Oxidative stress activates NORAD expression by H3K27ac and promotes oxaliplatin resistance in gastric cancer by enhancing autophagy flux via targeting the miR-433-3p

AbstractOxaliplatin resistance undermines its curative effects on cancer and usually leads to local recurrence. The oxidative stress induced DNA damage repair response is an important mechanism for inducing oxaliplatin resistance by activating autophagy. ELISA is used to detect target genes expression. TMT-based quantitative proteomic analysis was used to investigate the potential mechanisms involved in NORAD interactions based on GO analysis. Transwell assays and apoptosis flow cytometry were used for biological function analysis. CCK-8 was used to calculate IC50 and resistance index (RI) values. Dual-luciferase reporter gene assay, RIP and ChIP assays, and RNA pull-down were used to detect the interaction. Autophagy flux was evaluated using electron microscope and western blotting. Oxidative stress was enhanced by oxaliplatin; and oxaliplatin resistance gastric cancer cell showed lower oxidative stress. TMT labeling showed that NORAD may regulate autophagy flux. NORAD was highly expressed in oxaliplatin-resistant tissues. In vitro experiments indicate that NORAD knockdown decreases the RI (Resistance Index). Oxaliplatin induces oxidative stress and upregulates the expression of NORAD. SGC-7901 shows enhanced oxidative stress than oxaliplatin-resistant cells (SGC-7901-R). NORAD, activated by H3K27ac and CREBBP, enhanced the autophagy flux in SGC-7901-R to suppress the oxidative stress. NORAD binds to miR-433-3p and thereby stabilize the ATG5- ATG12 complex. Our findings illustrate that NORAD, activated by the oxidative stress, can positively regulate ATG5 and ATG12 and enhance the autophagy flux by sponging miR-433-3p. NORAD may be a potential biomarker for predicting oxaliplatin resistance and mediating oxidative stress, and provides therapeutic targets for reversing oxaliplatin resistance.

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PITPNA-AS1 Activated by H3K27ac Sponged miR-98-5p to Regulate Cisplatin Resistance in Gastric Cancer

10.21203/rs.3.rs-156344/v1 ◽

2021 ◽

Author(s):

Yaping Liu ◽

Xu Zhao ◽

Yinnan Chen ◽

Gang Guo ◽

Jiansheng Wang ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Platinum Resistance ◽

Luciferase Reporter Gene ◽

Gene Assay ◽

Proliferation And Apoptosis ◽

Gastric Cancer Cell Lines ◽

Cancer Tissues

Abstract To evaluate the expression of PITPNA-AS1 and miR-98-5p in gastric cancer tissues as well as their association with progression of gastric cancer, and investigate the role of PITPNA-AS1 and miR-98-5p in developing platinum resistance. RNA sequencing was used to identify candidate lncRNAs and microRNAs related to local recurrence of gastric cancer. qRT-PCR was used to investigate the expression of PITPNA-AS1 and miR-98-5p. CCK-8 and caspase3/7 activity were used to evaluate the cell proliferation and apoptosis rate. Dual luciferase reporter gene assay and RNA pull down were used to evaluate the cross talk between PITPNA-AS1 and miR-98-5p. PITPNA-AS1 and miR-98-5p could regulate cell proliferation and inhibit apoptosis in gastric cancer cell lines. Cisplatin and lobaplatin could significantly suppress the expression of PITPNA-AS1, which interacted with negatively regulated miR-98-5p expression. PITPNA-AS1 overexpression impaired the effect of platinum, which was partially reversed by downregulation of miR-98-5p knock down. In gastric cancer, PITPNA-AS1 and miR-98-5p could regulat cell growth, apoptosis and platinum resistance. They have the potential to be biomarkers and curative therapeutic targets. However, further research on molecular mechanisms are needed.

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Abstract 13141: Cellular Microrna 449b Upregulation Sensitizes Ischemia/reperfusion Injury via Inhibition of Nrf-1, Increasing Ros Production and Apoptosis in the Diabetic Heart

Circulation ◽

10.1161/circ.132.suppl_3.13141 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Hang Wang ◽

Wayne Lau ◽

Erhe Gao ◽

Walter Koch ◽

Xin Ma ◽

...

