Faster Experimental Validation of microRNA Targets Using Cold Fusion Cloning and a Dual Firefly-Renilla Luciferase Reporter Assay

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

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Hsa_circ_0123190 acts as a competitive endogenous RNA to regulate APLNR expression by sponging hsa-miR-483-3p in lupus nephritis

Arthritis Research & Therapy ◽

10.1186/s13075-020-02404-8 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Chunyi Zhang ◽

Congcong Gao ◽

Xueqi Di ◽

Siwan Cui ◽

Wenfang Liang ◽

...

Keyword(s):

Lupus Nephritis ◽

Lupus Erythematosus ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Circular Rnas ◽

Reporter Assay ◽

Clinical Value ◽

Chronicity Index

Abstract Background Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE). Circular RNAs (circRNAs) can act as competitive endogenous RNAs (ceRNAs) to regulate gene transcription, which is involved in mechanism of many diseases. However, the role of circRNA in lupus nephritis has been rarely reported. In this study, we aim to investigate the clinical value of circRNAs and explore the mechanism of circRNA involvement in the pathogenesis of LN. Methods Renal tissues from three untreated LN patients and three normal controls (NCs) were used to identify differently expressed circRNAs by next-generation sequencing (NGS). Validated assays were used by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The interactions between circRNA and miRNA, or miRNA and mRNA were further determined by luciferase reporter assay. The extent of renal fibrosis between the two groups was assessed by Masson-trichome staining and immunohistochemistry (IHC) staining. Results 159 circRNAs were significantly dysregulated in LN patients compared with NCs. The expression of hsa_circ_0123190 was significantly decreased in the renal tissues of patients with LN (P = 0.014). Bio-informatics analysis and luciferase reporter assay illustrated that hsa_circ_0123190 can act as a sponge for hsa-miR-483-3p, which was also validated to interact with APLNR. APLNR mRNA expression was related with chronicity index (CI) of LN (P = 0.033, R2 = 0.452). Moreover, the fibrotic-related protein, transforming growth factor-β1 (TGF-β1), which was regulated by APLNR, was more pronounced in the LN group (P = 0.018). Conclusion Hsa_circ_0123190 may function as a ceRNA to regulate APLNR expression by sponging hsa-miR-483-3p in LN.

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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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MicroRNA-16-5p Promoted Fibroblast-Like Synoviocyte Proliferation and Suppressed Apoptosis via Targeting Suppressor of Cytokine Signaling 6 (SOCS6) in Human Rheumatoid Arthritis

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2600 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1744-1751

Author(s):

Deqian Meng ◽

Wenyou Pan ◽

Ju Li

Keyword(s):

Rheumatoid Arthritis ◽

Cell Proliferation ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Cytokine Signaling ◽

Human Rheumatoid Arthritis ◽

Reporter Assay ◽

Functional Roles ◽

Proliferation And Apoptosis ◽

Fibroblast Like Synoviocytes

Accumulating evidence have indicated that MicroRNAs (miRNAs) are key regulators in human rheumatoid arthritis (RA). The aim of this study was to explore the functional roles of miR-16-5p in proliferation, inflammation, and apoptosis of fibroblast-like synoviocytes (FLS). The expression of miR-16-5p and SOCS6 in FLA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter assay was used to verify the direct target of miR-16-5p. Western blot analysis was performed to analysis the levels of SOCS6, Bcl-2, Bax and cleaved caspase 3. miR-16-5p expression was significantly upregulated while SOCS6 level was decreased in RA-FLS compared with normal FLS. In addition, luciferase reporter assay confirmed that SOCS6 was the target of miR-16-5p. Silencing of miR-16-5p inhibited cell proliferation, releases of TNF-α, IL-1β, IL-6 and IL-8, and induced the apoptosis. The effects of miR-16-5p silencing on RA-FLS were reversed by downregulation of SOCS6. In summary, knockdown of miR-16-5p could suppress cell proliferation and accelerate the apoptosis of RA-FLS through targeting SOCS6, which may provide a potential therapeutic target for patients with RA.

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