scholarly journals Ripasudil alleviated the inflammation of RPE cells by targeting the miR-136-5p/ROCK/NLRP3 pathway

2020 ◽  
Author(s):  
Zhao Gao ◽  
Qiang Li ◽  
Yunda Zhang ◽  
Haiyan Li ◽  
Xiaohong Gao ◽  
...  
Keyword(s):  
Western Blotting ◽  
Inflammatory Diseases ◽  
Normal Function ◽  
Eye Diseases ◽  
Luciferase Reporter ◽  
Rpe Cells ◽  
Reporter Assays ◽  
Related Proteins ◽  
Western Blotting Assay ◽  
Stimulation Of

Abstract Introduction Inflammation of RPE cells lead to different kinds of eye diseases and affect the normal function of the retina. Furthermore, higher levels of ROCK1 and ROCK2 induced injury of endothelial cells and many inflammatory diseases of the eyes. Ripasudil which was used for the treatment of glaucoma was one kind of the inhibitor of ROCK1 and ROCK2. But whether the ripasudil could relieved the LPS induced inflammation damage of RPE cells was not clear.Material and methods We used the LPS to stimulate ARPE-19 cells which is the RPE cell line. After that, we detected the levels of ROCK1 and ROCK2 by western-blotting assay after the stimulation of LPS and treatment of ripasudil. Then luciferase reporter assays were used to confirm the targeted effect of miR-136-5p on ROCK1 and ROCK2. At last, the levels of NLRP3, ASC, caspase1, IL-1β and IL-18 were detected with the western-blotting after the knockdown of miR-136-5p.Results The levels of ROCK1, ROCK2 and miR-136-5p in ARPE-19 cells was promoted after the stimulation of LPS. After the treatment of ripasudil, the ROCK1, ROCK2 and miR-136-5p was suppressed. The miR-136-5p targeted and inhibited the expression of ROCK1 and ROCK2. Inflammation related proteins NLRP3, ASC, caspase1, IL-1β and IL-18 was inhibited after the treatment of ripasudil. However, the expression of these proteins was rescued after the knockdown of the miR-136-5p.Conclusion Ripasudil relieved the inflammatory injury of RPE cells by upregulating miR-136-5p and therefore inhibiting the expression of ROCK1, ROCK2, NLRP3, ASC, caspase1, IL-1β and IL-18.

scholarly journals Ripasudil alleviated the inflammation of RPE cells by targeting the miR-136-5p/ROCK/NLRP3 pathway

2020 ◽  
Author(s):  
Zhao Gao ◽  
Qiang Li ◽  
Yunda Zhang ◽  
Xiaohong Gao ◽  
Haiyan Li ◽  
...  
Keyword(s):  
Western Blotting ◽  
Inflammatory Diseases ◽  
Normal Function ◽  
Eye Diseases ◽  
Luciferase Reporter ◽  
Rpe Cells ◽  
Reporter Assays ◽  
Related Proteins ◽  
Targeting Effect ◽  
Stimulation Of

Abstract Background: Inflammation of RPE cells led to different kinds of eye diseases and affected the normal function of the retina. Furthermore, higher levels of ROCK1 and ROCK2 induced injury of endothelial cell s and many inflammatory diseases of the eyes. Ripasudil, which was used for the treatment of glaucoma , was one kind of the inhibitor of ROCK1 and ROCK2, but whether ripasudil could relieve the LPS-induced inflammation and damage of RPE cells was not clear. Methods: We used LPS to stimulate ARPE-19 cells, the RPE cell line. After that, we detected the levels of ROCK1 and ROCK2 by western-blotting after the stimulation of LPS and treatment of ripasudil. Then luciferase reporter assays were used to confirm the targeting effect of miR-136-5p on ROCK1 and ROCK2. At last, the levels of NLRP3, ASC, caspase1, IL-1β and IL-18 were detected with the western-blotting after the knockdown of miR-136-5p. Results: The levels of ROCK1, ROCK2 and miR-136-5p in ARPE-19 cells were promoted after the stimulation of LPS. After the treatment of ripasudil, the expression levels of ROCK1, ROCK2 and miR-136-5p were suppressed. The expression of ROCK1 and ROCK2 was targeted and inhibited by the miR-136-5p. The levels of inflammation related proteins NLRP3, ASC, caspase1, IL-1β and IL-18 was also inhibited after the treatment of ripasudil. However, the expression of these proteins was rescued after the knockdown of miR-136-5p. Conclusion: Ripasudil relieved the inflammatory injury of RPE cells by upregulating miR-136-5p, therefore inhibiting the expression of ROCK1, ROCK2, NLRP3, ASC, caspase1, IL-1β and IL-18.