Keyword(s):

Oxidative Stress ◽

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Experimental Studies ◽

Diabetic Heart ◽

Luciferase Reporter ◽

Proteomics Data ◽

Luciferase Reporter Gene ◽

Gene Assay

Myocardial ischemic/reperfusion (MI/R) injury is significantly enhanced in diabetes by incompletely understood mechanisms. Recent clinical and experimental studies demonstrate that hypoadiponectinemia during diabetes enhances oxidative stress and exaggerates MI/R injury. However, molecular mechanisms responsible for hypoadiponectinemia-induced oxidative stress remain unknown. In a discovery-driven fashion, we determined the role of cardiac microRNAs in the MI/R response in adiponectin knockout (APNKO) mice. From 68 total miRNAs differentially expressed between APNKO and wild type (WT) mice, miRNA 449b was identified as the microRNA most relevant to oxidative stress and apoptosis. In cultured neonatal cardiomyocytes, miRNA 449b silencing inhibited hypoxia/reoxygenation-induced apoptosis, whereas miR-449b overexpression significantly increased oxidative stress and cardiomyocyte apoptosis. In APNKO mice, administration of anti-miR-449b decreased oxidative stress (-17.2±3.8%, p<0.05), reduced caspase-3 activity (-21.3±4.2%, p<0.05), attenuated myocardial apoptosis (-16.3±4.1%, p<0.05), and improved myocardial function (1.4±0.3 fold). To identify the downstream molecule regulated by miRNA 449b, we integrated transcriptomics and proteomics data with computational annotation data, and identified Nrf-1 as a miRNA 449b target. A luciferase reporter gene assay demonstrated that miRNA 449b inhibited Nrf-1 expression via Nrf-1 mRNA 3’UTR region binding. Finally, we demonstrated that miRNA 449b was significantly upregulated, Nrf-1 expression was significantly decreased, and the anti-oxidative molecule metallothionein (MT) was significantly inhibited in the diabetic heart subjected to MI/R. Administration of anti-miR-449b in diabetic animals upregulated Nrf-1 and MT expression, reduced oxidative stress, and improved cardiac function (P<0.01) after MI/R. Taken together, this study provides the first evidence that hypoadiponectinemia during diabetes causes cardiac miRNA-449b upregulation and subsequent downregulation of Nrf-1 and MT, thus enhancing oxidative stress and MI/R injury. MicroRNA 449b may represent a potential therapeutic target against diabetic heart disease.

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The lncRNA-MALAT1/miR-126-5p Axis Promotes Growth and Metastasis of Gastric Cancer through Regulation of VEGFA

10.21203/rs.3.rs-32205/v1 ◽

2020 ◽

Author(s):

Zhi-Li Hu ◽

Yang-zhi Hu ◽

Qing Li ◽

Tian-you Liao ◽

Hai-ping Jiang

Keyword(s):

Gastric Cancer ◽

Signaling Pathway ◽

Luciferase Reporter ◽

Mtor Signaling Pathway ◽

Luciferase Reporter Gene ◽

Ags Cells ◽

Luciferase Reporter Gene Assay ◽

Lncrna Malat1 ◽

Gene Assay ◽

Cancer Tissues

Abstract Background: It has been reported that reduction of miR-126 can promote the progression of gastric cancer (GC). However, the regulation of miR-126 in GC is still unclear. This study aims to explore the correlation between lncRNA MALAT1 and miR-126 in gastric cancer and disclose the underlying mechanisms.Methods: We analyzed the correlation of MALAT1 levels and clinical features by analysis of bioinformatic data and human samples. Then we down-regulate the expression of MALAT1 in AGS cells and examined the characteristics of cell proliferation, cycle, apoptosis, migration, invasion, and the effect on miR-126 as well as VEGFA and signaling pathway. In addition, we demonstrated the role of MALAT1/miR-126 axis in GC with dual-luciferase reporter gene assay and treatment of miR-126 inhibitor.Results: The expression of MALAT1 was higher in cancer tissues than para-cancer tissues. In addition, high MALAT1 level suggested greater malignancy and poorer prognosis. Down-regulating the expression of MALAT1 in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA, which is consistent with up-regulation of miR-126. According to dual-luciferase reporter gene assay and treatment of miR-126 inhibitor, we demonstrated that MALAT1 down-regulated miR-126 in GC, which leads to the up-regulation of VEGFA and activation of mTOR signaling pathway.Conclusions: MALAT1/miR-126 axis promotes growth and metastasis of gastric cancer through regulation of VEGFA via mTOR signaling pathway.Fund This article is supported by Science and Technology Funding Project of Hunan Province, China (No.2017SK4010)

