scholarly journals 5’UTR Variant rs6017916 Regulates SRC Expression and Contributes to the Susceptibility to Ischemic Stroke in the Chinese Han Population

2021 ◽  
Author(s):  
Jialei Yang ◽  
Xiaoxiao Song ◽  
Yibing Yang ◽  
Yan Yan ◽  
Baoyun Liang ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Mrna Expression ◽  
Genetic Association ◽  
Quantitative Polymerase Chain Reaction ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Chinese Han ◽  
Luciferase Reporter System

Abstract Previous studies reported that the SRC protein was involved in a variety of pathological mechanisms related to ischemic stroke (IS). In this study, we conducted a genetic association study between rs6017916 within the 5’UTR region of SRC gene and IS susceptibility. A total of 533 IS patients and 531 healthy controls were recruited to participate in the current study. The sequenom MassARRAY technology platform was used for genotyping. The quantitative polymerase chain reaction (qPCR) was conducted to detect SRC mRNA expression. The dual luciferase reporter system was used to verify the regulation of rs6017916 on SRC mRNA expression. Results showed that SRC mRNA expression was significantly increased in IS patients than that in controls (P<0.001). Receiver operating characteristic curve (ROC) analysis demonstrated that the signature of SRC mRNA expression differentiated between controls and IS patients with an area under the curve (AUC) of 0.935 corresponding to a specificity of 0.820 and sensitivity of 0.920. Genetic association analysis showed that rs6017916 was significantly associated with IS susceptibility under multiple genetic models, including additive [OR (95% CI)=0.76 (0.60,0.96), Padj=0.019] and dominant [OR (95% CI)=0.75(0.58,0.98), Padj=0.031]. In addition, the dual luciferase reporter system showed that the minor allele C of rs6017916 inhibited luciferase activity compared with the major allele A. In summary, we report that SRC mRNA expression was significantly increased in IS patients and was a potential diagnostic biomarker. Moreover, the 5’UTR variant rs6017916 of SRC was significantly associated with IS susceptibility. And rs6017916 might affect the pathological process of IS by regulating SRC expression.

scholarly journals miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blot Assay ◽  
Normal Tissues ◽  
Cell Progression ◽  
Luciferase Reporter System ◽  
Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

scholarly journals A Cell-Based High-Throughput Assay for the Screening of Small-Molecule Inhibitors of p53–MDM2 Interaction

2011 ◽  
Vol 16 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Jing Li ◽  
Shuyong Zhang ◽  
Linghuan Gao ◽  
Ying Chen ◽  
Xin Xie
Keyword(s):  
High Throughput ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Direct Inhibition ◽  
A Cell ◽  
Luciferase Reporter System ◽  
Novel Strategy ◽  
High Throughput Assay ◽  
Two Hybrid ◽  
Rule Out

The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using this assay, they screened a library consisting of 3840 compounds and identified one compound that activates p53 pathway and induces growth arrest in tumor cells.

Histopathological analysis of retrieved thrombi from patients with acute ischemic stroke with malignant tumors

2021 ◽  
pp. neurintsurg-2020-017195
Author(s):  
Yuko Kataoka ◽  
Kazutaka Sonoda ◽  
Jun C Takahashi ◽  
Hatsue Ishibashi-Ueda ◽  
Kazunori Toyoda ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Malignant Tumors ◽  
Characteristic Curve ◽  
Cerebral Arteries ◽  
Treatment Strategies ◽  
Area Under The Curve ◽  
Histopathological Analysis ◽  
Thrombotic Risk ◽  
Patients With Cancer ◽  
Auc Value

