scholarly journals Knockdown of Long Non-Coding RNA GAS5 Increases miR-23a by Targeting ATG3 Involved in Autophagy and Cell Viability

2018 ◽  
Vol 48 (4) ◽  
pp. 1723-1734 ◽  
Author(s):  
Lexing Li ◽  
Chunxiao Huang ◽  
Yulong He ◽  
Zhan Sang ◽  
Guoquan Liu ◽  
...  
Keyword(s):  
Complex Formation ◽  
Cell Viability ◽  
Mrna Level ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Protein Levels ◽  
Repressive Effect ◽  
Competing Endogenous Rnas ◽  
Reporter Assays ◽  
Direct Binding

Background/Aims: Autophagy is a process of evolutionarily conservative degradation, which could maintain cellular homeostasis and cope with various types of stress. LncRNAs are considered as competing endogenous RNAs (ceRNAs) contributing to autophagy. GAS5 has been suggested as a new potential factor to mediate autophagy pathway and the underlying mechanism remains to be further confirmed. This study was taken to identify the effect of GAS5/miR-23a/ATG3 axis on autophagy and cell viability. Methods: The western blotting assay was used to detecte the protein levels of LC3, mTOR, Beclin-1, ATG3, ATG5-ATG12 complex and p62. The mRNA level of Pre-miR-23a, Pri-miR-23a, miR-23a, GAS5, LC3, mTOR and ATG3 were quantified by real-time RT-PCR. Dual-luciferase reporter assays were performed to confirm the direct binding of miR-23a and ATG3 or GAS5. Cell viability was evaluated by CCK-8 and flow cytometry. Results: We showed that miR-23a could directly suppress ATG3 expression in 293T cells, which suggested that ATG3 was identified as a target of miR-23a. MiR-23a mimics could restrain LC3 II, Beclin1 levles and ATG5-ATG12 complex formation. Meanwhile, miR-23a also increased the expression of mTOR and p62. Notably, there was a putative miR-23a-binding site in GAS5. MiR-23a overexpression might suppress the GAS5 expression, but the repressive effect was abolished by mutation of binding sites. Importantly, overexpression of GAS5 could inhibit the mature miR-23a and has no effect on miR-23a precursors. Knockdown of GAS5 suppressed the expression of LC3 II, ATG3 and ATG5-ATG12 complex formation, whereas p62 and mTOR levels were promoted. The further results showed that miR-23a overexpression and GAS5 inhibition both significantly suppressed cell viability and promoted the apoptosis rate following LPS stimulation, and knockdown of miR-23a exhibited the opposite effects. Conclusions: Our study revealed that down-regulation GAS5 attenuated cell viability and inhibited autophagy through ATG3-dependent autophagy by regulating miR-23a expression. The results suggested that GAS5/miR-23a/ATG3 axis might be a novel regulatory network contributing to a better understanding of regulation on autophagy program and cell viability.

scholarly journals Hypertrophic adipocyte-derived exosomal miR-802-5p contributes to insulin resistance in cardiac myocytes through targeting HSP60

2020 ◽  
Author(s):  
Zhongyuan Wen ◽  
Junfeng Li ◽  
Yalin Fu ◽  
Yuyang Zheng ◽  
Mingke Ma ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Heat Shock Protein 60 ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Reporter Assays ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Epicardial adipose tissue (EAT) is implicated in insulin resistance, which has been recognized as a strongest predictor of the development of diabetic cardiomyopathy and subsequent heart failure. However, the underlying mechanism remains incompletely understood. Herein, we investigated the effect of hypertrophic adipocytes on cardiac insulin resistance. We found that hypertrophic adipocyte-derived exosomes (h-Exo) induced insulin resistance in NRVMs. Furthermore, h-Exo high-expressed miR-802-5p. Insulin sensitivity of NRVMs was impaired by miR-802-5p mimic but improved by its inhibitor. TargetScan and luciferase reporter assays revealed that heat shock protein 60 (HSP60) was a direct target of miR-802-5p. Both h-Exo and miR-802-5p mimic could downregulate HSP60 protein levels. In addition, HSP60 silencing induced insulin resistance and mitigated the insulin-sensitizing effects of adiponectin. HSP60 depletion also significantly increased the expression levels of CHOP, a marker of the unfolded protein response (UPR), and enhanced oxidative stress, accompanied by the increased phosphorylation of JNK and IRS-1 Ser307. Inhibition of both miR-802-5p and endocytosis abolished the impacts of HSP60 knockdown on the UPR and oxidative stress. In summary, hypertrophic adipocyte-derived exosomal miR-802-5p caused cardiac insulin resistance in NRVMs through downregulating HSP60. These findings provide a novel mechanism by which EAT impairs cardiac function.