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MicroRNA-448 targets SATB1 to reverse the cisplatin resistance in lung cancer via mediating Wnt/β-catenin signalling pathway

The Journal of Biochemistry ◽

10.1093/jb/mvaa024 ◽

2020 ◽

Vol 168 (1) ◽

pp. 41-51

Author(s):

Mei-Ying Ning ◽

Zhao-Lin Cheng ◽

Jing Zhao

Keyword(s):

Lung Cancer ◽

Target Gene ◽

A549 Cells ◽

Resistance Index ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Lung Cancer Patients ◽

Qrt Pcr ◽

Gene Assay

Abstract This study aims to examine whether miR-448 reverses the cisplatin (DDP) resistance in lung cancer by modulating SATB1. QRT-PCR and immunohistochemistry were used to examine the miR-448 and SATB1 expressions in DDP-sensitive and -resistant lung cancer patients. A microarray was used to investigate the cytoplasmic/nucleic ratio (C/N ratios) of genes in A549 cells targeted by miR-448, followed by Dual-luciferase reporter gene assay. A549/DDP cells were transfected with miR-448 mimics/inhibitors with or without SATB1 siRNA followed by MTT assay, Edu staining, flow cytometry, qRT-PCR and western blotting. MiR-448 was lower but SATB1 was increased in DDP-resistant patients and A549/DDP cells. And the patients showed low miR-448 expression or SATB1 positive expression had poor prognosis. SATB1, as a target gene with higher C/N ratios (>1), was found negatively regulated by miR-448. Besides, miR-448 inhibitors increased resistance index of A549/DDP cells, promoted cell proliferation, increased cell distribution in S phrase, declined cell apoptosis and activated Wnt/β-catenin pathway. However, SATB1 siRNA could reverse the above effect caused by miR-448 inhibitors. MiR-448 targeting SATB1 to counteract the DDP resistance of lung cancer cells via Wnt/β-catenin pathway.

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Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00402.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. H1133-H1140 ◽

Cited By ~ 106

Author(s):

Zoltan Ungvari ◽

Lora Bailey-Downs ◽

Tripti Gautam ◽

Rosario Jimenez ◽

Gyorgy Losonczy ◽

...

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

High Glucose ◽

Target Genes ◽

Cell Model ◽

Luciferase Reporter ◽

Standard Diet ◽

Related Factor ◽

Antioxidant Genes ◽

Gene Assay

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10–30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H2O2 mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2+/+ and Nrf2−/− mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2+/+ mice, but not Nrf2−/− mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2−/− than Nrf2+/+ mice. HFD-induced endothelial dysfunction was more severe in Nrf2−/− mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2+/+ mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.

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Comparative oxidative stress elicited by nanosilver in stable HSPA1A promoter-driven luciferase reporter HepG2 and A549 cells

Toxicology Research ◽

10.1039/c6tx00195e ◽

2016 ◽

Vol 5 (5) ◽

pp. 1298-1305 ◽

Cited By ~ 3

Author(s):

Lili Xin ◽

Jianshu Wang ◽

Guoqiang Fan ◽

Bizhong Che ◽

Kaiming Cheng ◽

...

Keyword(s):

Oxidative Stress ◽

Reporter Gene ◽

A549 Cells ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Gene Assay

HSPA1A promoter-driven luciferase reporter gene assay provides a novel tool for predictive screening of the oxidative stress elicited by nanosilver.

