scholarly journals miR-125 regulates PI3K/Akt/mTOR signaling pathway in rheumatoid arthritis rats via PARP2

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Kai Liu ◽  
Yingang Zhang ◽  
Liang Liu ◽  
Qiling Yuan
Keyword(s):  
Rheumatoid Arthritis ◽  
Signaling Pathway ◽  
Normal Group ◽  
Luciferase Reporter ◽  
Synovial Cells ◽  
Reporter Assay ◽  
Mtor Signaling Pathway ◽  
Model Group ◽  
Synovial Tissues ◽  
Model C

Abstract The present study aimed to explore miR-125 effects on rheumatoid arthritis (RA) development to provide a potential target for RA. Briefly, rat RA model was established (Model group) by injection of Freund’s Complete Adjuvant into the left hind toe. Normal rats injected with saline in the same location were set as Normal group. All rats’ secondary foot swelling degree, polyarthritis index score, spleen and thymus index were measured. Synovial tissues were subjected to Hematoxylin–Eosin (HE) staining and immunohistochemistry. Synovial cells of each group were isolated and named as Normal-C group and Model-C group, respectively. Synovial cells of Model-C group further underwent cotransfection with miR-125 mimics and PARP2-siRNA (mimics+siPARP2 group) or with miR-125 negative control (NC) and PARP2-siRNA NC (NC group). Quantitative reverse transcriptase PCR (qRT-PCR), Western blot, luciferase reporter assay, ELISA, and MTT assay were performed. As a result, compared with Normal group, rats of Model group showed significantly higher secondary foot swelling degree, polyarthritis index score, spleen and thymus index (P<0.01). Down-regulated miR-125 and up-regulated PARP2 was found in synovial tissues of Model group when compared with Normal group (P<0.01). Synovial tissues of Model-C group exhibited severe hyperplasia and inflammatory cell infiltration. Luciferase reporter assay indicated that PARP2 was directly inhibited by miR-125. Compared with NC group, cells of mimics+siPARP2 group had significantly lower IL-1β, MMP-1 and TIMP-1 levels, absorbance value, and p-PI3K, p-Akt and p-mTOR relative expression (P<0.01 or P<0.05). Thus, miR-125 might attenuate RA development by regulating PI3K/Akt/mTOR signaling pathway via directly inhibiting PARP2 expression.

scholarly journals CS nerve growth factor regulates sevoflurane anesthesiainduced nerve injury in nerve cells

2021 ◽  
Vol 19 (12) ◽  
pp. 2529-2536
Author(s):  
Naitian Wang ◽  
Duohui Liu ◽  
Xinyu Ning ◽  
Zhanjun Li ◽  
Lan Dong
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Mtor Signaling ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Mtor Signaling Pathway ◽  
Nerve Growth ◽  
Over Expression

Purpose: Sevoflurane is the most commonly used anesthetic agent for surgery. However, it is associated with deficiency in learning and memory abilities. The study was aimed at investigating the role of nerve growth factor (NGF) in sevoflurane anesthesia-induced nerve injury.Methods: RT-qPCR assay was applied to measure expressions of NGF, miR-98-5p and other factors related to apoptosis. CCK-8 assay was used for detecting cell viability while luciferase reporter assay was employed to measure binding condition between miR-98-5p and NGF. Expressions of proteins in PI3K/AKT/mTOR signaling pathway was measured with western blot.Results: Sevoflurane reduced cell viability of RGC-5 cells, promoted apoptosis and reduced the expression of NGF. In sevoflurane-induced RGC-5 cells, over-expression of NGF promoted cell viability with reduced apoptosis. Also, there was reduction in the protein expression of PI3K/AKT/mTOR signaling pathway by sevoflurane, while up-regulation of NGF promoted the expressions of these proteins. In the presence of PI3K inhibitor, reduction cell viability was reduced but apoptosis increased. Luciferase reporter assay detected MiR-98-5p as the target gene of NGF and its overexpression restored high cell viability in the over-expressed NGF. The rate of apoptosis and expressions of proteins was also restored with up-regulation of miR-98-5p.Conclusion: Sevoflurane caused damage to nerve cells, while over-expression of NGF reduced the injury through PI3K/AKT/mTOR signaling pathway and suppression of miR-98-5p. Keywords: Nerve growth factor, Sevoflurane, Nerve injury, Anesthesia, miR-98-5p

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.

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

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.

scholarly journals Gambogic acid suppresses inflammation in rheumatoid arthritis rats via PI3K/Akt/mTOR signaling pathway

2017 ◽  
Vol 16 (5) ◽  
pp. 7112-7118 ◽  
Author(s):  
Xiaodan Wu ◽  
Li Long ◽  
Jian Liu ◽  
Jin Zhang ◽  
Tong Wu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Signaling Pathway ◽  
Mtor Signaling ◽  
Gambogic Acid ◽  
Mtor Signaling Pathway

scholarly journals Dihydromyricetin promotes autophagy and attenuates renal interstitial fibrosis by regulating miR-155-5p/PTEN signaling in diabetic nephropathy

2019 ◽  
Author(s):  
Liming Guo ◽  
Kuibi Tan ◽  
Qun Luo ◽  
Xu Bai
Keyword(s):  
Diabetic Nephropathy ◽  
Signaling Pathway ◽  
Rat Model ◽  
Interstitial Fibrosis ◽  
Mtor Signaling ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Mtor Signaling Pathway ◽  
Renal Interstitial Fibrosis ◽  
Gene Assay

