scholarly journals tRNA-derived fragment TRF365 regulates the metabolism of anterior cruciate ligament cells by targeting IKBKB

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Dianbo Long ◽  
Yiyang Xu ◽  
Guping Mao ◽  
Ruobing Xin ◽  
Zengfa Deng ◽  
...  
Keyword(s):  
Anterior Cruciate Ligament ◽  
Cell Metabolism ◽  
Cruciate Ligament ◽  
Luciferase Reporter Assay ◽  
Interleukin 1Β ◽  
Luciferase Reporter ◽  
Fluorescence In Situ Hybridisation ◽  
Reporter Assay ◽  
Anterior Cruciate ◽  
Dual Luciferase Reporter Assay

AbstracttRNA-derived fragments (tRFs) are new noncoding RNAs, and recent studies have shown that tRNAs and tRFs have important functions in cell metabolism via posttranscriptional regulation of gene expression. However, whether tRFs regulate cellular metabolism of the anterior cruciate ligament (ACL) remains elusive. The aim of this study was to investigate the role and action mechanism of tRFs in ACL cell metabolism. A tRF array was used to determine tRF expression profiles in different human ACL cells, and quantitative real-time polymerase chain reaction and fluorescence in situ hybridisation were used to determine TRF365 expression. ACL cells were transfected with a TRF365 mimic or a TRF365 inhibitor to determine whether TRF365 regulates IKBKB expression. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3′-untranslated region (UTR) of IKBKB has a TRF365-binding site. TRF365 was weakly expressed in osteoarthritis (OA) ACL and interleukin-1β-treated ACL cells. IKBKB was highly expressed in OA ACL and interleukin-1β-treated ACL cells; transfection with the TRF365 mimic suppressed IKBKB expression, whereas transfection with the TRF365 inhibitor had the opposite effect. A dual-luciferase reporter assay showed that TRF365 silenced the expression of IKBKB by binding to its 3′-UTR. Thus, TRF365 regulates the metabolism of ACL cells by targeting IKBKB. In summary, TRF365 may provide a new direction for the study of ACL degeneration and on the pathophysiological process of OA.

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.

LncRNA PCAT19 Regulates Neuropathic Pain via Regulation of miR-182-5p/JMJD1A in a Rat Model of Chronic Constriction Injury

2021 ◽  
pp. 1-9
Author(s):  
Miao Huo ◽  
Xingxing Zheng ◽  
Ning Bai ◽  
Ruifen Xu ◽  
Guang Yang ◽  
...  
Keyword(s):  
Neuropathic Pain ◽  
Rat Model ◽  
Noncoding Rna ◽  
Thermal Hyperalgesia ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Reporter Assay ◽  
Withdrawal Threshold ◽  
Dual Luciferase Reporter Assay

<b><i>Introduction:</i></b> Neuropathic pain (NP) is one of the most severe chronic pain types. In recent years, more and more studies have shown that long noncoding RNA (LncRNA) plays a key role in a variety of human diseases, including NP. However, the role of LncRNA prostate cancer-associated transcript 19 (PCAT19) in NP and its specific mechanism remain unclear. <b><i>Methods:</i></b> A chronic constrictive injury (CCI) rat model was established. Rat paw withdrawal threshold and paw withdrawal latency were used to evaluate the neuronal pain behavior of rats in this model. mRNA expression of PCAT19, neuroinflammatory factor, microRNA (miR)-182-5p, and Jumonji domain containing 1A (JMJD1A) were detected by quantitative real-time PCR. ELISA analysis was used to detect inflammatory factor protein expression. Dual-luciferase reporter assay was used to evaluate the targeting relationship between genes. <b><i>Results:</i></b> PCAT19 was continuously upregulated in CCI rats. miR-182-5p was the target of PCAT19, and miR-182-5p was increased after PCAT19 knockdown. NP behaviors such as mechanical ectopic pain and thermal hyperalgesia as well as neuroinflammation can be reduced by knocking down PCAT19. However, the injection of miR-182-5p antagomir significantly reversed the level of the NP behaviors and neuroinflammation caused by PCAT19 knockdown. Besides, dual-luciferase reporter assay showed that JMJD1A was the target gene of miR-182-5p. The level of JMJD1A in CCI rats increased with time. After PCAT19 knockdown, JMJD1A was significantly decreased, but inhibition of miR-182-5p can reverse its levels. <b><i>Conclusion:</i></b> This study shows that PCAT19 plays a role in NP by targeting the miR-182-5p/JMJD1A axis, and PCAT19 can be used as a new therapeutic target for NP.

