scholarly journals Litopenaeus vannamei BMAL1 Is a Critical Mediator Regulating the Expression of Glucose Transporters and Can Be Suppressed by Constant Darkness

Animals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2893
Author(s):  
Lefei Jiao ◽  
Tianmeng Dai ◽  
Peng Sun ◽  
Min Jin ◽  
Qicun Zhou
Keyword(s):  
Gene Expression ◽  
Growth Performance ◽  
Litopenaeus Vannamei ◽  
Glucose Transporters ◽  
Luciferase Reporter ◽  
Constant Darkness ◽  
Carbohydrate Utilization ◽  
Dark Light

Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a core circadian transcription factor that controls the 24-h cycle of physiological processes. In shrimp, the role of BMAL1 in the regulating glucose metabolism remains unclear. Firstly, we observed that the daily profile of BMAL1, GLUT1 and SGLT1 expression were synchronized in the intestine and the hepatopancreas of Litopenaeus vannamei. Then we examined the effects of BMAL1 on the gene expression of glucose transporter type 1 (SGLT1) and sodium-glucose cotransporter 1 (GLUT1) in vivo and in vitro. BMAL1 in L. vannamei shares 70.91–96.35% of sequence identities with other shrimp species and possesses the conserved helix-loop-helix domain and polyadenylation site domain. The in vitro dual-luciferase reporter assay and in vivo RNA interference experiment demonstrated that BMAL1 exerted a positive regulation effect on the expression of glucose transporters in L. vannamei. Moreover, we conducted an eight-week treatment to investigate whether light/dark cycle change would influence growth performance, and gene expression of BMAL1, GLUT1 and SGLT1 in L. vannamei. Our result showed that compared with natural light treatment, constant darkness (24-hour darkness) significantly decreased (P < 0.05) serum glucose concentration, and suppressed (p < 0.05) the gene expression of BMAL1, GLUT1 and SGLT1 in the hepatopancreas and the intestine. Growth performance and survival rate were also decreased (p < 0.05) by constant darkness treatment. Our result identified BMAL1 as a critical mediator regulating the expression of glucose transporters, which could be suppressed by constant darkness in L. vannamei. It would be quite interesting to explore the mechanism of dark/light cycles on glucose transport and metabolism in L. vannamei, which might provide a feeding strategy for improving carbohydrate utilization in the future.

scholarly journals The Immunomodulatory Effects of AlbuminIn VitroandIn Vivo

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Derek S. Wheeler ◽  
John S. Giuliano ◽  
Patrick M. Lahni ◽  
Alvin Denenberg ◽  
Hector R. Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lethal Dose ◽  
Intraperitoneal Administration ◽  
Peritoneal Macrophages ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Dose Dependent

Albumin appears to have proinflammatory effectsin vitro. We hypothesized that albumin would induce a state of tolerance to subsequent administration of lipopolysaccharide (LPS)in vitroandin vivo. RAW264.7 and primary peritoneal macrophages were treated with increasing doses of bovine serum albumin (BSA) and harvested for NF-κB luciferase reporter assay or TNF-αELISA. In separate experiments, RAW264.7 cells were preconditioned with 1 mg/mL BSA for 18 h prior to LPS (10 μg/mL) treatment and harvested for NF-κB luciferase reporter assay or TNF-αELISA. Finally, C57Bl/6 mice were preconditioned with albumin via intraperitoneal administration 18 h prior to a lethal dose of LPS (60 mg/kg body wt). Blood was collected at 6 h after LPS administration for TNF-αELISA. Albumin produced a dose-dependent and TLR-4-dependent increase in NF-κB activation and TNF-αgene expressionin vitro. Albumin preconditioning abrogated the LPS-mediated increase in NF-κB activation and TNF-αgene expressionin vitroandin vivo. The clinical significance of these findings remains to be elucidated.

scholarly journals Downregulation of circ-PSMB6 suppresses NSCLC progression and metastasis through sponging miR-532-5p and regulating EZH1 expression

2021 ◽  
Author(s):  
Mingming Jin ◽  
Junqian Zhang ◽  
Yue Wu ◽  
Yitian Dai ◽  
Gang Huang
Keyword(s):  
Gene Expression ◽  
High Throughput Sequencing ◽  
Stem Cell Differentiation ◽  
Nsclc Cell ◽  
Cell Counting ◽  
Regulatory Mechanisms ◽  
Luciferase Reporter ◽  
Circular Rnas