scholarly journals Ripasudil alleviated the inflammation of RPE cells by targeting the miR-136-5p/ROCK/NLRP3 pathway

2020 ◽  
Author(s):  
Zhao Gao ◽  
Qiang Li ◽  
Yunda Zhang ◽  
Xiaohong Gao ◽  
Haiyan Li ◽  
...  
Keyword(s):  
Western Blotting ◽  
Inflammatory Diseases ◽  
Normal Function ◽  
Eye Diseases ◽  
Luciferase Reporter ◽  
Rpe Cells ◽  
Reporter Assays ◽  
Related Proteins ◽  
Targeting Effect ◽  
Stimulation Of

Abstract Background: Inflammation of RPE cells led to different kinds of eye diseases and affected the normal function of the retina. Furthermore, higher levels of ROCK1 and ROCK2 induced injury of endothelial cell s and many inflammatory diseases of the eyes. Ripasudil, which was used for the treatment of glaucoma , was one kind of the inhibitor of ROCK1 and ROCK2, but whether ripasudil could relieve the LPS-induced inflammation and damage of RPE cells was not clear. Methods: We used LPS to stimulate ARPE-19 cells, the RPE cell line. After that, we detected the levels of ROCK1 and ROCK2 by western-blotting after the stimulation of LPS and treatment of ripasudil. Then luciferase reporter assays were used to confirm the targeting effect of miR-136-5p on ROCK1 and ROCK2. At last, the levels of NLRP3, ASC, caspase1, IL-1β and IL-18 were detected with the western-blotting after the knockdown of miR-136-5p. Results: The levels of ROCK1, ROCK2 and miR-136-5p in ARPE-19 cells were promoted after the stimulation of LPS. After the treatment of ripasudil, the expression levels of ROCK1, ROCK2 and miR-136-5p were suppressed. The expression of ROCK1 and ROCK2 was targeted and inhibited by the miR-136-5p. The levels of inflammation related proteins NLRP3, ASC, caspase1, IL-1β and IL-18 was also inhibited after the treatment of ripasudil. However, the expression of these proteins was rescued after the knockdown of miR-136-5p. Conclusion: Ripasudil relieved the inflammatory injury of RPE cells by upregulating miR-136-5p, therefore inhibiting the expression of ROCK1, ROCK2, NLRP3, ASC, caspase1, IL-1β and IL-18.

scholarly journals Oncoprotein LAMTOR5 activates GLUT1 via upregulating NF-κB in liver cancer

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 264-270 ◽  
Author(s):  
Jing Zhou ◽  
Yajun Li ◽  
Danhua Li ◽  
Zhi Liu ◽  
Jie Zhang
Keyword(s):  
Liver Cancer ◽  
Cancer Cells ◽  
Western Blotting ◽  
Glucose Transporter ◽  
Luciferase Reporter ◽  
Pcr Assay ◽  
Glucose Transporter 1 ◽  
Over Expression ◽  
Liver Cancer Cells ◽  
Reporter Assays

