miR-18a-3p and Its Target Protein HuR May Regulate Myogenic Differentiation in Immune-Mediated Necrotizing Myopathy

Immune-mediated necrotizing myopathy (IMNM) is characterized by manifestation of myonecrosis and regeneration of muscle fibers; however, the underlying pathogenesis remains unclear. This study aimed to investigate the role and mechanism of miR-18a-3p and its target RNA-binding protein HuR in IMNM. HuR and miR-18a-3p levels were detected in the skeletal muscles of 18 patients with IMNM using quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) and western blotting analysis. Human myoblasts were transfected with small interfering RNA targeting HuR and miR-18a-3p mimic or inhibitor. Myogenic differentiation markers, myogenin and myosin heavy chain, were analyzed by qRT-PCR, western blotting analysis, and immunofluorescence staining. The results showed that miR-18a-3p was upregulated (p=0.0002), whereas HuR was downregulated (p=0.002) in the skeletal muscles of patients with IMNM. The expression of miR-18a-3p in patients with IMNM was negatively correlated with those of HuR (r = -0.512, p = 0.029). We also found that disease activity was positively correlated with HuR expression (r = 0.576, p = 0.012) but muscle activity was negatively correlated with miR-18a-3p expression (r = -0.550, p = 0.017). Besides, bioinformatics analysis and dual-luciferase reporter assays suggested that miR-18a-3p could directly target HuR. Cellular experiments showed that overexpression of miR-18a-3p inhibited myogenic differentiation by targeting HuR, whereas inhibition of miR-18a-3p led to opposite results. Therefore, miR-18a-3p and its target protein HuR may be responsible for modulating the myogenic process in IMNM and can thus be therapeutic targets for the same.

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12-Deacetyl-12-epi-Scalaradial, a Scalarane Sesterterpenoid from a Marine Sponge Hippospongia sp., Induces HeLa Cells Apoptosis via MAPK/ERK Pathway and Modulates Nuclear Receptor Nur77

Marine Drugs ◽

10.3390/md18070375 ◽

2020 ◽

Vol 18 (7) ◽

pp. 375

Author(s):

Mi Zhou ◽

Bo-Rong Peng ◽

Wenjing Tian ◽

Jui-Hsin Su ◽

Guanghui Wang ◽

...

Keyword(s):

Cervical Cancer ◽

Western Blotting ◽

Marine Sponge ◽

Hela Cells ◽

Luciferase Reporter ◽

Dependent Manner ◽

Erk Pathway ◽

Orphan Nuclear Receptors ◽

Western Blotting Analysis ◽

Blotting Analysis

12-Deacetyl-12-epi-scalaradial, a scalarane sesterterpenoid from a marine sponge Hippospongia sp, has been reported to possess cytotoxic activity on HepG2, MCF-7, and HCT-116 cells. However, there is no research to indicate that 12-deacetyl-12-epi-scalaradial exhibited anticancer effect on cervical cancer HeLa cells. The aim of this study was to investigate the anticancer activity of 12-deacetyl-12-epi-scalaradial against HeLa cells and to explore the mechanism. The results from a methylthiazolyldiphenyl-tetrazolium (MTT) assay suggested that 12-deacetyl-12-epi-scalaradial suppressed the proliferation of HeLa cells and flow cytometry analysis showed 12-deacetyl-12-epi-scalaradial could induce the apoptosis of HeLa cells in dose- and time-dependent manner. Western blotting analysis demonstrated that 12-deacetyl-12-epi-scalaradial triggered apoptosis via mediating the extrinsic pathway and was found to suppress MAPK/ERK pathway which was associate with cancer cell death. Nur77, a critical number of orphan nuclear receptors, plays diverse roles in tumor development as a transcription factor and has been considered as a promising anticancer drug target. The dual-luciferase reporter assays suggested that 12-deacetyl-12-epi-scalaradial could selectively enhance the trans-activation activity of Nur77. Furthermore, Western blotting analysis and fluorescence quenching showed that 12-deacetyl-12-epi-scalaradial could induce the phosphorylation of Nur77 and interact with the ligand-binding domain (LBD) of Nur77. Our research confirmed 12-deacetyl-12-epi-scalaradial as a potential agent for cervical cancer therapy and provided a view that 12-deacetyl-12-epi-scalaradial may be a modulator of Nur77.