Abstract Background: Accumulating reports showed how circular RNAs (circRNAs) act importantly during tumor progression via regulating gene expression, but regulatory mechanisms remain largely unknown. Current investigation clarified circRNA regulatory mechanisms in non-small cell lung cancer (NSCLC).Methods: High-throughput sequencing and quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection were utilized to explore circRNA expression in NSCLC tissues and cells. Our lab did statistical analyses and luciferase reporter analysis to validate correlations between circRNA, miRNA and gene expression. We transfected NSCLC cells with different vectors, and transwell migration, Cell Counting Kit-8 (CCK-8) proliferation along with colony formation assays were performed. In vivo tumorigenesis and metastasis assays were utilized to validate the circRNA role in NSCLC.Results: Data illustrated that hsa_circ_0041595 (circ-PSMB6) incremented in NSCLC cell lines and tissues, while circ-PSMB6 downregulation suppressed NSCLC cell proliferation and invasion in vitro and in vivo. Bioinformatics analysis and luciferase reporter data verified that miR-532-5p and Enhancer Of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) were circ-PSMB6 downstream targets in NSCLC cells. Overexpression of EZH1 or miR-532-5p inhibition reversed NSCLC cell invasion and proliferation after silencing circ-PSMB6. Further experiments discovered that circ-PSMB6 can influence cancer stem cell differentiation by regulating miR-532-5p/EZH1.Conclusions: Taken together, we found that circ-PSMB6 suppressed NSCLC metastasis and progression via sponging miR-532-5p and regulating EZH1 expression.

scholarly journals Functional analysis of haplotypes and promoter activity at the 5' region of the DRD2 gene and associations with schizophrenia.

2020 ◽  
Author(s):  
Xicen Zhang ◽  
Mei Ding ◽  
Yi Liu ◽  
Yongping Liu ◽  
Jiaxin Xing ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Region ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Drd2 Gene ◽  
Functional Regions

Abstract Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia.Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system.Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.

scholarly journals Orphan Nuclear Receptor ERRγ Is a Novel Transcriptional Regulator of IL-6 Mediated Hepatic BMP6 Gene Expression in Mice

2020 ◽  
Vol 21 (19) ◽  
pp. 7148
Author(s):  
Kamalakannan Radhakrishnan ◽  
Yong-Hoon Kim ◽  
Yoon Seok Jung ◽  
Jina Kim ◽  
Don-Kyu Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Molecular Mechanism ◽  
Nuclear Receptor ◽  
Iron Homeostasis ◽  
Luciferase Reporter ◽  
Orphan Nuclear Receptor ◽  
Knock Out

Bone morphogenetic protein 6 (BMP6) is a multifunctional growth factor involved in organ development and homeostasis. BMP6 controls expression of the liver hormone, hepcidin, and thereby plays a crucial role in regulating iron homeostasis. BMP6 gene transcriptional regulation in liver is largely unknown, but would be of great help to externally modulate iron load in pathologic conditions. Here, we describe a detailed molecular mechanism of hepatic BMP6 gene expression by an orphan nuclear receptor, estrogen-related receptor γ (ERRγ), in response to the pro-inflammatory cytokine interleukin 6 (IL-6). Recombinant IL-6 treatment increases hepatic ERRγ and BMP6 expression. Overexpression of ERRγ is sufficient to increase BMP6 gene expression in hepatocytes, suggesting that IL-6 is upstream of ERRγ. In line, knock-down of ERRγ in cell lines or a hepatocyte specific knock-out of ERRγ in mice significantly decreases IL-6 mediated BMP6 expression. Promoter studies show that ERRγ directly binds to the ERR response element (ERRE) in the mouse BMP6 gene promoter and positively regulates BMP6 gene transcription in IL-6 treatment conditions, which is further confirmed by ERRE mutated mBMP6-luciferase reporter assays. Finally, an inverse agonist of ERRγ, GSK5182, markedly inhibits IL-6 induced hepatic BMP6 expression in vitro and in vivo. Taken together, these results reveal a novel molecular mechanism on ERRγ mediated transcriptional regulation of hepatic BMP6 gene expression in response to IL-6.

scholarly journals Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets

2001 ◽  
Vol 170 (1) ◽  
pp. 91-98 ◽  
Author(s):  
P Fragner ◽  
SL Lee ◽  
S Aratan de Leon
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Neoplastic Cells

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.

scholarly journals Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2

2004 ◽  
Vol 32 (3) ◽  
pp. 975-986 ◽  
Author(s):  
A Menuet ◽  
Y Le Page ◽  
O Torres ◽  
L Kern ◽  
O Kah ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Transcription Unit ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Estrogen Regulation ◽  
Potential Implication ◽  
Hepatic Expression