Association study of a functional variant of TNF-α gene and serum TNF-α level with the susceptibility of congenital heart disease in a Chinese population

2019 ◽  
Vol 95 (1128) ◽  
pp. 547-551
Author(s):  
Jun Pan ◽  
Jiang Hu ◽  
Xusheng Qi ◽  
Liqin Xu
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Congenital Heart ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Reporter Assay ◽  
Tnf Α ◽  
Α Level ◽  
Dual Luciferase Reporter Assay

BackgroundCongenital heart disease (CHD) is among the leading causes of infant death worldwide. Although shortage of folate has been found potentially to contribute to CHD in the embryo, the aetiology of CHD was not completely understood. Inflammation and altered immune processes are involved in all forms of cardiac malformation, including CHD. Tumour necrosis factor-α (TNF-α), was involved in the pathogenesis of multiple kinds of heart diseases. However, no studies have systematically evaluated the associations of genetic variants of TNF-α with susceptibility of CHD.MethodsA case-control study was conducted to evaluate the associations between tagSNPs of TNF-α and CHD susceptibility. Serum level of TNF-α was assessed using ELISA. The dual luciferase reporter assay was used to evaluate the functional significance of variant rs1800629 on TNF-α transcriptional activity.ResultsWe found rs1800629 was significantly correlated with increased CHD susceptibility (OR: 1.72, 95% CI 1.26 to 2.36, p=0.001). Serum levels of TNF-α were significantly higher in CHD group (9.09±1.90 pg/mL) than that in control group (6.12±1.56 pg/mL, p<0.001). The AA genotype and AG genotype of rs1800629 was associated with higher serum TNF-α level, compared with GG genotype. The dual luciferase reporter assay showed that promoter activity was significantly increased by 57% and 76% for plasmids containing the minor A allele compared with the major G allele in H9c2 and HEK 293T, respectively.ConclusionThese results indicate that higher level of serum TNF-α increases risk of CHD, while TNF-α rs1800629 A allele might contribute to higher risk for CHD due to the increase in TNF-α expression.

scholarly journals MiR-28-5p mediates the anti-proliferative and pro-apoptotic effects of curcumin on human diffuse large B-cell lymphoma cells

2020 ◽  
Vol 48 (7) ◽  
pp. 030006052094379
Author(s):  
Tian Kang ◽  
Wei-Li Sun ◽  
Xiao-Fei Lu ◽  
Xin-Liang Wang ◽  
Lian Jiang
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Large B Cell Lymphoma ◽  
Apoptotic Effects ◽  
Large B Cell ◽  
Dual Luciferase Reporter Assay

Objective To investigate the anti-proliferative and pro-apoptotic effects of curcumin on diffuse large B-cell lymphoma (DLBCL) cells and explore the mechanism. Methods OCI-LY7 cells were treated with curcumin (2.5, 5, 10, 20, and 40 μM) for 24, 48, or 72 hours. Cell viability and apoptosis were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide assay and TdT-mediated dUTP nick-end labeling staining, respectively. MiR-28-5p expression was detected via qRT-PCR. The binding site of miR-28-5p was predicted using online databases and verified using the dual-luciferase reporter assay. MiR-28-5p overexpression and inhibition were achieved via transfection with an miR-28-5p mimic and inhibitor, respectively. Results Curcumin decreased the viability of OCI-LY7 cells in a concentration- and time-dependent manner, and these effects were attenuated by miR-28-5p inhibition. MiR-28-5p expression was upregulated by curcumin. Curcumin increased the numbers of apoptotic cells and upregulated cleaved caspase-3 expression, and these effects were attenuated by miR-28-5p inhibition. The dual-luciferase reporter assay confirmed that miR-28-5p directly targets the 3′-untranslated region of BECN1. Curcumin downregulated BECN1 and microtubule-associated protein 1 light chain 3 beta-II/I expression and upregulated p62 expression. Conclusions Our results described the curcumin exerted anti-proliferative and pro-apoptotic effects on OCI-LY7 cells through a mechanism potentially involving miR-28-5p.

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 Down-regulation of miR-206 is associated with Hirschsprung disease and suppresses cell migration and proliferation in cell models

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Ankur Sharan ◽  
Hairong Zhu ◽  
Hua Xie ◽  
Hongxing Li ◽  
Junwei Tang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Hirschsprung Disease ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Down Regulation ◽  
Tissue Samples ◽  
Qrt Pcr ◽  
Cell Migration And Proliferation ◽  
Dual Luciferase Reporter Assay