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Long Non-coding RNA LINC02474 Affects Metastasis and Apoptosis of Colorectal Cancer by Inhibiting the Expression of GZMB

Frontiers in Oncology ◽

10.3389/fonc.2021.651796 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tiantian Du ◽

Qinglun Gao ◽

Yinghui Zhao ◽

Jie Gao ◽

Juan Li ◽

...

Keyword(s):

Colorectal Cancer ◽

Western Blotting ◽

Noncoding Rna ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Nude Mouse Model ◽

Western Blotting Analysis ◽

Blotting Analysis

BackgroundColorectal cancer (CRC) is one of the most frequently diagnosed malignancies. Metastasis is the main event that impedes the therapeutic effect on CRC, and its underlying mechanisms remain largely unclear. LINC02474 is a novel long noncoding RNA (lncRNA) associated with metastasis of CRC, while little is known about how LINC02474 regulates these malignant characteristics.MethodsExpressions of LINC02474 and granzyme B (GZMB) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blotting analysis. Cell metastasis was detected by transwell assay and metastatic nude mouse model, and apoptosis was determined by Western blotting analysis and flow cytometry. Besides, the interaction between LINC02474 and GZMB was detected by dual-luciferase reporter assays.ResultsThe expression of LINC02474 was significantly up-regulated in CRC tissues. Moreover, depletion of LINC02474 damaged the metastatic abilities of CRC cells in vivo and in vitro while boosting apoptosis. Besides, up-regulation of LINC02474 could promote migration and invasion, while apoptosis was inhibited in CRC cells. Besides, down-regulation of LINC02474 promoted the expression of GZMB, and interference of GZMB could increase the metastatic abilities of CRC cells while reducing apoptosis. Furthermore, LINC02474 was related to the transcriptional repression of GZMB in CRC cells determined by the dual-luciferase reporter assay.ConclusionsThe findings revealed that a novel lncRNA, LINC02474, as an oncogene, could promote metastasis, but limit apoptosis partly by impeding GZMB expression in CRC. Besides, LINC02474 had the potential to be used as a biomarker in the prognosis of CRC.

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Analysis of the physical activity effects and measurement of pro-inflammatory cytokines in irradiated lungs in rats

Acta Cirurgica Brasileira ◽

10.1590/s0102-86502012000300004 ◽

2012 ◽

Vol 27 (3) ◽

pp. 223-230 ◽

Cited By ~ 1

Author(s):

Renata Cristiane Gennari Bianchi ◽

Eduardo Rochete Ropelle ◽

Carlos Kiyoshi Katashima ◽

José Barreto Campello Carvalheira ◽

Luiz Roberto Lopes ◽

...

Keyword(s):

