scholarly journals CCL2 is Upregulated by Decreased miR-122 Expression in Iron-Overload-Induced Hepatic Inflammation

2017 ◽  
Vol 44 (3) ◽  
pp. 870-883 ◽  
Author(s):  
Yuxiao Tang ◽  
Wentao Jia ◽  
Xiaowen Niu ◽  
Lusha Wu ◽  
Hui Shen ◽  
...  
Keyword(s):  
Vitamin E ◽  
Iron Overload ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Hepatic Inflammation ◽  
Ccl2 Expression ◽  
Ccl2 Mrna ◽  
Dual Luciferase Reporter Assay

Background/Aims: Iron overload (IO) is accompanied by hepatic inflammation. The chemokine (C-C motif) ligand 2 (CCL2) mediates inflammation, and its overexpression is associated with IO. However, whether IO results in CCL2 overexpression in the liver and the underlying mechanisms are unclear. Methods: We subjected mice to IO by administering intraperitoneal injections of dextran-iron or by feeding mice a 3% dextran-iron diet to observe the effects of IO on miR-122/CCL2 expression through real-time qPCR and Western blot analysis. We also used indicators, including the expression of the inflammatory cytokine, the inflammation score based on H&E staining and the serum content of ALT and AST to evaluate the effects of IO on hepatic inflammation. Meanwhile, we observed the effects of vitamin E on IO-induced hepatic inflammation. In cells, we used 100 µΜ FeSO4 or 30 µΜ Holo-Tf to produce IO and observed the roles of miR-122 in regulating CCL2 expression by using miR-122 mimics or inhibitors to overexpress or inhibit miR-122. Then, we used a dual-luciferase reporter assay to prove that miR-122 regulates CCL2 expression through direct binding to its complementary sequence in the CCL2 mRNA 3’UTR. Results: IO induces the downregulation of miR-122 and the upregulation of CCL2, as well as inflammatory responses both in vitro and in vivo. Although IO-induced oxidative stress is eliminated by the antioxidant vitamin E, IO-induced hepatic inflammation still exists, which probably can be explained by the fact that vitamin E has no effects on the miR-122/CCL2 pathway. In in vitro experiments, the overexpression and inhibition of miR-122 significantly reduced and increased CCL2 expression, respectively. The dual-luciferase reporter assay indicates that miR-122 binds CCL2 mRNA 3’UTR. Conclusion: We propose the roles of miR-122/CCL2 in IO-induced hepatic inflammation. Our studies should provide a new clue for developing clinical strategies for patients with IO.

scholarly journals FENDRR Sponges miR-424-5p to Inhibit Cell Proliferation, Migration and Invasion in Colorectal Cancer

2020 ◽  
Vol 19 ◽  
pp. 153303382098010
Author(s):  
Chuan Cheng ◽  
Huixia Li ◽  
Jiujian Zheng ◽  
Jie Xu ◽  
Peng Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Differential Analysis ◽  
Inhibit Cell Proliferation ◽  
Dual Luciferase Reporter Assay

Objective: LncRNAs are non-coding RNAs exerting vital roles in the occurrence and development of various cancer types. This study tended to describe the expression pattern of FENDRR in colorectal cancer (CRC), and further investigate the role of FENDRR in CRC cell biological behaviors. Methods: Gene expression profile of colon cancer was accessed from the TCGA database, and then processed for differential analysis for identification of differentially expressed lncRNAs and miRNAs. Some in vitro experiments like qRT-PCR, MTT, colony formation assay, wound healing assay and Transwell assay were performed to assess the effect of FENDRR on cell biological behaviors. Dual-luciferase reporter assay was conducted to further validate the targeting relationship between FENDRR and miR-424-5p, and rescue experiments were carried out for determining the mechanism of FENDRR/miR-424-5p underlying the proliferation, migration and invasion of CRC cells. Results: Bioinformatics analysis suggested that FENDRR was significantly down-regulated in CRC tissue, and low FENDRR was intimately correlated to poor prognosis. FENDRR overexpression could greatly inhibit cell proliferation, migration and invasion. Besides, there was a negative correlation between FENDRR and miR-424-5p. Dual-luciferase reporter assay indicated that miR-424-5p was a direct target of FENDRR. Rescue experiments discovered that FENDRR exerted its role in cell proliferation, migration and invasion in CRC via targeting miR-424-5p. Conclusion: FENDRR is poorly expressed in CRC tissue and cells, and low FENDRR is responsible for the inhibition of cell proliferation, migration and invasion of CRC by means of targeting miR-424-5p.

scholarly journals Knockdown of LincRNA PADNA Promotes Bupivacaine-Induced Neurotoxicity by miR-194/FBXW7 Axis

