scholarly journals Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5′-UTR splice variants with altered translational activities

2005 ◽  
Vol 387 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Neville J. BUTCHER ◽  
Ajanthy ARULPRAGASAM ◽  
Hui Li GOH ◽  
Tamara DAVEY ◽  
Rodney F. MINCHIN
Keyword(s):  
Splice Variant ◽  
Expressed Sequence Tag ◽  
The Body ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Type I ◽  
Alternative Promoters ◽  
Reporter Assay ◽  
Coding Region ◽  
Mrna Isoforms

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5′ non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9–11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.

scholarly journals MicroRNA-513b-5p targets COL1A1 and COL1A2 associated with the formation and rupture of intracranial aneurysm

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zheng Zheng ◽  
Yan Chen ◽  
Yinzhou Wang ◽  
Yongkun Li ◽  
Qiong Cheng
Keyword(s):  
Cell Death ◽  
Intracranial Aneurysm ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Mrna Levels ◽  
Reporter Assay ◽  
Over Expression ◽  
Tnf Α

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

scholarly journals Multiple regions within the promoter of the murine Ifnar-2 gene confer basal and inducible expression

2002 ◽  
Vol 365 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Matthew P. HARDY ◽  
Paul J. HERTZOG ◽  
Catherine M. OWCZAREK
Keyword(s):  
Splice Variants ◽  
Regulatory Region ◽  
Distal Region ◽  
Inducible Expression ◽  
Luciferase Reporter ◽  
Type I ◽  
Type I Ifn ◽  
Mrna Isoforms ◽  
Gene Encoding ◽  
Multiple Regions

The (murine) type I interferon (IFN) receptor, muIfnar-2, is expressed ubiquitously, and exists as both transmembrane and soluble forms. In the present study we show that the gene encoding muIfnar-2 spans approx. 33kb on mouse chromosome 16, and consists of nine exons and eight introns. The three mRNA splice variants resulting in one transmembrane (muIfnar-2c) and two soluble (muIfnar-2a/2a′) mRNA isoforms are generated by alternative RNA processing of the muIfnar-2 gene. Treatment of a range of murine cell lines with a combination of type I and II IFN showed that the muIfnar-2a and −2c mRNA isoforms were up-regulated independently of each other in L929 fibroblasts and hepa-1c1c7 hepatoma cells, but not in M1 myeloid leukaemia cells. Analysis of the 5′ flanking region of muIfnar-2 using promoter—luciferase reporter constructs defined three regulatory regions: a region proximal to exon 1, conferring high basal expression, a distal region conferring inducible expression, and a negative regulatory region between the two. These data represent the first promoter analysis of a type I IFN receptor and, taken together with our previous data demonstrating high expression levels and dual biological functions for muIfnar-2a protein, suggests that the regulation of muIfnar-2 isoform expression may be an important way of modulating type I IFN responses.

scholarly journals Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Dong-Kyung Lee ◽  
Mingyun Lee ◽  
Jae Yeon Hwang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Promoter Region ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Fluorescence Signal ◽  
Reporter Assay ◽  
Reporter System

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.

scholarly journals miR-410-3P inhibits adipocyte differentiation by targeting IRS-1 in cancer-associated cachexia patients

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Diya Sun ◽  
Zuoyou Ding ◽  
Lei Shen ◽  
Fan Yang ◽  
Jun Han ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Large Population ◽  
Predictive Marker ◽  
Luciferase Reporter Assay ◽  
Differentially Expressed ◽  
Tissue Loss ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Coding Region ◽  
Serum Exosomes