Physical Activity ◽

Western Blotting ◽

Inflammatory Cytokines ◽

Lower Lobe ◽

Whole Body ◽

Control Group ◽

Western Blotting Analysis ◽

Blotting Analysis ◽

Tnf Α ◽

Pro Inflammatory Cytokines

PURPOSE: To study if the pre-radiotherapy physical activity has radio-protective elements, by measuring the radio-induced activation of pro-inflammatory cytokines as interleukin-6 (il-6), transforming growth factor -β (tgf -β), tumor necrosis factor -α (tnf-α) and protein beta kinase β (ikkβ), through western blotting analysis. METHODS: A randomized study with 28 Wistar hannover rats, males, with a mean age of 90 days and weighing about 200 grams. The animals were divided into three groups: (GI, GII and GIII). GIII group were submitted to swimming for eight weeks (zero load, three times a week, about 30 minutes). Then, the groups (except the control group) were submitted to irradiation by cobalt therapy, single dose of 3.5 gray in the whole body. All animals were sacrificed by overdose of pentobarbital, according to the time for analysis of cytokines, and then a fragment of the lower lobe of the right lung went to western blotting analysis. RESULTS: The cytokines IKK β, TNF-α and IL-6 induced by radiation in the lung were lower in the exercised animals. However, exercise did not alter the radiation-induced increase in tgf-β. CONCLUSION: The results show a lower response in relation to inflammatory cytokines in the group that practiced the exercise pre-radiotherapy, showing that exercise can protect tissues from tissue damage due to irradiation.

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Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02762002000900024 ◽

2002 ◽

Vol 97 (suppl 1) ◽

pp. 115-116 ◽

Cited By ~ 4

Author(s):

Nilton Thaumaturgo ◽

Mônica Magno Vilar ◽

Ricardo Edelenyi ◽

Miriam Tendler

Keyword(s):

Western Blotting ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Western Blotting Analysis ◽

Blotting Analysis

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Mechanisms Involved in the Anti-Tumor Effects of Toosendanin in Glioma Cells

10.21203/rs.3.rs-614743/v1 ◽

2021 ◽

Author(s):

haiyan huang ◽

Chaochao Zhang ◽

Haijun Gao ◽

Ziqiang Liu ◽

Jiacheng Lai ◽

...

Keyword(s):

Flow Cytometry ◽

Wound Healing ◽

Western Blotting ◽

Colony Formation ◽

Glioma Cell ◽

Glioma Cells ◽

Migration And Invasion ◽

Antitumor Effects ◽

Western Blotting Analysis ◽

Blotting Analysis

Abstract Background: Toosendanin (TSN) is a triterpenoid compound mainly used as an ascaris repellant. Recent studies have shown that it possesses antitumor effects in many types of tumor cells. However, the effects of TSN on glioma cells have rarely been reported. Methods: Different assays were performed to investigate the effects of TSN on the different glioma cell lines including U87MG and LN18. The assays included colony formation, wound healing, and transwell assays. Furthermore, Hoechst 3342 staining, flow cytometry, and western blotting analysis were performed to investigate the apoptotic activities of TSN. Finally, the results were confirmed using a xenograft tumor model that comprised of nude mice. Results: In vitro, the CCK-8 and colony formation assays showed that TSN effectively inhibited glioma cell proliferation. Moreover, the inhibitory effects on glioma cell migration and invasion were demonstrated through the wound healing and transwell assays, respectively. Hoechst 33342 staining, flow cytometry, and western blotting assays demonstrated the significant effect of TSN in the apoptosis induction of glioma cells. Furthermore, the anti-glioma effect of TSN was exerted through the inhibition of the PI3K/Akt/mTOR signaling pathways as demonstrated by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model confirmed the suppressive effect of TSN on tumor growth in vivo. Conclusion: Our results suggest that TSN is a promising chemotherapeutic drug for patients with glioma.

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Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation

Methods in Molecular Biology - Kidney Research ◽

10.1007/978-1-4939-3353-2_18 ◽

2015 ◽

pp. 267-277 ◽

Cited By ~ 16

Author(s):

Takahiro Horinouchi ◽

Koji Terada ◽

Tsunehito Higashi ◽

Soichi Miwa

Keyword(s):

Protein Phosphorylation ◽

Western Blotting ◽

Western Blotting Analysis ◽

Blotting Analysis

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So-Ochim-Tang-Gamibang, a Traditional Herbal Formula, Ameliorates Depression by Regulating Hyperactive Glucocorticoid Signaling In Vitro and In Vivo

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/8834556 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Mirim Jin ◽

Sun Young Park ◽

Hye Jin Choi ◽

Younmin Shin ◽

Eunho Chun ◽

...

