MicroR-26b Targets High Mobility Group, AT-hook 2 to Ameliorate Myocardial Infarction-induced Fibrosis by Suppression of Cardiac Fibroblasts Activation

Background: Myocardial Fibrosis (MF) is an important physiological change after myocardial infarction (MI). MicroRNA-26b (MiR-26b) has a certain inhibitory effect on pulmonary fibrosis. However, the role of miR-26b in MI-induced MF rats and underlying molecular mechanisms remain unknown. Methods: Forty male Sprague Dawley (SD) rats weighing 200-250 g were divided into four groups (n=10): Sham group, MF group, MF + negative control (NC) agomir group and MF + miR-26b agomir group. Cardiac fibroblasts were isolated from cardiac tissue. Fibrosis levels were detected by MASSON staining, while the expression of related genes was detected by RT-qPCR, Western blotting and Immunohistochemistry, respectively. TargetScan and dual-luciferase reporter assay were utilized to predict the relationship between miR-26b and high mobility group, AT-hook 2 (HMGA2). Results: The study found the expression of miR-26b to be down-regulated in the myocardium of MF rats (P<0.01). miR-26b overexpression in vitro significantly reduced the survival rate of cardiac fibroblasts and inhibited the expression of the fibrillar-associated protein (α-SMA alphasmooth muscle actin (α-SMA) and collagen I) (P<0.01). TargetScan indicated that HMGA2 was one of the target genes of miR-26b; dual-luciferase reporter assay further confirmed the targeted regulatory relationship (P<0.01). Moreover, miR-26b overexpression significantly reduced the expression of HMGA2 (P<0.01). Notably, HMGA2 overexpression reversed the inhibitory effect of miR-26b overexpression on cardiac fibroblast viability and the expression of α-SMA and collagen I (P<0.01). Animal experiments further indicated that miR-26b overexpression inhibited MIinduced rat MF by inhibiting the expression of HMGA2 (P<0.05, P<0.01). Conclusion: In short, these findings indicate that miR-26b targets HMGA2 to ameliorate MI-induced fibrosis by suppression of cardiac fibroblasts activation.

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Circular RNA SIPA1L1 Promotes Osteogenesis via Regulating the MIR-617/SMAD3 Axis in Dental Pulp Stem Cells

10.21203/rs.3.rs-32816/v1 ◽

2020 ◽

Author(s):

Xingyun Ge ◽

Zehan Li ◽

Zhou Zhou ◽

Yibo Xia ◽

Minxia Bian ◽

...

Keyword(s):

Stem Cells ◽

Bone Regeneration ◽

Dental Pulp ◽

Sanger Sequencing ◽

Inhibitory Effect ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Dual Luciferase Reporter Assay

Abstract Background: Bone regeneration is preferred for bone loss caused by tumors, bone defects, fractures, etc. Recently, mesenchymal stem cells are considered as optimistic tools for bone defect therapy. Dental pulp stem cells (DPSCs) are a promising candidate for regenerative medicine and bone regeneration. Our previous study showed that up-regulated circSIPA1L1 during osteogenesis of DPSCs is of significance. In this paper, the potential role of circSIPA1L1 in osteogenesis of DPSCs and its underlying mechanisms are explored.Methods: The circular structure of circSIPA1L1 was identified by Sanger sequencing and PCR. Regulatory effects of circSIPA1L1 and miR-617 on mineral deposition in DPSCs were assessed by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and in vivo bone formation assay were conducted to verify the biological inﬂuences of circSIPA1L1 on DPSCs. Western blot was performed to detect the protein expression of Smad3. Localization of circSIPA1L1 and miR-617 was confirmed by FISH. Dual-luciferase reporter assay and rescue experiments were conducted to investigate the role of the circSIPA1L1/miR-617/Smad3 regulatory axis in osteogenesis of DPSCs.Results: Sanger sequencing and back-to-back primer experiments confirmed the closed loop structure of circSIPA1L1. CircSIPA1L1 could promote the committed differentiation of DPSCs. MiR-617 was predicted to be the target binding circSIPA1L1 through MiRDB, miRTarBase, and TargetScan database analyses, which was further confirmed by dual-luciferase reporter assay. FISH results showed that circSIPA1L1 and miR-617 colocalize in the cytoplasm of DPSCs. MiR-617 exerted an inhibitory effect on osteogenesis of DPSCs. Knockdown of circSIPA1L1 or upregulation of miR-617 down-regulated phosphorylated Smad3. In addition, rescue experiments showed that knockdown of miR-617 reversed the inhibitory effect of circSIPA1L1 on osteogenesis of DPSCs.Conclusion: CircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3.

