microRNA-422a Inhibits DCC Expression in a Manner Dependent on SNP rs12607853

2020 ◽  
Vol 160 (2) ◽  
pp. 63-71
Author(s):  
Yunxiao Li ◽  
Xugang Shi ◽  
Xintong Cai ◽  
Yongsheng Zhu ◽  
Yuanyuan Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Opioid Addiction ◽  
Heroin Addiction ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay ◽  
New Biomarkers ◽  
And Function

DCC netrin 1 receptor (DCC) affects the structure and function of the dopamine circuitry, which in turn affects the susceptibility to developing addiction. In a previous study, we found that single nucleotide polymorphism (SNP) rs12607853 in the 3′ untranslated region (3′-UTR) of DCC was significantly associated with heroin addiction. In the current study, we first used bioinformatics prediction to identify the DCC rs12607853 C allele as a potential hsa-miR-422a and hsa-miR-378c target site. We then used vector construction and dual-luciferase reporter assays to investigate the targeting relationship of DCC rs12607853 with hsa-miR-422a and hsa-miR-378c. The dual-luciferase reporter gene assay confirmed that the C allele of rs12607853 in combination with hsa-miR-422a led to repressed dual-luciferase gene expression. Moreover, gene expression assays disclosed that hsa-miR-422a inhibited DCC expression at both the mRNA and protein levels. We also found that morphine inhibited the expression of hsa-miR-422a but increased the expression of DCC mRNA, and this change in the expression of hsa-miR-422a could not be reversed by naloxone, which suggested that the role of DCC in opioid addiction might be regulated by hsa-miR-422a. In summary, this study improves our understanding of the role of hsa-miR-422a and identifies the genetic basis of rs12607853, which might contribute to the discovery of new biomarkers or therapeutic targets for opioid addiction.

Bone Marrow Stromal Cells (BMSCs) Inhibit Retinal Ganglion Cell Apoptosis and Alleviate Inflammation Through Up-Regulation of MicroRNA-43 and Brain-Derived Neurotrophic Factor in Glaucoma

2021 ◽  
Vol 11 (12) ◽  
pp. 2478-2483
Author(s):  
Xiang Ji ◽  
Kai-Wen Zhou
Keyword(s):  
Neurotrophic Factor ◽  
Vision Loss ◽  
Luciferase Reporter ◽  
Retinal Ganglion ◽  
Luciferase Reporter Gene ◽  
Down Regulation ◽  
Related Factors ◽  
Gene Assay

Glaucoma is a leading cause of vision loss mainly due to retinal ganglion cells (RGC) loss. MicroRNAs (miRNAs) are highlighted as potential biomarkers in diseases. This study aims to investigate the role of miR-43 and BMSCs in the RGC apoptosis and glaucoma.RGCs were transfected with miR-43 inhibitors and mimics, and then co-cultured with BMSCs. RT-qPCR analysis was conducted to determine miR-43 expression, whilst Western blot, and flow cytometry were carried out to assess the role of miR-43 in apoptosis and inflammation. The interaction between miR-43 and BDNF, a neurotrophic factor, was detected by dual-luciferase reporter gene assay. Overexpression of miR-43 promoted RGC proliferation and decreased apoptosis. Furthermore, miR-43 overexpression diminished the contents of apoptosis- and inflammatory-related factors, and elevated the expression of BDNF. Down-regulation of BDNF exerted similar effect as down-regulation of miR-43, enhancing apoptosis and aggravating inflammation. Importantly, BMSC treatment reversed the in vitro inhibitory effect of si-BDNF on RGC with enhancement of miR-43 expression. Mechanically, miR-43 was indicated to target BDNF in glaucoma. Collectively, miR-43 delivered by BMSCs plays an important role in the inflammatory injury and abnormal apoptosis of RGC by regulating the expression of BDNF. These findings might help development of new treatment for glaucoma and provide a promising biomarker for diagnosis and treatment.