Abstract Backgrounds Cancer-associated cachexia (CAC) is a metabolic syndrome characterized by progressive depletion of adipose and muscle tissue that cannot be corrected by conventional nutritional therapy. Adipose tissue, an important form of energy storage, exhibits marked loss in the early stages of CAC, which affects quality of life and efficacy of chemotherapy. MicroRNAs (miRNAs) are a class of noncoding RNAs that widely exist in all kinds of eukaryotic cells and play regulatory roles in various biological processes. However, the role of miRNAs in adipose metabolism in CAC has rarely been reported. This study attempted to identify important miRNAs in adipose metabolism in CAC and explore their mechanism to identify a new predictive marker or therapeutic target for CAC-related adipose tissue loss (CAL). Methods In this study, miRNA sequencing was firstly used to identify differentially expressed miRNAs related to CAL and the reliability of the conclusions was verified in large population samples. Furthermore, functional experiments were performed by up and down regulating miR-410-3p in adipocytes. The binding of miR-410-3p to Insulin Receptor Substrate 1 (IRS-1) was verified by Luciferase reporter assay and functional experiments of IRS-1 were performed in adipocytes. Finally, the expression of miR-410-3p in serum exosomes was detected. Results miR-410-3p was selected as differentially expressed miRNA through screening and validation. Adipogenesis was suppressed in miR-410-3p upregulation experiment and increased in downregulation experiment. Luciferase reporter assay showed that miR-410-3p binds to 3′ non-coding region of IRS-1 and represses its expression and ultimately inhibits adipogenesis. miR-410-3p was highly expressed in serum exosomes of CAC patients, which was consistent with results in adipose tissue. Conclusions The expression of miR-410-3p was higher in subcutaneous adipose tissues and serum exosomes of CAC patients, which significantly inhibits adipogenesis and lipid accumulation. The study shows that miR-410-3p could downregulate IRS-1 and downstream adipose differentiation factors including C/EBP-a and PPAR-γ by targeting 3′ noncoding region.

scholarly journals A Novel FOXL2 Mutation Implying Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type I

2018 ◽  
Vol 45 (1) ◽  
pp. 203-211 ◽  
Author(s):  
Fang Li ◽  
Peiwei Chai ◽  
Jiayan Fan ◽  
Xi Wang ◽  
Wenjuan Lu ◽  
...  
Keyword(s):  
Early Recognition ◽  
Molecular Genetic ◽  
Gene Mutations ◽  
Luciferase Reporter ◽  
Functional Study ◽  
Chinese Family ◽  
Type I ◽  
Coding Region ◽  
Mobility Shift ◽  
Female Patients

Background/Aims: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal dominant disease caused by FOXL2 gene mutations, and it is clinically characterized by an eyelid malformation associated (type I) or not (type II) with premature ovarian failure (POF). Functional study of novel mutations is especially critical for female patients, as it may allow the prediction of infertility and early planning of an appropriate therapy. Methods: A clinical and molecular genetic investigation was performed in all members of a Chinese family with BPES. Genomic DNA was extracted, and the FOXL2 coding region was sequenced. Subcellular localization was performed by confocal microscopy. Transactivation studies were performed by real-time PCR, dual luciferase reporter assays and electrophoretic mobility shift assays. Results: A novel deletion mutation (C.634_641 del, CCCATGC) between the forkhead domain and the polyalanine domain was found, resulting in a frameshift mutation and a truncated protein. Functional studies showed a strong cytoplasmic mislocalization and abnormal transactivation activity, implying a type I kind mutation with a large chance of infertility. Conclusion: This study identifies that this mutation indicates the probability of developing into POF and shows the importance and necessity of early recognition of BPES type through mutation testing for female patients. Prompt personalized therapy and follow-up is of great clinical significance for female patients carrying this kind of mutation.

THYROID DYSFUNCTION IN PATIENTS WITH CHRONIC KIDNEY DISEASE: THE STATE OF THE PROBLEM AND THE WAYS OF SOLVING

2018 ◽  
Vol 22 (4) ◽  
pp. 40-49 ◽  
Author(s):  
A. R. Volkova ◽  
O. D. Dygun ◽  
B. G. Lukichev ◽  
S. V. Dora ◽  
O. V. Galkina
Keyword(s):  
Chronic Kidney Disease ◽  
Thyroid Gland ◽  
Kidney Disease ◽  
Thyroid Hormones ◽  
Thyroid Function ◽  
Thyroid Dysfunction ◽  
The Body ◽  
Type I ◽  
Low T3 ◽  
Iodine Excretion

Disturbance of the thyroid function is often detected in patients with different profiles. A special feature of patients with chronic kidney  disease is the higher incidence of various thyroid function  disturbances, especially hypothyroidism. It is known that in patients  with chronic kidney disease (CKD) iodine excretion from the body is  violated, since normally 90% of iodine is excreted in urine.  Accumulation of high concentrations of inorganic iodine leads to the  formation of the Wolf-Chaikoff effect: suppression of iodine  organization in the thyroid gland and disruption of the thyroid  hormones synthesis. Peripheral metabolism of thyroid hormones is  also disturbed, namely, deiodinase type I activity is suppressed and  peripheral conversion of T4 into T3 is inhibited (so-called low T3  syndrome). Therefore, patients with CKD are often diagnosed with  hypothyroidism, and the origin of hypothyroidism is not always  associated with the outcome of autoimmune thyroiditis. The article  presents an overview of a large number of population studies of  thyroid gland dysfunction in patients with CKD, as well as  experimental data specifying the pathogenetic mechanisms of  thyroid dysfunction in patients with CKD. Therapeutic tactics are still  not regulated. However, in a number of studies, replacement therapy with thyroid hormones in patients with CKD had some advantages.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis

2020 ◽  
Vol 25 (45) ◽  
pp. 4806-4812 ◽  
Author(s):  
Zhibo Sun ◽  
Fei Wu ◽  
Yue Yang ◽  
Feng Liu ◽  
Fengbo Mo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Translocation ◽  
Bone Diseases ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Alizarin Red ◽  
Over Expression ◽  
Negative Effect ◽  
Proliferation Ability

Background: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. Objective: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. Method: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. Results: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. Conclusion: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Hsa_circ_0123190 acts as a competitive endogenous RNA to regulate APLNR expression by sponging hsa-miR-483-3p in lupus nephritis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Chunyi Zhang ◽  
Congcong Gao ◽  
Xueqi Di ◽  
Siwan Cui ◽  
Wenfang Liang ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Lupus Erythematosus ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Reporter Assay ◽  
Clinical Value ◽  
Chronicity Index

Abstract Background Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE). Circular RNAs (circRNAs) can act as competitive endogenous RNAs (ceRNAs) to regulate gene transcription, which is involved in mechanism of many diseases. However, the role of circRNA in lupus nephritis has been rarely reported. In this study, we aim to investigate the clinical value of circRNAs and explore the mechanism of circRNA involvement in the pathogenesis of LN. Methods Renal tissues from three untreated LN patients and three normal controls (NCs) were used to identify differently expressed circRNAs by next-generation sequencing (NGS). Validated assays were used by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The interactions between circRNA and miRNA, or miRNA and mRNA were further determined by luciferase reporter assay. The extent of renal fibrosis between the two groups was assessed by Masson-trichome staining and immunohistochemistry (IHC) staining. Results 159 circRNAs were significantly dysregulated in LN patients compared with NCs. The expression of hsa_circ_0123190 was significantly decreased in the renal tissues of patients with LN (P = 0.014). Bio-informatics analysis and luciferase reporter assay illustrated that hsa_circ_0123190 can act as a sponge for hsa-miR-483-3p, which was also validated to interact with APLNR. APLNR mRNA expression was related with chronicity index (CI) of LN (P = 0.033, R2 = 0.452). Moreover, the fibrotic-related protein, transforming growth factor-β1 (TGF-β1), which was regulated by APLNR, was more pronounced in the LN group (P = 0.018). Conclusion Hsa_circ_0123190 may function as a ceRNA to regulate APLNR expression by sponging hsa-miR-483-3p in LN.

Selection on Rapidly Evolving Proteins in the Arabidopsis Genome

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 723-733 ◽  
Author(s):  
Marianne Barrier ◽  
Carlos D Bustamante ◽  
Jiaye Yu ◽  
Michael D Purugganan
Keyword(s):  
Expressed Sequence Tag ◽  
Amino Acid Replacement ◽  
Adaptive Divergence ◽  
Coding Region ◽  
Protein Coding ◽  
Hierarchical Bayesian Analysis ◽  
Brassicaceae Species ◽  
Replacement Site ◽  
Orthologous Loci ◽  
Selection Intensities

Abstract Genes that have undergone positive or diversifying selection are likely to be associated with adaptive divergence between species. One indicator of adaptive selection at the molecular level is an excess of amino acid replacement fixed differences per replacement site relative to the number of synonymous fixed differences per synonymous site (ω = Ka/Ks). We used an evolutionary expressed sequence tag (EST) approach to estimate the distribution of ω among 304 orthologous loci between Arabidopsis thaliana and A. lyrata to identify genes potentially involved in the adaptive divergence between these two Brassicaceae species. We find that 14 of 304 genes (∼5%) have an estimated ω &gt; 1 and are candidates for genes with increased selection intensities. Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes. A hierarchical Bayesian analysis of protein-coding region evolution within and between species also indicates that the selection intensities of these genes are elevated compared to previously studied A. thaliana nuclear loci.

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