AbstractObjectiveAccumulating reports reveal that serving as an oncogenic factor LAMTOR5 is involved in the progression of many specific cancers. Glucose transporter 1 (GLUT1) is frequently identified in many cancers. However, it remains unexplored whether GLUT1 plays a role in LAMTOR5-enhanced liver cancer. Here, we aim to decipher the function of LAMTOR5 in the regulation of GLUT1 in liver cancer.MethodsThe effect of LAMTOR5 on GLUT1 was analyzed using Western blotting and RT-PCR assay. Dose-increased over-expression or silencing of LAMTOR5 was performed through transient transfection. LAMTOR5-activated GLUT1 promoter was revealed by luciferase reporter assay. The regulation of GLUT1 by LAMTOR5/NF-κB was examined via Western blotting and luciferase reporter assays.ResultsThe data showed that in liver cancer cells under the administration with dose-increased LAMTOR5, the level of mRNA and protein of GLUT1 was obviously raised. Our data revealed that the activities of GLUT1 promoter were induced by LAMTOR5. Then, we found that the elevation of GLUT 1 mediated by LAMTOR5 slowed when the inhibitor or siRNAs of NF-κB was introduced into the liver cancer cells. Conclusion. LAMTOR5 is responsible for the activation of GLUT1 via transcription factor NF-κB in liver cancer.

scholarly journals Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway

2021 ◽  
Vol 41 (1) ◽  
Author(s):  
Wuquan Li ◽  
Wentao Zhang ◽  
Jun Liu ◽  
Yalong Han ◽  
He Jiang ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Inflammatory Diseases ◽  
Pulmonary Inflammation ◽  
Lavage Fluid ◽  
Luciferase Reporter ◽  
Cytokine Signaling ◽  
Down Regulation ◽  
Reporter Assays

Abstract Excessive pulmonary inflammatory response is critical in the development of acute lung injury (ALI). Previously, microRNAs (miRNAs) have been recognized as an important regulator of inflammation in various diseases. However, the effects and mechanisms of miRNAs on inflammatory response in ALI remain unclear. Herein, we tried to screen miRNAs in the processes of ALI and elucidate the potential mechanism. Using a microarray assay, microRNA let-7e (let-7e) was chose as our target for its reported suppressive roles in several inflammatory diseases. Down-regulation of let-7e by antagomiR-let-7e injection attenuated LPS-induced acute lung injury. We also found that antagomiR-let-7e could obviously improve the survival rate in ALI mice. Moreover, antagomiR-let-7e treatment reduced the production of proinflammatory cytokines (i.e., TNF-α, IL-1β and IL-6) in bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice. Luciferase reporter assays confirmed that suppressor of cytokine signaling 1 (SOCS1), a powerful attenuator of nuclear factor kappa B (NF-κB) signaling pathway, was directly targeted and suppressed by let-7e in RAW264.7 cells. In addition, it was further observed that SOCS1 was down-regulated, and inversely correlated with let-7e expression levels in lung tissues of ALI mice. Finally, down-regulation of let-7e suppressed the activation of NF-κB pathway, as evidenced by the reduction of p-IκBα, and nuclear p-p65 expressions in ALI mice. Collectively, our findings indicate that let-7e antagomir protects mice against LPS-induced lung injury via repressing the pulmonary inflammation though regulation of SOCS1/NF-κB pathway, and let-7e may act as a potential therapeutic target for ALI.

scholarly journals LncRNA SNHG6 promotes chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in colorectal cancer cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xinke Wang ◽  
Zhixian Lan ◽  
Juan He ◽  
Qiuhua Lai ◽  
Xiang Yao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blotting ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Chemotherapy Resistance ◽  
Micro Rna ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Reporter Assays

Abstract Background Chemotherapy resistance is one of the main causes of recurrence in colorectal cancer (CRC) patients and leads to poor prognosis. Long noncoding RNAs (lncRNAs) have been reported to regulate chemoresistance. We aimed to determine the role of the lncRNA small nucleolar RNA host gene 6 (SNHG6) in CRC cell chemoresistance. Methods Cell drug sensitivity tests and flow cytometry were performed to analyze CRC cell chemoresistance. Animal models were used to determine chemoresistance in vivo, and micro RNA (miRNA) binding sites were detected by dual-luciferase reporter assays. Bioinformatics analysis was performed to predict miRNAs binding to SNHG6 and target genes of miR-26a-5p. SNHG6/miR-26a-5p/ULK1 axis and autophagy-related proteins were detected by qRT-PCR and western blotting. Furthermore, immunofluorescence was employed to confirm the presence of autophagosomes. Results SNHG6 enhanced CRC cell resistance to 5-fluorouracil (5-FU), promoted autophagy, inhibited 5-FU-induced apoptosis, and increased 5-FU resistance in vivo. Bioinformatics analysis showed that miR-26a-5p might bind to SNHG6 and target ULK1, and dual-luciferase reporter assays confirmed this activity. qRT-PCR and western blotting showed that SNHG6 was able to negatively regulate miR-26a-5p but correlated positively with ULK1. Conclusion SNHG6 may promote chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in CRC cells.