Keyword(s):

Western Blotting ◽

Hpa Axis ◽

Plasma Levels ◽

Molecular Mechanisms ◽

Western Blotting Analysis ◽

Blotting Analysis ◽

Wky Rats ◽

Glucocorticoid Signaling

So-ochim-tang-gamibang (SOCG) is a Korean traditional medicine; it has previously been shown to be safe and effective against depression. Persistently increased levels of circulating glucocorticoids have been considered as a pathological mechanism for depression and associated with decreased neurotrophic factors in the hippocampus. This study investigated whether SOCG controls the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and the molecular mechanisms underlying its effects in vivo and in vitro. Wistar Kyoto (WKY) rats were subjected to restraint stress, where SOCG was orally administered to the animals for 2 weeks. An open field test (OFT), forced swimming test (FST), and sucrose preference test (SPT) were performed to explore the antidepressant activity of SOCG in WKY rats. Plasma levels of HPA axis hormones were measured by ELISA or western blotting analysis. The expression levels or activation of HPA axis-related signaling molecules such as brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), extracellular regulated kinase (ERK), and glucocorticoid receptors (GRs) in the brain were determined by real-time PCR and western blotting analysis. Furthermore, a corticosterone- (CORT-) induced cell injury model was established using SH-SY5Y cells to explore the antidepressive effects of SOCG in vitro. The results of the OFT, FST, and SPT revealed that SOCG ameliorated depressive-like behaviors in the WKY rats. The blood plasma levels of HPA axis hormones such as CORT, CORT-releasing hormone (CRH), and adrenocorticotrophic hormone were downregulated by SOCG. On the other hand, SOCG upregulated the phosphorylation of CREB and ERK in both the rat hippocampus and CORT-treated SH-SY5Y cells. Moreover, it also increased the GR expression. These results suggested that SOCG may improve depression by controlling hyperactive glucocorticoid signaling via the downregulation of HPA axis hormones and upregulation of GR.

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Effects of topical mechanical stability on the formation of Masquelet membrane in a rabbit radial defect model

Scientific Reports ◽

10.1038/s41598-020-76112-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jie Xie ◽

Donghao Liu ◽

Haoyi Wang ◽

Haitao Long ◽

Yong Zhu ◽

...

Keyword(s):

Western Blotting ◽

Mechanical Stability ◽

Mechanical Test ◽

Control Group ◽

Rigid Fixation ◽

X Ray ◽

Western Blotting Analysis ◽

Blotting Analysis ◽

Fixation Group ◽

Masquelet Technique

Abstract The exact mechanism of Masquelet technique is unknown. This study intends to explore the effects of topical mechanical stability on the formation of Masquelet membrane. Segmental radius shaft defect was created in all rabbits, which were filled with polymethylmethacrylate (PMMA) in Non-fixation group, and with PMMA fixed with plates in Fixation group, and subjected to no disposal in control group. The topical stability of PMMA and plates were monitored via X-ray and mechanical test. And the membranes were excised for further Histological, IHC and Western-Blotting analysis 4 and 6 weeks post-operatively. X-ray revealed no sign of plates loosening, or shift of PMMA. Mechanical tests revealed superior topical stability by plates. Pathological examinations suggested that vascularized and osteogenic membranes were formed around PMMA. IHC and Western-Blotting analysis revealed that both Fixation and Non-fixation group exerted significant effects on the expression of Ki67, COL I, and CD31 positive cells, as well as the protein expression of osteogenic (RUNX2, ALP) and angiogenic (VEGFA, TGF-β1) factors. And compared with membrane in Non-fixation group, Fixing PMMA spacer with plates caused a significant increase in osteogenic and angiogenic expression. This study indicates that rigid fixation provided by plate in Masquelet technique positively alters the quality of membrane formed surrounding PMMA, in terms of significantly osteogenic and angiogenic potential.

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