We have previously cloned and characterized three estrogen receptors (ER) in the zebrafish (zfERalpha, zfERbeta1 and zfERbeta2). We have also shown that they are functional in vitro and exhibit a distinct expression pattern, although partially overlapping, in the brain of zebrafish. In this paper, we have shown that the hepatic expression of these zfER genes responds differently to estradiol (E2). In fact, a 48-h direct exposure of zebrafish to E2 resulted in a strong stimulation of zfERalpha gene expression while zfERbeta1 gene expression was markedly reduced and zfERbeta2 remained virtually unchanged. To establish the potential implication of each zfER in the E2 upregulation of the zfERalpha gene, the promoter region of this gene was isolated and characterized. Transfection experiments with promoter-luciferase reporter constructs together with different zfER expression vectors were carried out in different cell contexts. The data showed that in vivo E2 upregulation of the zfERalpha gene requires ERalpha itself and a conserved transcription unit sequence including at least an imperfect estrogen-responsive element (ERE) and an AP-1/ERE half site at the proximal transcription initiation site. Interestingly, although in the presence of E2 zfERalpha was the most potent at inducing the expression of its own gene, the effect of E2 mediated by zfERbeta2 represented 50% of the zfERalpha activity. In contrast, zfERbeta1 was unable to upregulate the zfERalpha gene whereas this receptor form was able to tightly bind E2 and activate a reporter plasmid containing a consensus ERE. Altogether, these results indicated that the two ERbeta forms recently characterized in teleost fish could have partially distinct and not redundant functions.

scholarly journals Role of Methylation in Period2 (PER2) Transcription in the Context of the Presence or Absence of Light Signals: Natural and Chemical—Studies on the Pig Model

2021 ◽  
Vol 22 (15) ◽  
pp. 7796
Author(s):  
Przemysław Gilun ◽  
Krzysztof Flisikowski ◽  
Tatiana Flisikowska ◽  
Joanna Kwiatkowska ◽  
Barbara Wąsowska ◽  
...  
Keyword(s):  
Biological Clock ◽  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Constant Darkness ◽  
Chemical Studies ◽  
Differential Pattern ◽  
E Boxes

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of Period2 gene (PER2), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the PER2 promoter was performed. qPCR showed a differential pattern of PER2 mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine PER2 in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of PER2 expression.

scholarly journals miR-5000-3p confers oxaliplatin resistance by targeting ubiquitin-specific peptidase 49 in colorectal cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yan-yan Zhuang ◽  
Wa Zhong ◽  
Zhong-sheng Xia ◽  
Shu-zhen Lin ◽  
Man chung Chan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Drug Resistance ◽  
Critical Role ◽  
Treatment Strategies ◽  
Luciferase Reporter ◽  
Multi Drug Resistance ◽  
Gastrointestinal Malignancies

AbstractColorectal cancer (CRC) is the most common form of gastrointestinal malignancies. A growing number of reports focusing on oxaliplatin (OXA) resistance in CRC treatment have revealed that drug resistance is an urgent issue in clinical applications, especially for finding effective therapeutic targets. Recently, microRNAs (miRNAs) are reported to play a critical role in tumor progressions and multi-drug resistance. The main aim of this study is to establish whether miR-5000-3p is an oncogene that is resistant to OXA and further confirm its underlying regulatory role in CRC. The OXA-associated gene expression dataset in CRC cells was downloaded from Gene Expression Omnibus (GEO) database. Statistical software R was used for significance analysis of differentially expressed genes (DEGs) between OXA-resistant (OR)-CRC cells and CRC cells, and results indicated ubiquitin-specific peptidase 49 (USP49) was upregulated in OR-CRC cells. Luciferase reporter assay showed that USP49 was verified to act as a downstream target gene of miR-5000-3p. From the results of TCGA database, miR-5000-3p expression was upregulated and USP49 was downregulated in patients with CRC. The function of miR-5000-3p was detected using MTT assay, wound healing, Transwell, and flow cytometry assays. Moreover, through in vitro and in vivo experiments, miR-5000-3p expression was confirmed to be upregulated in CRC cells or OR-CRC cells comparing to normal cell lines. Molecular mechanism assays revealed that USP49 binds to the miR-5000-3p promoter to increase the expression of miR-5000-3p, resulting in cancer cells sensitized to OXA. To sum up, these results suggest that miR-5000-3p may be a novel biomarker involved in drug-resistance progression of CRC. Moreover, the drug-resistance mechanism of miR-5000-3p/USP49 axis provides new treatment strategies for CRC in clinical trials.