scholarly journals Circular RNA circASPM Promotes the Progression of Glioblastoma by Acting as a Competing Endogenous RNA to Regulate miR-130b–3p/E2F1 Axis

2020 ◽  
Author(s):  
Dianqi Hou ◽  
Zhenlin Wang ◽  
Haimeng Li ◽  
Juan Liu ◽  
Yaohua Liu ◽  
...  
Keyword(s):  
Western Blotting ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Primary Malignancy ◽  
Rna Immunoprecipitation ◽  
Western Blotting Assay

Abstract background: Glioblastoma Multiform (GBM) is the primary malignancy with the highest incidence and worst prognosis in the adult CNS. Circular RNAs (circRNAs) are a novel and widely diverse class of endogenous non-coding RNAs that can promote or inhibit gliomagenesis. Our study aimed to explore the role of circASPM in GBM and its molecular mechanism.Methods: Levels of circASPM, miR-130b-3p and E2F1 were determined by quantitative real-time PCR (qRT-PCR) or western blotting assay. MTS, Edu, neurospheres formation and extreme limiting dilution assays were used to detect the tumorigenesis and proliferation of GSCs in vitro. The interactions between miR-130b-3p and circASPM or E2F1 was demonstrated via qPCR, western blotting, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Xenograft experiments was used to analyze tumor growth in vivo.Results: CircASPM was overexpressed in GBM and could promote the tumorigenesis and proliferation of GSCs both in vitro and in vivo. Mechanistically, circASPM up-regulated the expression of E2F1 in GSCs via miR-130b-3p sponging. We furtherly demonstrated that circAPSM could promote the GSCs proliferation via E2F1 up-regulating. Therefore, our study identified a novel circRNA and its possible mechanism in the development and tumorigenesis of GBM.Conclusions: CircASPM can promote GBM progression via regulating miR-130b-3p/E2F1 axis, suggesting that circAPSM could provide an effective biomarker for GBM diagnosis and prognostic evaluation and possibly being used for molecular targeted therapy.

scholarly journals Down-Regulation of TMPO-AS1 Induces Apoptosis in Lung Carcinoma Cells by Regulating miR-143-3p/CDK1 Axis

2021 ◽  
Vol 20 ◽  
pp. 153303382094888
Author(s):  
Qiu Li ◽  
Yuan Bian ◽  
Qiaolian Li
Keyword(s):  
Cell Viability ◽  
Cell Apoptosis ◽  
Carcinoma Cells ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Novel Therapy ◽  
Lung Carcinoma Cells ◽  
Kaplan Meier ◽  
Reporter Assays ◽  
Related Proteins