scholarly journals Administration of simvastatin halts progression of cirrhosis via up-regulating expression of miR-34a and interleukin-10 in rats

2021 ◽  
Author(s):  
Hui Yang ◽  
Xiaorong Zhou ◽  
Yonghua Wang ◽  
Yan Cheng ◽  
Zhao [email protected] ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Computational Analysis ◽  
Mrna Level ◽  
Underlying Mechanism ◽  
Interleukin 10 ◽  
Luciferase Assay ◽  
Protein Levels ◽  
Treatment Dose ◽  
Active Caspase ◽  
Complementary Binding

IntroductionSimvastatin (SIM) treatment has been found to be able to reduce the expression of miR-34a, and we found that interleukin-10 (IL-10) is a potential target gene of miR-34a by searching the online microRNA (miRNA) database. Furthermore, it has been shown that IL10 up-regulation could halt the progression of cirrhosis. The objective of this study was to explore the underlying mechanism of Simvastatin/miR-34a/IL-10 involved in HBV associated cirrhosis.Material and methodsReal-time PCR, western-blot analysis, immunohistochemistry, computational analysis, luciferase assay was carried out to explore the underlying mechanism of miR-34a involved in HBV associated cirrhosis.ResultsSIM treatment dose-dependently decreased the levels of miR-34a while increasing the levels of IL-10 mRNA and protein. Levels of IL-10 mRNA and protein were remarkably decreased, while miR-34a mRNA level and active caspase-3 protein level was apparently increased in Cirrhosis group compared with sham group. Accordingly, SIM treatment obstructed the dysregulated miR-34a expression and IL-10 expression in cirrhosis animals. By performing computational analysis, we identified that a complementary binding site of miR-34a was located in IL-10 3’ untranslated region (3’UTR), and miR-34a reduced luciferase activity of wild-type IL-10 3’UTR.ConclusionsOur data also suggested that SIM may become a new therapeutic strategy for HBV-associated cirrhosis via targeting the miR-34a/IL-10 axis.

scholarly journals MicroRNA-224 Promotes Tumorigenesis through Downregulation of Caspase-9 in Triple-Negative Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Li Zhang ◽  
Xin Zhang ◽  
Xin Wang ◽  
Miao He ◽  
Shixing Qiao
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Breast Cancer Cell Lines ◽  
Protein Levels ◽  
Reporter Assays ◽  
Novel Therapeutic Strategies

Triple-negative breast cancer (TNBC) harbors genetic heterogeneity and generally has more aggressive clinical outcomes. As such, there is urgency in identifying new prognostic targets and developing novel therapeutic strategies. In this study, miR-224 was overexpressed in breast cancer cell lines and TNBC primary cancer samples. Knockdown of miR-224 in MDA-MB-231 cancer cells reduced cell proliferation, migration, and invasion. Through integrating in silico prediction algorithms with KEGG pathway and Gene Ontology analyses, CASP9 was identified to be a potential target of miR-224. miR-224 knockdown significantly increased CASP9 transcript and protein levels. Furthermore, luciferase reporter assays confirmed a direct interaction of miR-224 with CASP9. Our findings have demonstrated that the miR-224/CASP9 axis plays an important role in TNBC progression, providing evidence in support of a promising therapeutic strategy for this disease.

scholarly journals Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by downregulating Cyp19A1 expression

2019 ◽  
Vol 316 (6) ◽  
pp. E1071-E1080 ◽  
Author(s):  
Hyeonhae Choi ◽  
Ki-Young Ryu ◽  
Jaesook Roh
Keyword(s):  
Luciferase Reporter ◽  
Luteal Cell ◽  
Rapid Decline ◽  
Binding Motif ◽  
Steroidogenic Factor 1 ◽  
Estrogen Production ◽  
Reporter Assays ◽  
Direct Binding ◽  
Serum Gonadotropin ◽  
Specificity Protein

The transition from granulosa cell (GC) to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 ( Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone (LH)/human chorionic gonadotropin treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore, we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory GCs obtained from pregnant mare serum gonadotropin-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG- Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and chromatin immunoprecipitation assays. The results revealed that the steroidogenic factor-1 (SF1)-binding motif, but not the specificity protein 1 (Sp1) binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs and that it has an essential role in the luteal transition in steroidogenesis.

scholarly journals EIF4A3-induced circular RNA MMP9 (circMMP9) acts as a sponge of miR-124 and promotes glioblastoma multiforme cell tumorigenesis

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Renjie Wang ◽  
Sai Zhang ◽  
Xuyi Chen ◽  
Nan Li ◽  
Jianwei Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Glioblastoma Multiforme ◽  
Aurora Kinase ◽  
Underlying Mechanism ◽  
Circular Rna ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Reporter Assays