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Increased mitochondrial H2O2 production promotes endothelial NF-κB activation in aged rat arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01346.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. H37-H47 ◽

Cited By ~ 213

Author(s):

Zoltan Ungvari ◽

Zsuzsanna Orosz ◽

Nazar Labinskyy ◽

Aracelie Rivera ◽

Zhao Xiangmin ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Endothelial Cells ◽

Vascular System ◽

Vascular Inflammation ◽

Luciferase Reporter ◽

H2o2 Production ◽

Luciferase Reporter Gene ◽

Mitochondrial Oxidative Stress ◽

Gene Assay

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-κB activity (assessed by luciferase reporter gene assay and NF-κB binding assay), which was attenuated by scavenging H2O2. Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-κB. In endothelial cells of aged vessels there was an increased production of H2O2 (assessed by 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H2O2 generation, which was associated with activation of NF-κB. In aged vessels inhibition of NF-κB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-κB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H2O2 production and/or directly inhibiting NF-κB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.

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ADSC Exosomes Mediate lncRNA-MIAT Alleviation of Endometrial Fibrosis by Regulating miR-150-5p

Frontiers in Genetics ◽

10.3389/fgene.2021.679643 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiaowen Shao ◽

Jinlong Qin ◽

Chendong Wan ◽

Jiajing Cheng ◽

Lian Wang ◽

...

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Target Genes ◽

Expression Level ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Injury Model ◽

Gene Assay ◽

Tgf Β1

BackgroundSecondary infertility remains a major complication of endometrial fibrosis in women. The use of exosomes from adipose-derived mesenchymal stem cells (ADSCs) has shown promising results for the treatment of endometrial fibrosis. However, the mechanisms of action of ADSC-exosome (ADSC-Exo) therapy remain unclear.Materials and MethodsAn endometrial fibrosis model was established in mice treated with alcohol and endometrial epithelial cells (ESCs) treated with TGF-β1. ADSCs were isolated from Sprague Dawley (SD) rats, and exosomes were isolated from ADSCs using ExoQuick reagent. Exosomes were identified by transmission electron microscopy (TEM), NanoSight, and Western blot analysis. The expression level of lncRNA-MIAT was detected by qPCR analysis. Western blot analysis was carried out to determine the protein levels of fibrosis markers (TGFβR1, α-SMA, and CK19). A dual-luciferase reporter gene assay was used to verify the relationship between target genes. The endometrial tissues of the endometrial fibrosis model were stained with HE and Masson’s trichrome.ResultsADSCs and ADSC-Exos were successfully isolated, and the expression level of lncRNA-MIAT was significantly down-regulated in endometrial tissue and the TGF-β1-induced ESC injury model, whereas ADSC-Exos increased the expression of lncRNA-MIAT in the TGF-β1-induced ESC model. Functionally, ADSC-Exo treatment repressed endometrial fibrosis in vivo and in vitro by decreasing the expression of hepatic fibrosis markers (α-SMA and TGFβR1) and increasing the expression of CK19. Moreover, miR-150-5p expression was repressed by lncRNA-MIAT in the TGF-β1-induced ESC injury model. The miR-150-5p mimic promoted TGF-β1-induced ESC fibrosis.ConclusionADSC-Exos mediate lncRNA-MIAT alleviation of endometrial fibrosis by regulating miR-150-5p, which suggests that lncRNA-MIAT from ADSC-Exos may be a viable treatment for endometrial fibrosis.

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Screening and validation of differentially expressed microRNAs and target genes in hypertensive mice induced by cytomegalovirus infection

Bioscience Reports ◽

10.1042/bsr20202387 ◽

2020 ◽

Vol 40 (12) ◽

Cited By ~ 1

Author(s):

YunZhong Shi ◽

DongMei Xi ◽

XiaoNi Zhang ◽

Zhen Huang ◽

Na Tang ◽

...