scholarly journals miR-495-3p Depresses Cell Proliferation and Migration By Down-Regulating HMGB1 in Colorectal Cancer

2021 ◽  
Author(s):  
Zhang Jieling ◽  
Li Kai ◽  
Zheng Huifen ◽  
Zhu Yiping
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Migration ◽  
Cancer Tissues

Abstract Background: MicroRNAs play an important role in the genesis and progression of tumors, including colorectal cancer (CRC), which has a high morbidity and mortality rate. In this research, the role of miR-495-3p and HMGB1 in CRC was investigated.Methods: We performed qRT-PCR to detect the expression of miR-495-3p in colorectal cancer tissues and cell lines. Functional experiments such as CCK-8 assay, EDU assay, Transwell assay and apoptosis assay were conducted to explore the effects of miR-495-3p on the proliferation, migration and apoptosis of CRC cells in vitro. Then, the use of database prediction, dual-luciferase reporter gene assay and functional experiments verified the role of miR-495-3p target gene HMGB1 in CRC. Finally, rescue experiments was performed to investigate whether overexpression of HMGB1 could reverse the inhibitory effect of miR-495-3p on CRC cell proliferation in vivo and in vitro.Results: miR-495-3p was down-regulated in colorectal cancer tissues and cell lines, and could inhibit the proliferation and migration of colorectal cancer cells, and promote cell apoptosis. The database prediction and dual-luciferase reporter gene assay showed that HMGB1 was the downstream target gene of miR-495-3p. We finally demonstrated that miR-495-3p inhibited CRC cell proliferation by targeting HMGB1 in vitro and in vivo.Conclusion: Our research shows that miR-495-3p inhibits the progression of colorectal cancer by down-regulating the expression of HMGB1, which indicates that miR-495-3p may become a potential therapeutic target for colorectal cancer.

scholarly journals Circular RNA circANKS1B as the Sponge of miR-152-3p Promotes Prostate Cancer Progression by up-Regulating TGF-α Expression

2020 ◽  
Author(s):  
Liangjun Tao ◽  
Xinyuan Pan ◽  
Jiawei Wang ◽  
Li Zhang ◽  
Lingsong Tao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Progression ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Prostate Cancer Progression ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Function ◽  
The Impact

Abstract Background: Growing studies indicate that circRNAs play critical roles in human diseases, and show great potential as biomarkers and therapeutic targets. This study aims to investigate the expression and function of circANKS1B in prostate cancer (PC).Methods: The expression of circANKS1B and miRNA-152-3p were determined by real-time qRT-PCR. The cell migration and invasion were measured by transwell assay. The interaction between circANKS1B and miR-152-3p was confirmed by dual-luciferase reporter gene assay. Rescue experiments were conducted to demonstrate whether circANKS1B regulated the migration and invasion of PC cells by the circANKS1B-miR-152-3p-TGF-α pathway.Results: The expression of circANKS1B was dramatically up-regulated both in PC cells and tissues. Moreover, high circANKS1B expression was associated with a poor prognosis of PC patients. Dual-luciferase reporter assay indicated that circABKS1B directly bound to miRNA-152-3p. Furthermore, circANKS1B negatively regulated miR-152-3p expression. Knockdown of circANKS1B remarkably suppressed PC cells invasion and TGF-α expression, while the effects of circANKS1B silencing were reversed by miR-152-3p deficiency. In addition, the impact of miR-152-3p silencing on PC cell invasion was also abrogated by TGF-α deficiency. In all, circANKS1B as the sponge of miR-152-3p promotes prostate cancer progression by up-regulating TGF-α expression.Conclusion: Our findings reveal that circANKS1B could be a potential prognostic biomarker and therapeutic target of PC.

scholarly journals A novel peptide RIFV suppresses human adipocyte differentiation through the inhibition of C/EBP-β expression

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Wen Zhang ◽  
Dan Shen ◽  
Yun Li ◽  
Hong Zhong ◽  
Xing Wang ◽  
...  
Keyword(s):  
Adipocyte Differentiation ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Epidemic Disease ◽  
Global Epidemic ◽  
Protein Levels ◽  
Therapeutic Drugs ◽  
Peptide Database ◽  
Gene Assay