Abstract Background Circular RNAs (circRNAs) have been found to play critical roles in the development and progression of various cancers. However, little is known about the effects of the circular RNA network on glioblastoma multiforme (GBM). Methods A microarray was used to screen circRNA expression in GBM. Quantitative real-time PCR was used to detect the expression of circMMP9. GBM cells were transfected with a circMMP9 overexpression vector or siRNA, and cell proliferation, migration and invasion, as well as tumorigenesis in nude mice, were assessed to examine the effect of circMMP9 in GBM. Biotin-coupled miRNA capture, fluorescence in situ hybridization and luciferase reporter assays were conducted to confirm the relationship between circMMP9 and miR-124. Results In this study, we screened differentially expressed circRNAs and identified circMMP9 in GBM. We found that circMMP9 acted as an oncogene, was upregulated in GBM and promoted the proliferation, migration and invasion abilities of GBM cells. Next, we verified that circMMP9 served as a sponge that directly targeted miR-124; circMMP9 accelerated GBM cell proliferation, migration and invasion by targeting miR-124. Furthermore, we found that cyclin-dependent kinase 4 (CDK4) and aurora kinase A (AURKA) were involved in circMMP9/miR-124 axis-induced GBM tumorigenesis. Finally, we found that eukaryotic initiation factor 4A3 (eIF4A3), which binds to the MMP9 mRNA transcript, induced circMMP9 cyclization and increased circMMP9 expression in GBM. Conclusions Our findings indicate that eIF4A3-induced circMMP9 is an important underlying mechanism in GBM cell proliferation, invasion and metastasis through modulation of the miR-124 signaling pathway, which could provide pivotal potential therapeutic targets for the treatment of GBM. Graphical abstract

scholarly journals Modulation of FoxO1 Expression by miR-21 to Promote Growth of Pancreatic Ductal Adenocarcinoma

2015 ◽  
Vol 35 (1) ◽  
pp. 184-190 ◽  
Author(s):  
Weifeng Song ◽  
Qi Li ◽  
Lei Wang ◽  
Liwei Wang
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Underlying Mechanism ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Primary Tumors ◽  
Protein Levels ◽  
Infusion System

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal primary tumors in humans, with undetermined tumorigenesis. Although previous work by us, and by others, has clearly demonstrated an involvement of miR-21 in the growth of PDAC, the underlying mechanism has not been clarified. Methods: Here we analyzed the regulation of FoxO1 by miR-21 in vitro and in vivo, using luciferase-reporter assay and pancreatic intraductal infusion of antisense of miR-21, respectively. Results: We found that overexpression of miR-21 in PDAC cells decreased FoxO1 protein levels, whereas inhibition of miR-21 increased FoxO1 levels. Further, miR-21 bound to FoxO1 mRNA to prevent its translation through its 3'UTR. Moreover, administration of antisense of miR-21 through an intraductal infusion system significantly decreased miR-21 levels and increased FoxO1 levels in implanted PDAC, resulting in a significant decrease in PDAC growth. Conclusion: Taken together, our data highlight miR-21/FoxO1 axis as a novel therapeutic target for inhibiting the growth of PDAC.

Mechanism of cardiomyocyte PGC-1α gene regulation by ERRα

2013 ◽  
Vol 91 (3) ◽  
pp. 148-154 ◽  
Author(s):  
Angela Ramjiawan ◽  
Rushita A. Bagchi ◽  
Laura Albak ◽  
Michael P. Czubryt
Keyword(s):  
Cardiac Metabolism ◽  
Luciferase Reporter ◽  
Acid Oxidation ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Ppar Γ ◽  
Impaired Metabolism ◽  
Repressive Effect ◽  
Evolutionarily Conserved ◽  
Reporter Assays

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) regulates critical genes involved in cardiac mitochondrial biogenesis and fatty acid oxidation, and its loss is associated with impaired metabolism and various cardiac pathologies. Estrogen-related receptor α (ERRα) targets many of the same genes as PGC-1α, and extensive cross talk exists between these 2 regulators. Here we report the identification of an evolutionarily conserved ERRα binding site within the PGC-1α promoter. Using luciferase reporter assays and overexpression, inhibition, or knockdown of ERRα, we show that PGC-1α expression is critically dependent upon ERRα in primary cardiomyocytes. We demonstrate that short-term hypoxia results in reduced ERRα mRNA expression, which precedes a similar loss of PGC-1α mRNA. However, chromatin immunoprecipitation reveals that despite a key role for ERRα in regulating PGC-1α in normoxic cardiomyocytes, ERRα loss is not responsible for PGC-1α loss in hypoxia. Histone deacetylase 5 (HDAC5) has previously been demonstrated to strongly inhibit expression of PGC-1α, and we show that overexpression of ERRα is sufficient to overcome this repressive effect. Our data elucidates the mechanism by which ERRα regulates cardiac PGC-1α gene expression, and suggests that ERRα may provide a means to normalize PGC-1α expression that could be useful in the development of strategies aimed at improving cardiac metabolism in disease.