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Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells

10.21203/rs.3.rs-32816/v2 ◽

2020 ◽

Author(s):

Xingyun Ge ◽

Zehan Li ◽

Zhou Zhou ◽

Yibo Xia ◽

Minxia Bian ◽

...

Keyword(s):

Stem Cells ◽

Bone Regeneration ◽

Dental Pulp ◽

Sanger Sequencing ◽

Inhibitory Effect ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Dual Luciferase Reporter Assay

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

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666191210115614 ◽

2020 ◽

Vol 21 (8) ◽

pp. 720-726 ◽

Cited By ~ 2

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.

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Demethylated miR-216a Regulates High Mobility Group Box 3 Promoting Growth of Esophageal Cancer Cells Through Wnt/β-Catenin Pathway

Frontiers in Oncology ◽

10.3389/fonc.2021.622073 ◽

2021 ◽

Vol 11 ◽

Author(s):

Cheng-Xi Sun ◽

Feng Zhu ◽

Lei Qi

Keyword(s):

Esophageal Cancer ◽

Cell Lines ◽

High Mobility ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Methylation Inhibitor ◽

Underlying Mechanisms ◽

Dual Luciferase Reporter Assay

BackgroundEsophageal cancer (EC) is the eighth most common cause of cancer-associated mortality in humans. Recent studies have revealed the important roles of microRNAs (miRs) in mediating tumor initiation and progression. miR-216a has been found to be involved in the progression of EC, but the underlying mechanisms remain largely unknown. The aim of this study is to explore the mechanism of miR-216a and the downstream molecules in esophageal cancer.Materials and MethodsThe degree of methylation of miR-216a promoter in EC tissues and cell lines was determined with methylation specific polymerase chain reaction (MSP). The levels of miR-216a and HMGB3 in EC cells were quantified by quantitative PCR (qPCR) and Western blot (WB). EC cell lines were treated with DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-AZ), miR-216a mimics, and HMGB3 siRNA to explore the effects of miR-216a and HMGB3 on the proliferation, migration, invasion, and apoptosis of cells. Dual-luciferase reporter assay was employed to verify the binding of miR-216a to the 3’UTR of HMGB2 mRNA.ResultsThe promoter of MiR-216a was hypermethylated and the expression of miR-216a was down-regulated in EC, while HMGB3 was up-regulated. Dual luciferase reporter assay confirmed the binding of miR-216a to the 3’UTR of HMGB3 mRNA. Demethylated miR-216a and miR-216a mimics elevated miR-216a expression and down-regulated HMGB3, as well as inhibited cell proliferation, migration, and invasion. Inhibiting the expression of HMGB3 played an important role in inducing apoptosis, suppressing cell expansion, and down-regulating the activity of Wnt/β-catenin pathway.ConclusionsHypermethylation in the promoter of miR-216a upregulated HMGB3 and the Wnt/β-catenin pathway, resulting in enhanced EC progression.

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

Molecular Biotechnology ◽

10.1007/s12033-021-00341-8 ◽

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.

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

Technology in Cancer Research & Treatment ◽

10.1177/1533033820985868 ◽

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.

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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

Journal of Animal Science ◽

10.1093/jas/skab235.149 ◽

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.

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

Cell Death Discovery ◽

10.1038/s41420-021-00806-4 ◽

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.

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

NeuroImmunoModulation ◽

10.1159/000518847 ◽

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

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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.

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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

Postgraduate Medical Journal ◽

10.1136/postgradmedj-2019-136621 ◽

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.

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