2020 ◽  
Author(s):  
Fan Yuning ◽  
Chen Liang ◽  
Wang Tenghuan ◽  
Nan Zhenhua ◽  
Shengkai Gong
Keyword(s):  
Functional Analysis ◽  
Western Blot ◽  
Cell Viability ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Drg Neurons ◽  
Dual Luciferase Reporter Assay

Abstract The aim of the study was to explore the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Mouse DRG neurons were cultured in vitro and treated with bupivacaine to establish the neurotoxicity model. Caspase3 activity, cell viability, tunel assay were analyzed to assess the role of lincRNA PADNA. Dual-luciferase reporter assay was used to determine the binding target of lincRNA PANDA. The expression of lincRNA PADNA was significantly increased with the increasing concentration of bupivacaine. Functional analysis revealed that knockdown of lincRNA PADNA accelerated the caspase3 activity and inhibited the cell viability. Western blot showed that knockdown of lincRNA PADNA promoted the occurrence of cleaved-caspase3. We also proved that lincRNA PADNA may bind with miR-194. Overexpression of miR-194 could rescued the function of lincRNA PADNA, suggesting that lincRNA PADNA may sponge miR-194. In addition, we provided new evidences that lincRNA PADNA/miR-194/FBXW7 axis play an important role in the neurotoxicity process. We performed comprehensive experiments to verify the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Our study provided new evidences and clues for prevention of neurotoxicity.

scholarly journals miRNA-1290 Promotes Aggressiveness in Pancreatic Ductal Adenocarcinoma by Targeting IKK1

2018 ◽  
Vol 51 (2) ◽  
pp. 711-728 ◽  
Author(s):  
Na Ta ◽  
Xiaoyi Huang ◽  
Kailian Zheng ◽  
Yunshuo Zhang ◽  
Yisha Gao ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Western Blotting ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Low Grade ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Background/Aims: MicroRNAs (miRNAs) are a group of non-coding RNAs that play diverse roles in pancreatic carcinogenesis. In pancreatic ductal adenocarcinoma (PDAC), NF-kB is constitutively activated in most patients and is linked to a mutation in KRAS via IkB kinase complex 1 (IKK1, also known as IKKa). We investigated the link between PDAC aggressiveness and miR-1290. Methods: We used miRCURYTM LNA Array and in situ hybridization to investigate candidate miRNAs and validated the findings with PCR. The malignant behavior of cell lines was assessed with Cell Counting Kit-8, colony formation, and Transwell assays. A dual-luciferase reporter assay was used to evaluate the interaction between miR-1290 and IKK1. Protein expression was observed by western blotting. Results: In this study, 36 miRNAs were dysregulated in high-grade pancreatic intraepithelial neoplasia (PanIN) and PDAC tissues compared with low-grade PanIN tissues. The area under the curve values of miR-1290 and miR-31-5p were 0.829 and 0.848, respectively (95% confidence interval, 0.722–0.936 and 0.749–0.948, both P < 0.001). There was a significant correlation between miR-1290 and histological differentiation (P = 0.029), pT stage (P = 0.006), and lymph node metastasis (P = 0.001). In addition, the in vitro work showed that miR-1290 promoted PDAC cell proliferation, invasion, and migration. Western blotting and the dual-luciferase reporter assay showed that miR-1290 promoted cancer aggressiveness by directly targeting IKK1. The synergist effect of miR-1290 on the proliferation and metastasis of PDAC cells was attenuated and enhanced by IKK1 overexpression and knockdown, respectively. Consistent with the in vitro results, a subcutaneous tumor mouse model showed that miR-1290 functioned as a potent promoter of PDAC in vivo. Conclusion: MiR-1290 may act as an oncogene by directly targeting the 3’-untranslated region of IKK1, and the miR-1290/IKK1 pathway may prove to be a novel diagnostic and therapeutic target for PDAC.

scholarly journals Brucine Inhibits Proliferation of U87 Glioblastoma Cells by Targeting the G-quadruplexes in the c-Myb Promoter