Abstract Background Obesity is a global epidemic disease that increases the risk of metabolic syndrome. However, therapeutic drugs for obesity are still scarce. In recent years, peptides have been identified as new biological regulators. RIFV (R-I-F-V-P-I-K-G-R-P-A-P), a novel active peptide from our peptide database. Methods We performed oil red O staining and triglyceride measurement to analyze the influence of RIFV on white preadipocytes differentiation. Then the effects of RIFV on cell proliferation, apoptosis and cell cycle were determined by using CCK-8 assay and flow cytometry. The mRNA and protein levels of adipogenesis-related genes were respectively detected by qRT-PCR and western blot. Rescue experiment was conducted to confirm whether RIFV could regulate adipocytes differentiation via targeting C/EBP-β. Finally, the luciferase reporter gene assay was performed to verify the regulation of RIFV on C/EBP-β gene. Results RIFV was revealed to inhibit the differentiation of human white adipocytes without affecting their proliferation. Additionally, RIFV could also suppress the differentiation of mouse primary white preadipocytes isolated from inguinal fat tissues. Furthermore, RIFV may have an inhibitory effect on adipogenesis by inhibiting the regulation of the adipogenic gene C/EBP-β. Conclusions Our results indicated that RIFV may be a novel essential regulator of adipocyte differentiation and represents a therapeutic strategy for obesity and related complications.

Bioengineered microRNA-1291 effects on pancreatic cancer cell proliferation and xenograft tumor growth.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 307-307
Author(s):  
Mei-Juan Tu ◽  
Zhijian Duan ◽  
Qianyu Zhang ◽  
Jing-Xin Qiu ◽  
Frank J Gonzalez ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Therapeutic Potential ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay

307 Background: MicroRNAs (miR) have proved to be vital regulators in the control of tumor progression. Our recent studies have revealed miR-1291 is downregulated in patient pancreatic cancer (PC) specimens and re-introduction of miR-1291 suppresses tumorigenesis of PC cells. We have developed a novel ncRNA bioengineering technology to produce a miR-1291 prodrug. In this study, we aimed to assess the effectiveness of this miR-1291 prodrug as a monotherapy, as well as in combination with chemotherapy, for treatment of PC. Methods: Sensitivity of PC cells to miR-1291 prodrug alone, gemcitabine plus nab-paclitaxel (Gem-nP) alone, and their combination was evaluated by CellTiter-Glo assay. Mature miR-1291 and ARID3B mRNA levels were determined by quantitative real-time PCR (q-PCR) assay. A luciferase reporter gene assay was used to validate interaction between miR-1291 and ARID3B 3’UTR. Target protein expression was examined by Western blot and immunofluorescence analyses. PANC-1 and PC patient-derived xenograft (PDX) mouse models were established and used to assess anti-tumor effects of miR-1291 monotherapy and combination therapy with Gem-nP. Results: Cytotoxicity assays showed that miR-1291 prodrug enhanced the sensitivity of PANC-1 and AsPC-1 cells to Gem-nP. Luciferase assays confirmed ARID3B as a target for miR-1291 as predicted by computational analysis. qPCR analysis demonstrated that miR-1291 prodrug was readily processed to mature miR-1291 and subsequently upregulated ARID3B mRNA levels. miR-1291 prodrug also elevated the protein levels of ARID3B. Co-administration of miR-1291 prodrug and Gem-nP increased caspase-3/7 and γH2AX levels in PC cells, compared to miR-1291 or Gem-nP treatment alone. In addition, systemic administration of in vivo-jet PEI formulated miR-1291 prodrug suppressed tumor growth in both a PANC-1 xenograft model and three PDX models, and largely enhanced the efficacy of Gem-nP. All treatments were well tolerated in mice in vivo. Conclusions: Our bioengineered miR-1291 prodrug has therapeutic potential as a monotherapy but also can act as a sensitizing agent to chemotherapy. This novel treatment approach should be further explored for PC.