BackgroundThe procoagulant state in cancer increases the thrombotic risk, and underlying cancer could affect treatment strategies and outcomes in patients with ischemic stroke. However, the histopathological characteristics of retrieved thrombi in patients with cancer have not been well studied. This study aimed to assess the histopathological difference between thrombi in patients with and without cancer.MethodsWe studied consecutive patients with acute major cerebral artery occlusion who were treated with endovascular therapy between October 2010 and December 2016 in our single-center registry. The retrieved thrombi were histopathologically investigated with hematoxylin and eosin and Masson’s trichrome staining. The organization and proportions of erythrocyte and fibrin/platelet components were studied using a lattice composed of 10×10 squares.ResultsOf the 180 patients studied, 17 (8 women, age 76.5±11.5 years) had cancer and 163 (69 women, age 74.1±11.2 years) did not. Those with cancer had a higher proportion of fibrin/platelets (56.6±27.4% vs 40.1±23.9%, p=0.008), a smaller proportion of erythrocytes (42.1±28.3% vs 57.5±25.1%, p=0.019), and higher serum D-dimer levels (5.9±8.2 vs 2.4±4.3 mg/dL, p=0.005) compared with the non-cancer cases. Receiver operating characteristic curve analysis showed the cut-off ratio of fibrin/platelet components related to cancer was 55.7% with a sensitivity of 74.8%, specificity 58.8% and area under the curve (AUC) value of 0.67 (95% CI 0.53 to 0.81), and the cut-off ratio of erythrocyte components was 44.7% with a sensitivity of 71.2%, specificity 58.9% and AUC value of 0.66 (95% CI 0.51 to 0.80).ConclusionsThromboemboli of major cerebral arteries in patients with cancer were mainly composed of fibrin/platelet-rich components.

scholarly journals Circulating Circular RNAs as Biomarkers for the Diagnosis and Prediction of Outcomes in Acute Ischemic Stroke

Stroke ◽  
2020 ◽  
Vol 51 (1) ◽  
pp. 319-323 ◽  
Author(s):  
Lei Zuo ◽  
Lin Zhang ◽  
Juan Zu ◽  
Zan Wang ◽  
Bing Han ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Quantitative Polymerase Chain Reaction ◽  
Area Under The Curve ◽  
Circular Rna ◽  
Population Based ◽  
Circular Rnas ◽  
Healthy Controls ◽  
Change Rate ◽  
Stroke Outcomes

Background and Purpose— Circular RNAs (CircRNAs) show promise as stroke biomarkers because of their participation in various pathophysiological processes associated with acute ischemic stroke (AIS) and stability in peripheral blood. Methods— A circRNA microarray was used to identify differentially expressed circulating circRNAs in a discovery cohort (3 versus 3). Validation (36 versus 36) and replication (200 versus 100) were performed in independent cohorts by quantitative polymerase chain reaction. Platelets, lymphocytes, and granulocytes were separated from blood to examine the origins of circRNAs. Results— There were 3 upregulated circRNAs in Chinese population–based AIS patients compared with healthy controls. The combination of 3 circRNAs resulted in an area under the curve of 0.875, corresponding to a specificity of 91% and a sensitivity of 71.5% in AIS diagnosis. Furthermore, the combination of change rate in 3 circRNAs within the first 7 days of treatment showed an area under the curve of 0.960 in predicting stroke outcome. There was significant increase in lymphocytes and granulocytes for circPDS5B (circular RNA PDS5B) and only in granulocytes for circCDC14A (circular RNA CDC14A) in AIS patients compared with healthy controls. Conclusions— Three circRNAs could serve as biomarkers for AIS diagnosis and prediction of stroke outcomes. The elevated levels of circPDS5B and circCDC14A after stroke might be because of increased levels in lymphocytes and granulocytes.

scholarly journals Cyp11b1 Is Induced in the Murine Gonad by Luteinizing Hormone/Human Chorionic Gonadotropin and Involved in the Production of 11-Ketotestosterone, a Major Fish Androgen: Conservation and Evolution of the Androgen Metabolic Pathway

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1786-1792 ◽  
Author(s):  
Takashi Yazawa ◽  
Miki Uesaka ◽  
Yoshihiko Inaoka ◽  
Tetsuya Mizutani ◽  
Toshio Sekiguchi ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Metabolic Pathway ◽  
Leydig Cells ◽  
Plasma Concentrations ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Mouse Ovary ◽  
Luciferase Reporter System