scholarly journals miR-135b Plays a Neuroprotective Role by Targeting GSK3β in MPP+-Intoxicated SH-SY5Y Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Jianlei Zhang ◽  
Wei Liu ◽  
Yabo Wang ◽  
Shengnan Zhao ◽  
Na Chang
Keyword(s):  
Cell Proliferation ◽  
Glycogen Synthase ◽  
Underlying Mechanism ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Protective Effects ◽  
Inflammatory Cytokine Production ◽  
Protein Levels

miR-135a-5p was reported to play a crucial role in the protective effects of hydrogen sulfide against Parkinson’s disease (PD) by targeting rho-associated protein kinase 2 (ROCK2). However, the role of another member of miR-135 family (miR-135b) and the underlying mechanism in PD are still unclear. qRT-PCR and western blot showed that miR-135 was downregulated and glycogen synthase kinase 3β (GSK3β) was upregulated at mRNA and protein levels in MPP+-intoxicated SH-SY5Y cells in a dose- and time-dependent manner. MTT, TUNEL, and ELISA assays revealed that miR-135b overexpression significantly promoted cell proliferation and inhibited apoptosis and production of TNF-α and IL-1β in SH-SY5Y cells in the presence of MPP+. Luciferase reporter assay demonstrated that GSK3β was a direct target of miR-135b. Moreover, sodium nitroprusside (SNP), a GSK3β activator, dramatically reversed the effects of miR-135b upregulation on cell proliferation, apoptosis, and inflammatory cytokine production in MPP+-intoxicated SH-SY5Y cells. Taken together, miR-135b exerts a protective role via promotion of proliferation and suppression of apoptosis and neuroinflammation by targeting GSK3β in MPP+-intoxicated SH-SY5Y cells, providing a potential therapeutic target for the treatment of PD.

scholarly journals LncRNA HOXA11-AS Aggravates The Keloid Formation By Targeting miR-148b-3p/IGFBP5 Axis

2021 ◽  
Author(s):  
Juan Wang ◽  
Jing Shen
Keyword(s):  
Cell Proliferation ◽  
Caspase 3 ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Western Blots ◽  
Rna Immunoprecipitation ◽  
Keloid Formation ◽  
Reporter Assays ◽  
Migration Assays ◽  
Keloid Fibroblasts

Abstract Background: LncRNA homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism is not fully understood.Methods: HOXA11-AS, miR-148b-3p and IGFBP5 expression were detected by RT-qPCR or western blots. CCK8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p HOXA11-AS and IGFBP5 was confirmed by luciferase reporter assays or RNA immunoprecipitation.Results: The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis.Conclusion: These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid.

scholarly journals Down-Regulation of miR-212-5p Facilitates Homocysteine-Induced Hepatocytes Apoptosis Via Targeting PSMD10

2020 ◽  
Author(s):  
Huiping Zhang ◽  
Kun Xiao ◽  
Shengchao Ma ◽  
Long Xu ◽  
Ning Ding ◽  
...  
Keyword(s):  
Liver Injury ◽  
Er Stress ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Over Expression ◽  
Direct Binding ◽  
Induced Apoptosis ◽  
Insight Into

Abstract Background: Increasing evidences supported that elevated homocysteine (Hcy) levels contribute to cell apoptosis is implicated in the pathogenesis of liver injury, it correlates with liver disease severity. However, the underlying mechanism of apoptosis in Hcy-mediated liver injury remains obscure. Results: In this study, we found that homocysteine increases ER stress-mediated apoptosis and aggravates liver injury through up-regulation of PSMD10 expression in cbs+/- mice mice fed with high methionine diet and hepatocytes treated with homocysteine in vitro. Knockdown of PSMD10 expression remarkably reduced ER stress or apoptosis-associated protein in hepatocytes exposed to homocysteine. Moreover, bioinformatics analysis revealed that PSMD10 is a potential target gene of miR-212-5p, and luciferase reporter assay also confirmed that miR-212-5p negatively regulated PSMD10 expression by direct binding to its 3’-UTR regions. Subsequently, over-expression of miR-212-5p inhibited ER stress-mediated hepatocytes apoptosis though targeting PSMD10, all of which were abrogated by knockdown of miR-212-5p expression. Further study showed that the interaction between PSMD10 and GRP78 accelerated ER stress-mediated hepatic apoptosis induced by homocysteine. Conclusion: Taken together, these results demonstrated that down-regulation of miR-212-5p facilitates homocysteine-induced hepatocytes apoptosis via targeting PSMD10, which provides novel insight into the mechanism of homocysteine induced apoptosis in liver injury.

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