2020 ◽  
Author(s):  
Qiaochu Liu ◽  
Di Huo ◽  
Qunhui Wang ◽  
Chuanqi Lv ◽  
Ziqiang Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Promoter Region ◽  
Luciferase Reporter Assay ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Ionization Mass ◽  
Reporter Assay ◽  
U87 Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background: The proto-oncogene c-Myb plays an important role in the proliferation of cells and its upregulation affects the development of glioblastomas. G-quadruplexes are secondary structures of DNA or RNA that usually form in the promoter region of oncogenes, including c-Myb, and regulate the expression of these genes. The traditional Chinese medicine brucine is a ligand of G-quadruplexes located in the promoter region of c-Myb. In this study, the U87 cell line was used both in vitro and in vivo to investigate the therapeutic effect and mechanism of action of brucine. Methods: MTT assay and flow cytometry were used to determine the effect of brucine on the cell cycle, viability, and apoptosis of U87 cells. The effects of brucine on transcription and expression of c-Myb were determined through RT-PCR and western blotting. Dual-luciferase reporter assay and electrospray ionization-mass spectrometry were used to investigate whether brucine acts directly and binds G-quadruplexes in the promoter region of c-Myb, respectively. Results: The results showed that brucine suppressed the growth of U87 cells in vitro by arresting the cell cycle and reducing the expression of c-Myb. Through the dual luciferase reporter assay, brucine was found to inhibit the expression of c-Myb by targeting the guanine-rich sequence that forms G-quadruplexes in the c-Myb promoter. Moreover, U87 tumors were suppressed by brucine in a tumor xenograft nude mice model. Conclusion: The findings of the study indicate that brucine is a potentially effective medicine for treatment of glioblastomas.

miR-205 Suppresses Pulmonary Fibrosis by Targeting GATA3 Through Inhibition of Endoplasmic Reticulum Stress

2020 ◽  
Vol 21 (8) ◽  
pp. 720-726 ◽  
Author(s):  
Bingke Sun ◽  
Shumin Xu ◽  
Yanli Yan ◽  
Yusheng Li ◽  
Hongqiang Li ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Collagen I ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Sd Rats ◽  
Dual Luciferase Reporter Assay

Objective: To investigate the role of miR-205 and GATA3 in Pulmonary Fibrosis (PF). Methods: Bleomycin (BLM) was used to induce PF in SD rats and in vitro PF model was established by using TGFβ1-induced RLE-6TN cells. miR-205 mimics were used for the overexpression of miR- 205. The expression of miR-205, GATA3, α-SMA, Collagen I, CHOP and GRP78 were measured using RT-qPCR or western blotting. Dual-luciferase reporter assay was used to confirm binding between GATA3 3’-UTR and miR-205. Results: The expression of miR-205 was significantly down-regulated, while the expression of GATA3 was remarkably up-regulated in the model rats. GATA3 levels were remarkably decreased when miR-205 was overexpressed. When miR-205 was overexpressed, the lung injury by BLM-induced fibrosis was improved. The expression of α-SMA, Collagen I, as well as GRP78 and CHOP, was significantly up-regulated in both in vivo and in vitro PF models, and overexpression of miR-205 remarkably reversed the effects. Dual-luciferase reporter assay showed that miR-205 directly targeted and negatively regulated GATA3. Conclusion: miR-205 improved pulmonary fibrosis through inhibiting ER-stress by targeting GATA3.

scholarly journals miR-129-5p Inhibits Adipogenesis through Autophagy and May Be a Potential Biomarker for Obesity

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xue Fu ◽  
Lina Jin ◽  
Luyu Han ◽  
Yini Yuan ◽  
Qian Mu ◽  
...  
Keyword(s):  
Target Genes ◽  
Adipocyte Differentiation ◽  
Biologically Active ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Obese Mouse ◽  
Reporter Assay ◽  
Potential Biomarker ◽  
Dual Luciferase Reporter Assay

Introduction. Obesity has an unclear pathogenesis. MicroRNAs (miRNAs) may function as biologically active molecules for obesity through regulating adipocyte differentiation. This study aimed to identify how miR-129-5p (a specific miRNA) regulates adipogenesis in vitro and explore its possible role in the pathogenesis of obesity in humans. Materials and Methods. The miR-129-5p expression was detected in obese mouse models. The effect of miR-129-5p on adipocyte differentiation was observed, and the adipose markers were analyzed. Bioinformatics and dual-luciferase reporter assay were applied to predict and confirm the target genes of miR-129-5p. The human serum samples were detected and analyzed. Results. miR-129-5p is highly expressed in adipose tissues of db/db mice. Gain- and loss-of-function studies show that miR-129-5p could significantly inhibit adipocyte differentiation and white adipocyte browning in vitro and decreases the level of specific markers, such as FABP4, UCP1, and PPARγ, in mature white and brown adipocytes. miR-129-5p directly targets ATG7 which is predicted with bioinformatics and confirmed by dual-luciferase reporter assay. Serum miR-129-5p level was evidently elevated in patients with simple obesity (p<0.01) and correlates with obesity indices, including BMI (r = 0.407, p<0.029) and fat percentage (r = 0.394, p<0.038). Conclusion. miR-129-5p might target on the ATG7-related autophagy signaling network that regulates white and brown adipogenesis. Importantly, the aforementioned results suggest serum miR-129-5p might be a potential biomarker and therapeutic target for obesity.

