific loci.

Long non-coding RNA colon cancer-associated transcript 2:role and function in human cancers

Long non-coding RNAs(lncRNAs)are a family of non-protein-coding RNAs that span a length of over 200 nucleotides.Research reports have illustrated that lncRNAs are involved in various cellular processes and that their abnormal expression leads to the occurrence and development of various tumors.Colon cancer-associated transcript 2(CCAT2)was first reported as an oncogene in colon cancer.LncRNA CCAT2 is abnormally expressed in hepatocellular carcinoma,cholangiocarcinoma,lung cancer,breast cancer,ovarian cancer,glioma,and other tumors.In tumor tissues,abnormally overexpressed CCAT2 can affect cell proliferation,migration,epithelial-mesenchymal transition,apoptosis,and other biological behaviors through endogenous RNAs mechanisms,various signaling pathways,transcriptional regulation,and other complex mechanisms.Additionally,the overexpression of CCAT2 is also closely related to the tumor size,tumor node metastasis(TNM)stage,survival time,and other prognostic factors,suggesting that it is a potential prognostic indicator.This article reviews the biological functions of CCAT2 and its mechanisms of action in tumors from previous studies.In this review,we attempt to provide a molecular basis for future clinical applications of lncRNA CCAT2.

Long Non-coding RNAs and Their Biological Roles in Plants

With the development of genomics and bioinformatics, especially the extensive applications of high-throughput sequencing technology, more transcriptional units with little or no protein-coding potential have been discovered. Such RNA molecules are called non- protein-coding RNAs （npcRNAs or ncRNAs）. Among them, long npcRNAs or ncRNAs 0npcRNAs or lncRNAs） represent diverse classes of transcripts longer than 200 nucleotides. In recent years, the lncRNAs have been considered as important regulators in many essential biological processes. In plants, although a large number of lncRNA transcripts have been predicted and identified in few species, our current knowledge of their biological functions is still limited. Here, we have summarized recent studies on their identification, characteristics, classification, bioinformatics, resources, and current exploration of their biological functions in olants.

Cellular, physiological and pathological aspects of the long non-coding RNA NEAT1

BACKGROUND： The majority of mammalian genomes have been found to be transcribed into non-coding RNAs. One category of non-coding RNAs is classified as long non-coding RNAs （lncRNAs） based on their transcript sizes larger than 200 nucleotides. Growing evidence has shown that lncRNAs are not junk transcripts and play regulatory roles in multiple aspects of biological processes. Dysregulation of lncRNA expression has also been linked to diseases, in particular cancer. Therefore, studies of lncRNAs have attracted significant interest in the field of medical research. Nuclear enriched abundant transcript 1 （NEAT1）, a nuclear lncRNA, has recently emerged as a key regulator involved in various cellular processes, physiological responses, developmental processes, and disease development and progression. OBJECTIVE： This review will summarize and discuss the most recent findings with regard to the roles of NEAT1 in the function of the nuclear paraspeckle, cellular pathways, and physiological responses and processes. Particularly, the most recently reported studies regarding the pathological roles of deregulated NEAT1 in cancer are highlighted in this review. METHODS： We performed a systematic literature search using the Pubmed search engine. Studies published over the past 8 years （between January 2009 and August 2016） were the sources of literature review. The following keywords were used： ＂Nuclear enriched abundant transcript 1,＂ ＂NEATI,＂ and ＂paraspeckles.＂ RESULTS： The Pubmed search identified 34 articles related to the topic of the review. Among the identified literature, 13 articles report findings related to cellular functions of NEAT1 and eight articles are the investigations of physiological functions of NEAT1. The remaining 13 articles are studies of the roles of NEAT1 in cancers. CONCLUSION： Recent advances in NEAT1 studies reveal the multifimctional roles of NEAT1 in various biological processes, which are beyond its role in nuclear paraspeckles. Recent studies also indicate that dysregulation of NEAT1 function contributes to the development and progression of various cancers. More investigations will be needed to address the detailed mechanisms regarding how NEAT1 executes its cellular and physiological functions and how NEAT1 dysregulation results in tumorigenesis, and to explore the potential of NEAT1 as a target in cancer diagnosis, prognosis and therapy.

Study on the relationship between the expression of NFκB1 and LncRNA-PACER in peripheral blood mononuclear cells of patients with pulmonary tuberculosis