Keyword(s):

Target Genes ◽

Reporter Gene Assay ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Mcmv Infection ◽

Luciferase Reporter Gene Assay ◽

Qrt Pcr ◽

Gene Assay ◽

Differentially Expressed Mirnas

Abstract Introduction: Multiple studies have suggested an association between cytomegalovirus (CMV) infection and essential hypertension (EH). MicroRNAs (miRNAs) play a critical role in the development of EH by regulating the expression of specific target genes. However, little is known about the role of miRNAs in CMV-induced EH. In the present study, we compared the miRNA expression profiles of samples from normal and murine cytomegalovirus (MCMV)-infected C57BL/6 mice using high-throughput sequencing analysis. Methods: We collected the thoracic aorta, heart tissues, and peripheral blood from 20 normal mice and 20 MCMV-infected mice. We identified differentially expressed miRNAs in the peripheral blood samples and predicted their target genes using bioinformatics tools. We then experimentally validated them using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the target genes with double luciferase reporter gene assay. Results: We found 118 differentially expressed miRNAs, among which 9 miRNAs were identified as potential MCMV infection-induced hypertension regulators. We then validated the expression of two candidate miRNAs, mmu-miR-1929-3p and mcmv-miR-m01-4-5p, using qRT-PCR. Furthermore, the dual-luciferase reporter gene assay revealed that the 3′-untranslated region (UTR) of endothelin A receptor (Ednra) messenger RNA (mRNA) contained a binding site for mmu-miR-1929-3p. Collectively, our data suggest that MCMV infection can raise the blood pressure and reduce mmu-miR-1929-3p expression in C57BL/6 mice. Moreover, we found that mmu-miR-1929-3p targets the 3′-UTR of the Ednra mRNA. Conclusion: This novel regulatory axis could aid the development of new approaches for the clinical prevention and control of EH.

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Targeting ZFP64/GAL-1 axis promotes therapeutic effect of nab-paclitaxel and reverses immunosuppressive microenvironment in gastric cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02224-x ◽

2022 ◽

Vol 41 (1) ◽

Author(s):

Mengxuan Zhu ◽

Pengfei Zhang ◽

Shan Yu ◽

Cheng Tang ◽

Yan Wang ◽

...

Keyword(s):

Gastric Cancer ◽

Zinc Finger Protein ◽

Tumor Formation ◽

Humanized Mice ◽

Luciferase Reporter ◽

Tunel Staining ◽

Luciferase Reporter Gene ◽

Paclitaxel Resistance ◽

Gene Assay ◽

Immunosuppressive Microenvironment

Abstract Background Chemoresistance is a main obstacle in gastric cancer (GC) treatment, but its molecular mechanism still needs to be elucidated. Here, we aim to reveal the underlying mechanisms of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) resistance in GC. Methods We performed RNA sequencing (RNA-seq) on samples from patients who were resistant or sensitive to nab-paclitaxel, and identified Zinc Finger Protein 64 (ZFP64) as critical for nab-paclitaxel resistance in GC. CCK8, flow cytometry, TUNEL staining, sphere formation assays were performed to investigate the effects of ZFP64 in vitro, while subcutaneous tumor formation models were established in nude mice or humanized mice to evaluate the biological roles of ZFP64 in vivo. Chromatin immunoprecipitation sequencing (CHIP-seq) and double-luciferase reporter gene assay were conducted to reveal the underlying mechanism of ZFP64. Results ZFP64 overexpression was linked with aggressive phenotypes, nab-paclitaxel resistance and served as an independent prognostic factor in GC. As a transcription factor, ZFP64 directly binds to Galectin-1 (GAL-1) promoter and promoted GAL-1 transcription, thus inducing stem-cell like phenotypes and immunosuppressive microenvironment in GC. Importantly, compared to treatment with nab-paclitaxel alone, nab-paclitaxel plus GAL-1 blockade significantly enhanced the anti-tumor effect in mouse models, particularly in humanized mice. Conclusions Our data support a pivotal role for ZFP64 in GC progression by simultaneously promoting cellular chemotherapy resistance and tumor immunosuppression. Treatment with the combination of nab-paclitaxel and a GAL-1 inhibitor might benefit a subgroup of GC patients.

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