scholarly journals miR-126 Functions as a Tumor Suppressor by Targeting SRPK1 in Human Gastric Cancer

2018 ◽  
Vol 26 (9) ◽  
pp. 1345-1353 ◽  
Author(s):  
Qiaorong Li ◽  
Geng Wang ◽  
Hong Wang
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
In Vitro Assay ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Vitro Assay ◽  
Dual Luciferase Reporter Assay

The expression of miR-126 and serine‐arginine protein kinase 1 (SRPK1) are linked to tumor development; nevertheless, its role in the tumor growth and invasion of gastric cancer (GC) and the underlying mechanism have not been clarified. Here the expression and role of miR-126 and SRPK1 were investigated in GC tissues and cells by in vitro assay, and then targets of miR-126 were identified by dual-luciferase reporter assay. In this study, miR-126 expression was downregulated and associated with lymph node metastasis and poor prognosis as well as SRPK1 expression. In vitro assay revealed that miR-126 obviously inhibited the proliferative and invasive capabilities of GC cells. The dual-luciferase reporter assay showed that miR-126 targets the 3′-UTR of SRPK1 and downregulates its expression. SRPK1 overexpression promoted cell migration and invasion. In conclusion, the reduced expression of miR-126 is suggestive of the risk of GC recurrence and metastasis, and miR-126 functions as a tumor suppressor by targeting SRPK1 expression in the development of GC.

scholarly journals Micro-RNA-338-3p Promotes the Development of Atherosclerosis by Targeting Desmin and Promoting Proliferation

2021 ◽  
Author(s):  
Shiran Yan ◽  
Jing Chen ◽  
Teng Zhang ◽  
Jian Zhou ◽  
Ge Wang ◽  
...  
Keyword(s):  
Rat Model ◽  
Luciferase Reporter Assay ◽  
Immunofluorescent Staining ◽  
Luciferase Reporter ◽  
Cell Phenotype ◽  
Reporter Assay ◽  
Synthetic Cell ◽  
Incorporation Assay ◽  
Morphologic Changes ◽  
Dual Luciferase Reporter Assay

AbstractAtherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

159 In vitro Characterization of Potential Pig Calcium-sensing Receptor (CaSR) Ligands and Cell Signaling Pathways Related to CaSR Activation by a Dual-luciferase Reporter Assay

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 82-83
Author(s):  
Xiaoya Zhao ◽  
Qianru Hui ◽  
Paula Azevedo ◽  
Karmin O ◽  
Chengbo Yang
Keyword(s):  
Signaling Pathways ◽  
Signaling Pathway ◽  
Binding Pocket ◽  
Extracellular Calcium ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Biased Agonism ◽  
Calcium Sensing ◽  
Dual Luciferase Reporter Assay

Abstract The calcium-sensing receptor (CaSR) is a pivotal regulator of calcium homeostasis. Our previous study has found that pig CaSR (pCaSR) is widely expressed in intestinal segments in weaned piglets. To characterize the activation of pCaSR by potential ligands and related cell signaling pathways, a dual-luciferase reporter assay was employed for the ligands screening and molecular docking was utilized to predict the binding mode of identified ligands. Our results showed that the dual-luciferase reporter assay system was well suited for pCaSR research and its ligand screening. The extracellular calcium activated pCaSR in a concentration-dependent manner with a half-maximal effective concentration (EC50) = 4.74 mM through the Gq/11 signaling pathway, EC50 = 2.85 mM through extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation signaling pathway, and EC50 = 2.26 mM through the Ras homolog family member A (RhoA) activation signaling pathway. Moreover, the activation of pCaSR stimulated by extracellular calcium showed biased agonism through three main signaling pathways: ERK1/2 phosphorylation signaling, Gq/11 signaling, and G12/13 signaling. Both L-Tryptophan and α-casein (90–95) could activate the pCaSR in the presence of extracellular calcium. Furthermore, we characterized the L-tryptophan binding pocket formed by pCaSR residues TRP 70, SER 147, ALA168, SER 169, SER 170, ASP 190, GLU 297, ALA 298, and ILE 416, as well as the α-casein (90–95) binding pocket formed by pCaSR residues PRO188, ASN189, GLU191, HIS192, LYS225, LEU242, ASP480, VAL486, GLY487, VAL513, and TYR514. In conclusion, similar to the human CaSR, the pCaSR also shows biased agonism through three main signaling pathways and both α-casein (90–95) and L-tryptophan are agonists for pCaSR. Furthermore, the binding sites of α-casein (90–95) and L-tryptophan are mainly located within the extracellular domain of pCaSR.

Sign in / Sign up

Export Citation Format

Share Document