scholarly journals MicroRNA-141 Targets Sirt1 and Inhibits Autophagy to Reduce HBV Replication

2017 ◽  
Vol 41 (1) ◽  
pp. 310-322 ◽  
Author(s):  
Ying Yang ◽  
Yanning Liu ◽  
Jihua Xue ◽  
Zhenggang Yang ◽  
Yu Shi ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
B Virus

Background/Aims: About 400 million individuals are chronically infected with hepatitis B virus, at high risk of developing liver cirrhosis and hepatocellular carcinoma. Recent studies have demonstrated an interaction between hepatitis B virus replication and autophagy activity of hepatocytes. In the present study, we aimed to investigate the role of miR-141 in regulating autophagy and hepatitis B virus replication. Methods: The expression of HBV-DNA, miR-141 and Sirt1 mRNA was determined by quantitative real-time PCR analysis. The expression of HBsAg and HBeAg was determined by ELISA. Western blotting was performed to detect protein expression. The LC3 puncta was determined by immunofluorescence. To test whether miR-141 directly regulate the expression level of Sirt1 mRNA, dual-luciferase reporter gene assay was performed. Results: In vitro studies showed that miR-141 mimic inhibited the autophagic response, hepatitis B virus and the expression of Sirt1 in hepatocytes. And transfection with miR-141 inhibitor enhanced autophagic response and Sirt1 expression. The autophagy induced by overexpression of Sirt1 was inhibited by miR-141 mimic. In addition, miR-141 mimic also decreased the expression of Sirt1 mRNA. Sirt1 was predicted as a potential miR-141 target by bioinformatic analysis of its 3'-UTR, and confirmed by luciferase reporter assays which analyzing the interaction of miR-141 with the wild- type or the mutated Sirt1 3’-UTR. Conclusion: We have therefore demonstrated a role of miR-141 in regulating autophagy-mediated hepatitis B virus inhibition by targeting Sirt1, and may provide potential targets for drug development.

scholarly journals Curcumin induces paraoxonase 1 in cultured hepatocytes in vitro but not in mouse liver in vivo

2010 ◽  
Vol 105 (2) ◽  
pp. 167-170 ◽  
Author(s):  
Charlotte Schrader ◽  
Christina Schiborr ◽  
Jan Frank ◽  
Gerald Rimbach
Keyword(s):  
Cultured Cells ◽  
Paraoxonase 1 ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Huh7 Cells ◽  
Gene Assay ◽  
B6c3f1 Mice

Paraoxonase 1 (PON1) is an enzyme that is mainly synthesised in the liver and protects LDL from oxidation, thereby exhibiting antiatherogenic properties. Using a luciferase reporter gene assay, we tested curcumin for its ability to induce PON1 in Huh7 hepatocytes in culture. Curcumin ( ≥ 10 μmol/l) dose-dependently induced PON1 transactivation in Huh7 cells. However, dietary supplementation of female B6C3F1 mice with curcumin (500 mg/kg diet) for 2 weeks did not increase the hepatic PON1 mRNA and protein levels. No curcumin was detectable in the plasma of the 12 h fasted mice. In conclusion, curcumin may be a potent PON1 inducer in cultured cells in vitro, but not in the liver of curcumin-fed mice because of its low concentrations in vivo.

