scholarly journals Clock Gene Regulates Kainic Acid-Induced Seizures Through Inhibiting Ferroptosis in Mice

2021 ◽  
Author(s):  
Fei Wang ◽  
Lianxia Guo ◽  
Zhengping Wu ◽  
Tianpeng Zhang ◽  
Dong Dong ◽  
...  
Keyword(s):  
Kainic Acid ◽  
Specific Binding ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Mobility Shift ◽  
Protein Levels ◽  
Ppar Γ ◽  
N2a Cells ◽  
Molecular Events ◽  
Regulatory Effects

Abstract Temporal lobe epilepsy (TLE) is a common and intractable form of epilepsy. There is a strong need to better understand molecular events underlying TLE and to find novel therapeutic agents. Here we aimed to investigate the role of Clock gene and ferroptosis in regulating TLE. TLE model was established by treating mice with kainic acid (KA). Regulatory effects of Clock gene on KA-induced seizures and ferroptosis were evaluated using Clock knockout (Clock−/−) mice. mRNA and protein levels were determined by quantitative real-time PCR and western blotting, respectively. Ferroptosis was assessed by measuring the levels of iron, GSH and ROS. Transcriptional regulation was studied using a combination of luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays. We found that Clock ablation exacerbated KA-induced seizures in mice, accompanied by enhanced ferroptosis in the hippocampus. Furthermore, Clock ablation reduced the hippocampal expression of GPX4 and PPAR-γ, two ferroptosis-inhibitory factors, in mice and in N2a cells. Moreover, Clock regulates diurnal expression of GPX4 and PPAR-γ in mouse hippocampus and rhythmicity in KA-induced seizures. Consistently, Clock overexpression up-regulated GPX4 and PPAR-γ, and protected against ferroptosis in N2a cells. In addition, based on luciferase reporter, mobility shift and ChIP assays, we uncovered that CLOCK protein trans-activated Gpx4 and Ppar-γ through specific binding to an E-box element in gene promoters. In conclusion, CLOCK protests against KA-induced seizures through promoting expression of GPX4 and PPAR-γ and inhibiting ferroptosis.

Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter

2002 ◽  
Vol 362 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Rama K. MALLAMPALLI ◽  
Alan J. RYAN ◽  
James L. CARROLL ◽  
Timothy F. OSBORNE ◽  
Christie P. THOMAS
Keyword(s):  
Specific Binding ◽  
Core Promoter ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Luciferase Reporter ◽  
Standard Diet ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Ctp:Phosphocholine Cytidylyltransferase

Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268–1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal ∼ 1900bp of the 5′ flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between −169 and +71bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from −1867 to +71, was induced 2–3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5′ flanking region.

scholarly journals miR-27a Regulates Sheep Adipocyte Differentiation by Targeting CPT1B Gene

Animals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 28
Author(s):  
Bo Li ◽  
Xiaoyu Huang ◽  
Chen Yang ◽  
Ting Ge ◽  
Leiyun Zhao ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Target Gene ◽  
Lipid Synthesis ◽  
Ovis Aries ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Protein Levels ◽  
Ppar Γ ◽  
Expression Of Genes ◽  
Droplet Production

MiRNAs are vital regulators and play a major role in cell differentiation, biological development, and disease occurrence. In recent years, many studies have found that miRNAs are involved in the proliferation and differentiation of adipocytes. The objective of this study was to evaluate the effect of miR-27a and its target gene CPT1B on ovine preadipocytes differentiation in Small-tailed Han sheep (Ovis aries). Down-regulation of miR-27a significantly promoted the production of lipid droplets, while overexpression of miR-27a led to a reduction in lipid droplet production. In addition, inhibition of miR-27a led to a significant increase in the expression of genes involved in lipid synthesis, including PPAR γ, SCD, LPL, and FABP4. Target Scan software predicted that CPT1B is a new potential target gene of miR-27a. Further experiments revealed that CPT1B gene expression and protein levels were negatively correlated with miR-27a expression. Overexpression of miR-27a led to a significant decrease in CPT1B mRNA levels and inhibited the accumulation of lipid droplets and vice versa. Moreover, overexpression of CPT1B promoted the synthesis of lipid droplets in ovine preadipocytes. Furthermore, luciferase reporter assays confirmed CPT1B to be a miR-27a direct target gene. This study confirmed that miR-27a increases the expression of genes related to lipid synthesis in ovine preadipocytes by targeting CPT1B, thereby promoting the synthesis of lipid droplets. The results of this study can be used to be exploited in devising novel approaches for improving the IMF content of sheep.

scholarly journals NF-κB-Regulated miR-99a Modulates Endothelial Cell Inflammation