Evidence has shown that long non-coding RNAs (lncRNA) play pivotal roles in cancer promotion as well as suppression. But the molecular mechanism of lncRNA TMPO antisense transcript 1 (TMPO-AS1) in lung cancer (LC) remains unclear. This study mainly investigated the effect of TMPO-AS1 in LC treatment. TMPO-AS1 was tested by Kaplan-Meier method. Quantitative real time polymerase chain reaction (qRT-PCR) was employed to assess the expressions of TMPO-AS1, miR-143-3p, and CDK1 respectively in LC tissues and cell lines. TMPO-AS1, miR-143-3p and CDK1 expressions in LC cells were regulated through cell transfection, followed by MTT for cell viability detection. Besides, dual-luciferase reporter assays were performed to verify the interrelated microRNA of TMPO-AS1 and the target of miR-143-3p. Western blot experiments were used to examine the expressions of apoptosis-related proteins, and flow cytometry tested the cell apoptosis in treated cells. TMPO-AS1 and CDK1 were overexpressed in LC tissues and cells, while miR-143-3p level was suppressed. The decrease of TMPO-AS1 led to the increase of miR-143-3p, which further resulted in the reduction of CDK1. Down-regulating TMPO-AS1 reduced LC cell viability, motivated cell apoptosis, as well as promoted the expressions of Bcl and CCND1 and restrained Caspase-3 level, but all these consequences were abrogated by miR-143-3p inhibitor. Simultaneously, siCDK1 could reverse the anti-apoptosis and pro-activity functions of miR-143-3p inhibitor in LC cells. Down-regulation of TMPO-AS1 has the effects of pro-apoptosis in LC by manipulating miR-143-3p/CDK1, which is hopeful to be a novel therapy for LC patients.

scholarly journals MiR30a Inhibits Inflammation by Targeting Klf14 in Autoimmune Hepatitis

2021 ◽  
Author(s):  
Xiaofeng Yuan ◽  
Shunwen Pan ◽  
Mingliang Li ◽  
Li Quan ◽  
Kouxing Zhang ◽  
...  
Keyword(s):  
Autoimmune Hepatitis ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Liver Inflammation ◽  
Con A ◽  
Qrt Pcr ◽  
Reporter Assays ◽  
The Relationship

Abstract MiR30a plays diverse roles in inflammatory diseases, including autoimmune hepatitis (AIH). Klf14 is associated with the inflammation in AIH. We investigated whether miR30a exerts its regulatory function via Klf14. Concanavalin A (Con A)-induced AIH mice were infected with a miR30a agomir or antagomir. MiR30a expression was quantified using qRT-PCR. TargetScan and luciferase reporter assays were used to predict the relationship between miR30a and Klf14. Liver inflammation was evaluated by measuring serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, performing histology, and measuring mRNA expressions of inflammatory cytokines and Klf14 by qRT-PCR, protein of Klf14 by western blotting, and Tregs by FACS. MiR30a was downregulated in the hepatocytes (HCs) of AIH mice, which was negatively associated with the liver inflammation. MiR30a overexpression alleviated the inflammation, whereas downregulation of endogenous miR30a aggravated it. The mRNA and protein level of Klf14 were inversely correlated with the miR30a expression. The luciferase reporter assay validated the relationship between Klf14 and miR30a. Moreover, the frequency of Tregs was consistently correlated with the expression of miR30a. MiR30a may play an essential role in AIH, and its ability to regulate the inflammatory responses may, at least partially, be mediated by targeting Klf14 to modulate Tregs.

scholarly journals Sequential Activation of Hypoxia-Inducible Factor 1 and Specificity Protein 1 is Required for Hypoxia-Induced Transcriptional Stimulation of Abcc8

2011 ◽  
Vol 32 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Seung Kyoon Woo ◽  
Min Seong Kwon ◽  
Zhihua Geng ◽  
Zheng Chen ◽  
Alexander Ivanov ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Binding Sites ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Hypoxia Inducible Factor 1 ◽  
Specificity Protein 1 ◽  
Reporter Assays ◽  
Sequential Activation ◽  
Specificity Protein ◽  
Stimulation Of

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

scholarly journals Neural Crest Deletion of Dlx3 Leads to Major Dentin Defects through Down-regulation of Dspp

2012 ◽  
Vol 287 (15) ◽  
pp. 12230-12240 ◽  
Author(s):  
Olivier Duverger ◽  
Angela Zah ◽  
Juliane Isaac ◽  
Hong-Wei Sun ◽  
Anne K. Bartels ◽  
...  
Keyword(s):  
Mouse Model ◽  
Neural Crest ◽  
Matrix Protein ◽  
Tooth Development ◽  
Normal Function ◽  
Luciferase Reporter ◽  
Reporter Assays ◽  
Direct Binding ◽  
Dentin Formation

During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.

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