Abstract Hirschsprung disease (HSCR) is a well-known congenital digestive disease that originates due to the developmental disorder of neural crest cells. MiR-206 is kown to have a relationship with digestive malfunctions. Therefore, we investigated whether or not miR-206 was involved in the pathogenesis of HSCR. qRT-PCR and Western blot assays were used to detect the expression levels of miRNA and mRNAs and proteins in case and control tissue samples and two cell lines (293T and SH-SY5Y). The functions of miR-206 in vitro were measured by transwell assay, CCK8 assay and flow cytometry. Finally, we conducted dual-luciferase reporter assay to verify the connections between miR-206 and the target mRNA SDPR. Down-regulation of miR-206 was found in HSCR case tissue samples compared with controls, which was validated to be connected with the increased level of mRNA and protein of SDPR by qRT-PCR and dual-luciferase reporter assay. Moreover, miR-206 suppressed the cell migration and proliferation and silencing of SDPR could rescue the extent of the suppressing effects by miR-206 inhibitor. The findings suggest that miR-206 may play a significant role in the pathogenesis of HSCR, as well as inhibiting the cell migration and proliferation by targeting SDPR in disease models.

scholarly journals LncRNA ZEB1-AS1 regulates hepatocellular carcinoma progression by targeting miR-23c

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shuai Xue ◽  
Fengqin Lu ◽  
Chunhui Sun ◽  
Jingjing Zhao ◽  
Honghua Zhen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Correlation Analysis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Transwell Assay ◽  
Qrt Pcr ◽  
Proliferation And Invasion ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background It has been reported that long-chain non-coding RNA (lncRNA) zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) is an oncogene in various cancers, including hepatocellular carcinoma (HCC). We investigated the role and mechanism of ZEB1-AS1 as a competitive endogenous RNA (ceRNA) combined with miR-23c in HCC cell proliferation and invasion. Methods QRT-PCR was used to detect ZEB1-AS1 and miR-23c expressions in HCC tissues and cells. The dual luciferase reporter assay detected the targeted regulation of miR-23c and ZEB1-AS1. We also performed the correlation analysis of their expression in HCC tissues by the Spearman’s correlation analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of hepatoma cells. Cell invasion was assessed by the Transwell assay. Results QRT-PCR results indicated ZEB1-AS1 was upregulated and miR-23c was downregulated in HCC tissues and cell lines. ZEB1-AS1 knockdown hampered the proliferation and invasion of HCC cells. Dual luciferase reporter assay showed that miR-23c is a target of ZEB1-AS1, and ZEB1-AS1 was significantly negatively correlated with the miR-23c expression in HCC tissues. The results of MTT and Transwell assay showed that miR-23c inhibition restored the inhibitory effect of ZEB1-AS1 knockdown on HCC cells proliferation and invasion. Conclusions As a ceRNA, lncRNA ZEB1-AS1 may play a vital role in inhibiting HCC progression through miR-23c, which will provide new clues and theoretical basis for the HCC diagnosis and treatment.

scholarly journals MiR-18a-5p Promotes NPC Cell Proliferation, Invasion, Migration, and EMT by Targeting SMAD2

2020 ◽  
Author(s):  
Pengcheng Li ◽  
Junhui Xing ◽  
Jianwu Jiang ◽  
Xinyu Tian ◽  
Xuemeng Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
Metastasis Model ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Abstract Background: Nasopharyngeal carcinoma (NPC) is the most common malignant tumor in the head and neck that is characterized by high local malignant invasion and distant metastasis. miR-18a-5p reportedly plays an important role in tumorigenesis and development. However, little is known about the mechanism underlying miR-18a-5p’s role in NPC.Methods：Quantitative real-time PCR was used to detect the expression of miR-18a-5p in NPC tissues and cell lines. MTT assay and plate clone formation assay were used to detect the effect of miR-18a-5p on NPC cell proliferation. Woundhealing assays and Transwell assays were used to detect the effect of miR-18a-5p on NPC cell invasion and migration. The expressions of epithelialmesenchymal transition (EMT)-related proteins N-cadherin, Vimentin, and E-cadherin were detected by Westernblot. Bioinformatics and dual-luciferase reporter assay were used to detect the targeting interaction between miR-18a-5p and SMAD2. Xenotransplantation and metastasis model were used to detect the effect of miR-18a-5p on NPC growth and metastasis in vivo.Results:miR-18a-5p was highly expressed in NPC tissues and cell lines. Overexpression of miR-18a-5p promotedNPC cell proliferation, invasion, migration, and EMT process, whereas inhibition of miR-18a-5p expression led to the oppositeresults. Results of dual-luciferase reporter assay showed that SMAD2 was the target gene of miR-18a-5p, and SMAD2 could reverse the effect of miR-18a-5p on NPC cell line. Xenotransplantation and metastasis model experiments in nude mice showed that miR-18a-5p promotesNPC growth and metastasis in vivo.Conclusions:Targeting SMAD2 downregulated miR-18a-5p expression, thereby promoting NPC cell proliferation, invasion, migration, and EMT.

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