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Mei-hua Bao ◽  
Jian-Ming Li ◽  
Huai-qing Luo ◽  
Liang Tang ◽  
Qiao-li Lv ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Nuclear Translocation ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Software Analysis ◽  
Regulatory Effects ◽  
Electrophoretic Mobility Shift Assays ◽  
Insight Into ◽  
Lps Treatment

Objective. The present study was performed to investigate the effects and mechanisms of miR-99a on LPS-induced endothelial cell inflammation, as well as the regulation of NF-κB on miR-99a production.Methods and Results. ELISA showed that LPS treatment significantly promoted the secretion of inflammatory factors (TNF-α, IL-6, IL-1β, and MCP-1). LPS treatment also inhibited miR-99a production and promoted mTOR expression and NF-κB nuclear translocation. Overexpression of miR-99a suppressed the LPS-induced TNF-α, IL-6, IL-1β, and MCP-1 overproduction, mTOR upregulation, and NF-κB nuclear translocation. The PROMO software analysis indicated NF-κB binding site in the −1643 to −1652 region of miR-99a promoter. Dual luciferase reporter analysis, electrophoretic mobility shift assays (EMSA), and chromosome immunoprecipitation (ChIP) assays demonstrated that NF-κB promoted the transcription of miR-99a by binding to the −1643 to −1652 region of miR-99a promoter. Further studies on HUVECs verified the regulatory effects of NF-κB on miR-99a production.Conclusion. MiR-99a inhibited the LPS-induced HUVECs inflammation via inhibition of the mTOR/NF-κB signal. NF-κB promoted miR-99a production by binding to the −1643 to −1652 region of miR-99a promoter. Considering the importance of endothelial inflammation on cardiovascular diseases, such as atherosclerosis, our results may provide a new insight into the pathogenesis and therapy of atherosclerosis.

scholarly journals Transcriptional Regulation of the CLC-K1 Promoter by myc-Associated Zinc Finger Protein and Kidney-Enriched Krüppel-Like Factor, a Novel Zinc Finger Repressor

2000 ◽  
Vol 20 (19) ◽  
pp. 7319-7331 ◽  
Author(s):  
Shinichi Uchida ◽  
Yujiro Tanaka ◽  
Hiroshi Ito ◽  
Fumiko Saitoh-Ohara ◽  
Johji Inazawa ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Consensus Sequence ◽  
Interstitial Cells ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Finger Protein

ABSTRACT The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for “kidney-enriched Krüppel-like factor”) and the previously isolated MAZ (for “myc-associated zinc finger protein”) were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1–luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.

scholarly journals Impact of Promoter Polymorphisms on the Transcriptional Regulation of the Organic Cation Transporter OCT1 (SLC22A1)

2018 ◽  
Vol 8 (4) ◽  
pp. 42 ◽  
Author(s):  
Kristin Bokelmann ◽  
Jürgen Brockmöller ◽  
Mladen Tzvetkov
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Organic Cation ◽  
Specific Binding ◽  
Organic Cation Transporter ◽  
Luciferase Reporter ◽  
Potential Contribution ◽  
Luciferase Reporter Gene ◽  
Promoter Polymorphisms ◽  
Mobility Shift

The organic cation transporter 1 (OCT1, SLC22A1) is strongly expressed in the human liver and facilitates the hepatic uptake of drugs such as morphine, metformin, tropisetron, sumatriptan and fenoterol and of endogenous substances such as thiamine. OCT1 expression is inter-individually highly variable. Here, we analyzed SNPs in the OCT1 promoter concerning their potential contribution to the variability in OCT1 expression. Using electrophoretic mobility shift and luciferase reporter gene assays in HepG2, Hep3B, and Huh7 cell lines, we identified the SNPs −1795G>A (rs6935207) and −201C>G (rs58812592) as having effects on transcription factor binding and/or promoter activity. The A-allele of the −1795G>A SNP showed allele-specific binding of the transcription factor NF-Y leading to 2.5-fold increased enhancer activity of the artificial SV40 promoter. However, the −1795G>A SNP showed no significant effects on the native OCT1 promoter activity. Furthermore, the −1795G>A SNP was not associated with the pharmacokinetics of metformin, fenoterol, sumatriptan and proguanil in healthy individuals or tropisetron efficacy in patients undergoing chemotherapy. Allele-dependent differences in USF1/2 binding and nearly total loss in OCT1 promoter activity were detected for the G-allele of −201C>G, but the SNP is apparently very rare. In conclusion, common OCT1 promoter SNPs have only minor effects on OCT1 expression.

scholarly journals Transcriptional regulation of α1H T-type calcium channel under hypoxia

2014 ◽  
Vol 307 (7) ◽  
pp. C648-C656 ◽  
Author(s):  
Hassan Sellak ◽  
Chun Zhou ◽  
Bainan Liu ◽  
Hairu Chen ◽  
Thomas M. Lincoln ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Pc12 Cells ◽  
Promoter Region ◽  
Low Voltage ◽  
Significant Rise ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Gene Promoters ◽  
Mobility Shift

The low-voltage-activated T-type Ca2+ channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5′-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site −1,173cacgc−1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.

Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

scholarly journals Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhibin Liao ◽  
Hongwei Zhang ◽  
Chen Su ◽  
Furong Liu ◽  
Yachong Liu ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Experiments ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Downstream Target ◽  
Protein Levels ◽  
Elevated Expression ◽  
Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

scholarly journals MiR-489-3p inhibits cell proliferation, migration, and invasion, and induces apoptosis, by targeting the BDNF-mediated PI3K/AKT pathway in glioblastoma

2020 ◽  
Vol 15 (1) ◽  
pp. 274-283
Author(s):  
Bo Zheng ◽  
Tao Chen
Keyword(s):  
Cell Proliferation ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Bdnf Mrna ◽  
Protein Levels ◽  
Rna Immunoprecipitation ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Protein Cell

AbstractAmong astrocyte tumors, glioblastoma (GBM) is the most malignant glioma, highly aggressive and invasive, with extremely poor prognosis. Previous research has reported that microRNAs (miRNAs) participate in the progression of many cancers. Thus, this study aimed to explore the role and the underlying mechanisms of microRNA (miR)-489-3p in GBM progression. The expression of miR-489-3p and brain-derived neurotrophic factor (BDNF) mRNA was measured by quantitative real-time polymerase chain reaction. Western blot analysis was used to detect BDNF protein and the PI3K/AKT pathway-related protein. Cell proliferation, apoptosis, migration, and invasion were analyzed using CKK-8 assay, flow cytometry, and transwell assay, respectively. The interaction between BDNF and miR-489-3p was explored by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. MiR-489-3p was down-regulated and BDNF was up-regulated in GBM tissues and cells. MiR-489-3p re-expression or BDNF knockdown inhibited GBM cell proliferation, migration, and invasion, and promoted apoptosis. BDNF was a target of miR-489-3p, and BDNF up-regulation reversed the effects of miR-489-3p on GBM cells. The protein levels of p-AKT and p-PI3K were notably reduced in GBM cells by overexpression of miR-489-3p, but were rescued following BDNF up-regulation. Therefore, miR-489-3p inhibited proliferation, migration, and invasion, and induced apoptosis, by targeting the BDNF-mediated PI3K/AKT pathway in GBM, providing new strategies for clinical treatment of GBM.

scholarly journals Let-7a-5p regulates the inflammatory response in chronic rhinosinusitis with nasal polyps

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jianwei Zhang ◽  
Lei Han ◽  
Feng Chen
Keyword(s):  
Inflammatory Response ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Mapk Pathway ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Protein Levels ◽  
Tnf Α

Abstract Background Let-7a-5p is demonstrated to be a tumor inhibitor in nasopharyngeal carcinoma. However, the role of let-7a-5p in chronic rhinosinusitis with nasal polyps (CRSwNP) has not been reported. This study is designed to determine the pattern of expression and role of let-7a-5p in CRSwNP. Methods The expression level of let-7a-5p, TNF-α, IL-1β, and IL-6 in CRSwNP tissues and cells were detected by RT-qPCR. Western blot assay was carried out to measure the protein expression of the Ras-MAPK pathway. Dual luciferase reporter assay and RNA pull-down assay were used to explore the relationship between let-7a-5p and IL-6. Results Let-7a-5p was significantly downregulated in CRSwNP tissues and cells. Moreover, the mRNA expression of TNF-α, IL-1β and IL-6 was increased in CRSwNP tissues, while let-7a-5p mimic inhibited the expression of TNF-α, IL-1β and IL-6. Besides that, let-7a-5p was negatively correlated with TNF-α, IL-1β and IL-6 in CRSwNP tissues. In our study, IL-6 was found to be a target gene of let-7a-5p. Additionally, let-7-5p mimic obviously reduced the protein levels of Ras, p-Raf1, p-MEK1 and p-ERK1/2, while IL-6 overexpression destroyed the inhibitory effect of let-7a-5p on the Ras-MAPK pathway in CRSwNP. Conclusion We demonstrated that let-7a-5p/IL-6 interaction regulated the inflammatory response through the Ras-MAPK pathway in CRSwNP.

Sign in / Sign up

Export Citation Format

Share Document