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

2007 ◽  
Vol 293 (1) ◽  
pp. H37-H47 ◽  
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.

scholarly journals LncRNA DSCAM-AS1 Promotes Proliferation, Migration and Invasion of Colorectal Cancer Cells via Modulating miR-144-5p/CDKL1

2020 ◽  
Author(s):  
Jiaping Pei ◽  
Xiaozhao Deng
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Sw480 Cell ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Regulatory Relationship ◽  
Qrt Pcr ◽  
Gene Assay ◽  
And Function

Abstract Background LncRNA DSCAM-AS1 is oncogenic in several cancers. However, DSCAM-AS1 expression and function in colorectal cancer (CRC) remain far from being fully elucidated. Methods Paired CRC tissues/adjacent tissues were collected, and the expression levels of DSCAM-AS1, miR-144-5p and CDKL1 were examined by qRT-PCR; DSCAM-AS1 shRNA was transfected into HCT-116 and SW480 cell lines to establish cell models. The proliferation was detected through CCK-8 assay and plate colony formation assay. Transwell assay was used to evaluate the migration and invasion. QRT-PCR and western blot were adopted to analyze changes in miR-144-5p and CDKL1; luciferase reporter gene assay was performed to determine the regulatory relationship between miR-144-5p and DSCAM-AS1, miR-144-5p and CDKL1. Results DSCAM-AS1 was notably up-regulated in CRC samples, positively correlated with CDKL1 expression, while negatively correlated with miR-144-5p. After the transfection of DSCAM-AS1 shRNAs into cancer cells, the proliferative and metastatic ability of cancer cells were impeded. DSCAM-AS1 could reduce the expression level of miR-144-5p by binding with it. Additionally, CDKL1 was also validated as a target gene of miR-144-5p, and DSCAM-AS1 was proved to indirectly regulate CDKL1 expression. Conclusion DSCAM-AS1 was aberrantly up-regulated in CRC, and it can modulate the cells proliferative and metastatic ability. It has the ability to be the “ceRNA” to regulate CDKL1 expression via sponging miR-144-5p.

Abstract 289: Perm1 is a Novel Regulator of Cardiac Energetics and Function

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Shinichi Oka ◽  
Amira Sabry ◽  
Amanda Horiuchi ◽  
Keiko Cawley ◽  
Sean O'Very ◽  
...  
Keyword(s):  
Heart Failure ◽  
Complex I ◽  
Stress Test ◽  
Cardiac Metabolism ◽  
Luciferase Reporter ◽  
Pathway Enrichment Analysis ◽  
Luciferase Reporter Gene ◽  
Atp Production ◽  
Gene Assay ◽  
And Function

Regulation of mitochondrial energetics is key to maintain cardiac function. Perm1 ( P GC-1 and E RR- r egulator, m uscle 1 ) was previously shown to enhance exercise endurance in skeletal muscle through increasing mitochondrial bioenergetics. Despite the significant expression levels of Perm1 in the heart, its role in the healthy and diseased hearts has never been investigated. We found that cardiac Perm1 was downregulated in the mouse failing heart subjected to pressure overload for 4 weeks (24.4 ± 5.9 % of sham operated, p<0.05) and in patients with advanced heart failure (55.2 ± 13.1 % of donors, p<0.05) suggesting a role of Perm1 in cardiac pathology. Phenylephrine (PE)-induced hypertrophy in cardiomyocytes was accompanied by downregulation of Perm1 (55.7 ± 5.7 % of control, p <0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I), suggesting that downregulation of Perm1 contributes to the development of mitochondrial dysfunction in response to hypertrophic stimuli. Pathway enrichment analysis in cardiomyocytes where Perm1 was knocked-down by siRNA (siPerm1) revealed that the most downregulated pathway was metabolism, while upregulated pathways were mostly related to synthesis and assembly of collagen fibrils (i.e. Col3a1, Col5a1, Col11a1, Lox). Cell stress test using Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7 % and 23.6 % of scrambled-siRNA, respectively, both p <0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known targets of ERRα, Ndufv1 and Ndufs1 (Complex I). Furthermore, systemic Perm1-knockout mice developed heart failure (3 months old ejection fraction 38.8 %), concurrent with downregulation of proteins involved in oxidative phosphorylation, such as Ndufaf4 and Uqcr11 (-6.14 and -2.39 in fold change, respectively), and increased fibrosis. These results suggest that Perm1 is a novel regulator of cardiac metabolism and function, that enhances energetics and suppresses fibrosis.

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