We have shown previously that Cyp11b1, an 11β-hydroxylase responsible for glucocorticoid biosynthesis in the adrenal gland, was induced by cAMP in androgen-producing Leydig-like cells derived from mesenchymal stem cells. We found that Cyp11b1 was induced in male Leydig cells, or female theca cells, when human chorionic gonadotropin was administered in immature mice. Expression of Cyp11b1 in rodent gonads caused the production of 11-ketotestosterone (11-KT), a major fish androgen, which induces male differentiation or spermatogenesis in fish. As in teleosts, plasma concentrations of 11-KT were elevated in human chorionic gonadotropin-treated mice. In contrast to teleosts, however, plasma concentrations of 11-KT were similar in both sexes, despite levels of testosterone, a precursor substrate, being about 20 times higher in male mice. Because expression of 11β-hydroxysteroid dehydrogenase type 2, was much higher in the mouse ovary than in the testis, conversion of testosterone into 11-KT may occur more efficiently in the ovary. In a luciferase reporter system that was responsive to and activated by androgens, 11-KT efficiently activated mammalian androgen receptor-mediated transactivation. Our results suggest that the androgen metabolic pathway is conserved between teleosts and mammals, despite sexual dominance and reproductive functions of 11-KT being altered during evolution.

Optimal thresholds for ischemic penumbra predicted by computed tomography perfusion in patients with acute ischemic stroke treated with mechanical thrombectomy

2017 ◽  
Vol 10 (3) ◽  
pp. 279-284 ◽  
Author(s):  
Katsuharu Kameda ◽  
Junji Uno ◽  
Ryosuke Otsuji ◽  
Nice Ren ◽  
Shintaro Nagaoka ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Cerebral Blood Volume ◽  
Mechanical Thrombectomy ◽  
Ct Perfusion ◽  
Characteristic Curve ◽  
Mean Transit Time ◽  
Area Under The Curve ◽  
Ischemic Penumbra ◽  
Optimal Thresholds

Background and purposeOptimal thresholds for ischemic penumbra detected by CT perfusion (CTP) in patients with acute ischemic stroke (AIS) have not been elucidated. In this study we investigated optimal thresholds for salvageable ischemic penumbra and the risk of hemorrhagic transformation (HT).MethodsA total of 156 consecutive patients with AIS treated with mechanical thrombectomy (MT) at our hospital were enrolled. Absolute (a) and relative (r) CTP parameters including cerebral blood flow (aCBF and rCBF), cerebral blood volume (aCBV and rCBV), and mean transit time (aMTT and rMTT) were evaluated for their value in detecting ischemic penumbra in each of seven arbitrary regions of interest defined by the major supplying blood vessel. Optimal thresholds were calculated by performing receiver operating characteristic curve analysis in 47 patients who achieved Thrombolysis In Cerebral Infarction (TICI) grade 3 recanalization. The risk of HT after MT was evaluated in 101 patients who achieved TICI grade 2b–3 recanalization.ResultsAbsolute CTP parameters for distinguishing ischemic penumbra from ischemic core were as follows: aCBF, 27.8 mL/100 g/min (area under the curve 0.82); aCBV, 2.1 mL/100 g (0.75); and aMTT, 7.30 s (0.70). Relative CTP parameters were as follows: rCBF, 0.62 (0.81); rCBV, 0.83 (0.87); and rMTT, 1.61 (0.73). CBF was significantly lower in areas of HT than in areas of infarction (aCBF, p<0.01; rCBF, p<0.001).ConclusionsCTP may be able to predict treatable ischemic penumbra and the risk of HT after MT in patients with AIS.

The dual luciferase reporter system and RT‐qPCR strategies for screening of MicroRNA‐21 small‐molecule inhibitors

2019 ◽  
Vol 66 (5) ◽  
pp. 755-762 ◽  
Author(s):  
Yuan‐Yuan Hei ◽  
Yuan‐Xu Guo ◽  
Cong‐Shan Jiang ◽  
Si Wang ◽  
She‐Min Lu ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Luciferase Reporter System

scholarly journals Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

2011 ◽  
Vol 300 (4) ◽  
pp. C888-C895 ◽  
Author(s):  
Timothy B. Chaston ◽  
Pavle Matak ◽  
Katayoun Pourvali ◽  
Surjit K. Srai ◽  
Andrew T. McKie ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Promoter Activity ◽  
Quantitative Polymerase Chain Reaction ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Smad Signaling ◽  
Hepcidin Expression ◽  
Huh7 Cells ◽  
Hepcidin Mrna

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H2O2, hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

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