84 PORCINE EMBRYOS UTILIZE SMALL AMOUNTS OF PYRUVATE, LACTATE, AND GLUCOSE IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 171
Author(s):  
B. R. Redel ◽  
L. D. Spate ◽  
B. Elliott ◽  
M. Paczkowski ◽  
R. L. Krisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Culture ◽  
Glucose Transporters ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Energy Substrates ◽  
Stage Embryo

Porcine embryo culture systems are suboptimal to the in vivo environment, and significant effort has been made to improve development to the blastocyst stage in vitro. Since metabolism of the early embryo has many similarities to the Warburg effect, our goal was to determine the role of glucose on development, gene expression, and metabolism of other energy substrates in the blastocyst stage embryo. Pig embryos were in vitro produced and cultured in MU1 containing pyruvate, lactate, amino acids, and either 0, 7.5, 15, or 250 µM glucose, N = 1164, 4 replications. There was no difference in blastocyst percentage between the 0 µM and 7.5 µM glucose (34% ± 6.5 v. 29% ± 8.2), but there was a decrease in development in response to 15 and 250 µM compared with 0 µM glucose (25% ± 8.5, 23% ± 8.7 v. 34% ± 6.5; P ≤ 0.01). Glucose transporters (SLC2A1 and SLC2A2) and hexokinases (HK1 and HK2) were analysed by qPCR to detect differences in gene expression, 3 replicates containing 10 blastocyst pools. The abundance of both HK1 and HK2 was decreased in blastocysts cultured with 7.5 µM glucose compared with 0 µM (P ≤ 0.04). Glucose transporters were not affected by glucose supplementation (P ≥ 0.5). Metabolic data were collected to determine if embryos were adjusting their energy substrate use in response to glucose. Two assays were completed to determine lactate and pyruvate consumption or release into the media by embryos, in comparison with media without embryos. In vitro-produced embryos were cultured in MU1 with 0 or 7.5 µM glucose N = 360, 4 replications. Both treatments consumed lactate, but there were no differences between treatments (6.8 ± 9.4 pmol/blastocyst/h v. 12.5 ± 1.6 pmol/blastocyst/h; P = 0.6). Blastocysts cultured in 7.5 µM glucose consumed pyruvate, whereas blastocysts without glucose produced pyruvate (–0.34 ± 0.3 pmol/blastocyst/h v. 0.73 ± 0.2 pmol/blastocyst/h; P < 0.01). It has been suggested that fructose is a more efficient replacement for glucose in pig embryo culture. Therefore, we produced pig embryos in vitro and cultured these embryos in MU1, MU1 + 2 mM glucose, or MU1 + 2 mM fructose to the blastocyst stage, 4 replications, N = 389. Again, there was a decrease in embryos that developed to the blastocyst stage in 2 mM glucose compared with MU1 control blastocysts (26% ± 5.8 v. 11% ± 2.5; P = 0.001), but there was only a trend for a decrease in development in response to 2 mM fructose (17 ± 2.3%; P = 0.06). There was no difference in total cell number between MU1, 2 mM glucose, and 2 mM fructose (30.6 ± 2.2, 30.5 ± 3.7, and 32.6 ± 3.0, respectively; P ≥ 0.9) 3 replications, N = 32. Because there is very little consumption of lactate and very low levels of pyruvate are being consumed when glucose is present, it does not appear that any of these energy substrates are major players for the developing pig embryo. Future experiments should be conducted to determine other means of energy production and metabolism in these embryos. The research was funded by Food for the 21st Century.

scholarly journals Extrachromosomal circular DNA, microDNA, without canonical promoters produce short regulatory RNAs that suppress gene expression

2019 ◽  
Author(s):  
Teressa Paulsen ◽  
Yoshiyuki Shibata ◽  
Pankaj Kumar ◽  
Laura Dillon ◽  
Anindya Dutta
Keyword(s):  
Gene Expression ◽  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Circular Dna ◽  
Endogenous Gene ◽  
Mrna Targets ◽  
Small Regulatory Rna

ABSTRACTInterest in extrachromosomal circular DNA (eccDNA) molecules has increased recently because of their widespread presence in normal cells across every species ranging from yeast to humans, their increased levels in cancer cells, and their overlap with oncogenic and drug-resistant genes. However, the majority of eccDNA (microDNA) are too small to carry protein coding genes. We have tested functional capabilities of microDNA, by creating artificial microDNA molecules mimicking known microDNA sequences and have discovered that they express functional small regulatory RNA including microRNA and novel si-like RNA. MicroDNA is transcribed in vitro and in vivo independent of a canonical promoter sequence. MicroDNA which carry miRNA genes form transcripts which are processed into mature miRNA molecules, through the endogenous RNA-interference pathway, which repress a luciferase reporter gene as well as endogenous mRNA targets of the miRNA. Further, microDNA containing sequences of exons repress the endogenous gene from which the microDNA was derived through the formation of novel si-like RNA. We also show that endogenous microDNA associate with RNA polymerases subunits POLR2H and POLR3F. Together, these results suggest that microDNA may modulate gene expression through the production of both known and novel regulatory small RNA.

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