Objective: To investigate the expression relationship between nuclear transcription factor kappa B1 (NFκB1) and long non-coding RNA PACER (LncRNA-PACER) in peripheral blood mononuclear cells (PBMCs) of patients with pulmonary tuberculosis. Methods: From February 2018 to March 2019, 40 patients with pulmonary tuberculosis (tuberculosis group) and 40 healthy persons (control group) were collected, the levels of TNF-α, IL-6 and IL-8 in serum were detected by enzyme-linked immunosorbent assay (ELISA);the expressions of LncRNA-PACER and NFκB1 mRNAs in PBMCs were detected by real-time fluorescence quantitative PCR;Western blot was used to detect the expressions of NFκB1 and COX 2 in PBMCs;Pearson method was used to analyze the expressions of LncRNA-PACER and NFκB1 in PBMCs of patients with pulmonary tuberculosis, and the expressions of LncRNA-PACER and NFκB1 in PBMCs of patients with pulmonary tuberculosis were analyzed. Results: Compared with the control group, the expressions of TNF-α, IL-6 and IL-8 in the serum of patients with pulmonary tuberculosis was significantly increased (P<0.05), and the expressions of LncRNA-PACER, NFκB1 mRNAs, proteins and COX-2 protein in PBMCs were significantly increased (P<0.05). The expressions of LncRNA-PACER and NFκB1 proteins in PBMCs were related to the number of pulmonary lesions and pulmonary cavity (P<0.05), and there was a positive correlation between the expression of LncRNA-PACER and the expression of NFκB1 mRNA in PBMCs of patients with pulmonary tuberculosis (r = 0.873, P<0.05). Conclusions: The expressions of NFκB1 and LncRNA-PACER in PBMCs of patients with pulmonary tuberculosis are significantly increased, they are positively correlated and both of them are related to the occurrence and development of pulmonary tuberculosis.

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.

Functional impacts of non-coding RNA processing on enhancer activity and target gene expression

Tight regulation of gene expression is orchestrated by enhancers.Through recent research advancements,it is becoming clear that enhancers are not solely distal regulatory elements harboring transcription factor binding sites and decorated with specific histone marks,but they rather display sign atures of active transcription,showingdistinct degrees oftranscription unit organization.Thereby,a substantial fraction of enhancers give rise to different species of non-coding RNA transcripts with an unprecedented range of potential functions.In this review,we bring together data from recent studies indicating that non-coding RNA transcription from active enhancers,as well as enhancer-produced long non-coding RNA transcripts,may modulate or define the functional regulatory potential ofthe cognate enhancer.In addition,we summarize supporting evidence that RNA processing ofthe enhancer-associated long non-coding RNA transcripts may constitute an additional layer of regulation of enhancer activity,which contributes to the control and final outcome of enhancer-targeted gene expression.

Mechanisms of Huangqi Decoction Granules (黄芪汤颗粒剂) on Hepatitis B Cirrhosis Patients Based on RNA-Sequencing

Objective: To explore the action mechanisms of Huangqi Decoction Granules(黄芪汤颗粒剂, HQDG) on hepatitis B cirrhosis. Methods: A total of 85 patients with hepatitis B cirrhosis were randomly divided into HQDG group(42 cases) and control group(43 cases) by a random number table and were treated with HQDG or placebo for 48 weeks(6 g per times and orally for 3 times a day), respectively. After RNA-sequencing of serum samples extracted from the patients, the differentially expressed genes(DEGs) in HQDG and control groups before and after treatment were separately screened. The DEGs were then performed pathway enrichment analysis and proteinprotein interaction(PPI) network analysis. The expression levels of key genes were detected by quantitative realtime polymerase chain reaction(qRT-PCR). Results: After the investigation, 4 and 3 cases were respectively excluded from HQD and control groups because of the incomplete data. Additionally, 3 and 5 cases were lost to follow up in HQD and control groups, respectively. Finally, a total of 70 cases with good compliance were included for further DEGs analysis. A total of 1,070 DEGs(including 455 up-regulated genes and 615 down-regulated genes) in HQDG group and 227 DEGs(including 164 up-regulated genes and 63 down-regulated genes) in the control group were identified after treatment. Compared with the control group, 1,043 DEGs were specific in HQDG group. Besides, 1 up-regulated transcription factor(TF, such as GLI family zinc finger 1, GLI1) and 25 down-regulated TFs(such as drosophila mothers against decapentaplegic proteinfamily member 2, SMAD2) were identified. Pathway enrichment analysis showed that down-regulated Ras homolog gene family member A(RHOA) was enriched in pathogenic Escherichia coli infection. In the PPI network, up-regulated epidermal growth factor receptor(EGFR), and down-regulated cell division cycle 42(CDC42) as well as v-akt murine thymoma viral oncogene homolog 1(AKT1) had higher degrees. Moreover, long non-coding RNAs(lncR NA) growth arrest-specific 5(GAS5) was involved in the lncR NA-target regulatory network. Furthermore, qR T-PCR revealed that expression levels of CDC42 and GLI1 had significant differences in HQDG group before and after treatment(P<0.05). Conclusions: CDC42 and GLI1 may be the targets of HQDG in patients with hepatitis B cirrhosis. Additionally, SMAD2, EGFR, AKT1, RHOA and GAS5 might be associated with the curative effect of HQDG on hepatitis B cirrhosis patients.

Three-dimensional regulation of transcription

Cells can adapt to environment and development by reconstructing their transcriptional networks to regulate diverse cellular processes without altering the underly- ing DNA sequences. These alterations, namely epige- netic changes, occur during cell division, differentiation and cell death. Numerous evidences demonstrate that epigenetic changes are governed by various types of determinants, including DNA methylation patterns, his- tone posttranslational modification signatures, histone variants, chromatin remodeling, and recently discovered chromosome conformation characteristics and non- coding RNAs （ncRNAs）. Here, we highlight recent efforts on how the two latter epigenetic factors participate in the sophisticated transcriptional process and describe emerging techniques which permit us to uncover and gain insights into the fascinating genomic regulation.