Diabetic nephropathy (DN) is the most common complication of diabetes and is prone to kidney failure. Dihydromyricetin (DHM) has been reported to have a variety of pharmacological activities. This study aims to explore the effect of DHM on DN and the underlying molecular mechanism. An in vivo DN rat model was established. The degree of renal interstitial fibrosis (RIF) was detected by hematoxylin-eosin (HE) staining, Masson's trichrome staining, and immunohistochemistry (IHC). In vitro, NRK-52E cells were divided into four groups: normal glucose (NG), high glucose (HG), HG+DHM, and HG+rapamycin (autophagy inhibitor). The levels of autophagy- and fibrosis-related proteins were analyzed by western blotting. The expression of miR-155-5p and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and their relationship were assessed by quantitative reverse transcription (qRT)-PCR and dual luciferase reporter gene assay. Our results showed that RIF was increased in DN rat model and in HG-induced NRK-52E cells. DHM treatment attenuated the increased RIF and also increased autophagy. MiR-155-5p expression was increased, while PTEN expression was decreased in DN rat and cell model, and DHM reversed both effects. Dual luciferase assay showed that PTEN was the target gene of miR-155-5p. DHM inhibited HG-induced fibrosis and promoted autophagy by inhibiting miR-155-5p expression in NRK-52E cells. In addition, DHM promoted autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway. In conclusion, DHM promotes autophagy and attenuates RIF by regulating the miR-155-5p/PTEN signaling and PI3K/AKT/mTOR signaling pathway in DN.

scholarly journals A Network Pharmacology Approach to Explore Mechanism of Action of Longzuan Tongbi Formula on Rheumatoid Arthritis

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
An Huang ◽  
Gang Fang ◽  
Yuzhou Pang ◽  
Zongran Pang
Keyword(s):  
Rheumatoid Arthritis ◽  
Signaling Pathway ◽  
Active Rheumatoid Arthritis ◽  
Trp Channels ◽  
Network Pharmacology ◽  
Mtor Signaling Pathway ◽  
Active Ingredients ◽  
Major Pathway ◽  
Pharmacological Mechanism ◽  
Ppar Signaling

Longzuan Tongbi Formula (LZTB) is an effective proved prescription in Zhuang medicine for treating active rheumatoid arthritis (RA). However, its active ingredients, underlying targets, and pharmacological mechanism are still not clear in treating RA. We have applied network pharmacology to study LZTB and found that 8 herbs in LZTB and 67 compounds in the 8 herbs are involved in the regulation of RA-related genes; we have conducted pathway analysis of overlapping genes and found that 7 herbs participate in the regulations of 24 pathways associated with RA and that 5 herbs in the 7 herbs and 25 compounds in the 5 herbs participate in the regulation of hsa05323 (rheumatoid arthritis). The results indicated that all herbs in LZTB and some compounds in those herbs participate in the treatment of RA; 25 compounds are main active ingredients and hsa05323 (rheumatoid arthritis) is the major pathway in the treatment of RA. We have also found that three pathways (inflammatory mediator regulation of TRP channels, PPAR signaling pathway, and mTOR signaling pathway) might have some effect on the treatment of RA.

scholarly journals The lncRNA-MALAT1/miR-126-5p Axis Promotes Growth and Metastasis of Gastric Cancer through Regulation of VEGFA

2020 ◽  
Author(s):  
Zhi-Li Hu ◽  
Yang-zhi Hu ◽  
Qing Li ◽  
Tian-you Liao ◽  
Hai-ping Jiang
Keyword(s):  
Gastric Cancer ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Mtor Signaling Pathway ◽  
Luciferase Reporter Gene ◽  
Ags Cells ◽  
Luciferase Reporter Gene Assay ◽  
Lncrna Malat1 ◽  
Gene Assay ◽  
Cancer Tissues

Abstract Background: It has been reported that reduction of miR-126 can promote the progression of gastric cancer (GC). However, the regulation of miR-126 in GC is still unclear. This study aims to explore the correlation between lncRNA MALAT1 and miR-126 in gastric cancer and disclose the underlying mechanisms.Methods: We analyzed the correlation of MALAT1 levels and clinical features by analysis of bioinformatic data and human samples. Then we down-regulate the expression of MALAT1 in AGS cells and examined the characteristics of cell proliferation, cycle, apoptosis, migration, invasion, and the effect on miR-126 as well as VEGFA and signaling pathway. In addition, we demonstrated the role of MALAT1/miR-126 axis in GC with dual-luciferase reporter gene assay and treatment of miR-126 inhibitor.Results: The expression of MALAT1 was higher in cancer tissues than para-cancer tissues. In addition, high MALAT1 level suggested greater malignancy and poorer prognosis. Down-regulating the expression of MALAT1 in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA, which is consistent with up-regulation of miR-126. According to dual-luciferase reporter gene assay and treatment of miR-126 inhibitor, we demonstrated that MALAT1 down-regulated miR-126 in GC, which leads to the up-regulation of VEGFA and activation of mTOR signaling pathway.Conclusions: MALAT1/miR-126 axis promotes growth and metastasis of gastric cancer through regulation of VEGFA via mTOR signaling pathway.Fund This article is supported by Science and Technology Funding Project of Hunan Province, China (No.2017SK4010)

scholarly journals Lnc RNA ZFAS1 regulates the proliferation, apoptosis, inflammatory response and autophagy of fibroblast-like synoviocytes via miR-2682-5p/ADAMTS9 axis in rheumatoid arthritis

2020 ◽  
Vol 40 (8) ◽  
Author(s):  
Shanshan Yang ◽  
Wei Yin ◽  
Yan Ding ◽  
Fan Liu
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Inflammatory Response ◽  
Large Subunit ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Behaviors ◽  
Synovial Tissues ◽  
Related Proteins ◽  
Fibroblast Like Synoviocytes

Abstract Backgrounds: Rheumatoid arthritis (RA) is a frequent autoimmune disease. Emerging evidence indicated that ZNFX1 antisense RNA1 (ZFAS1) participates in the physiological and pathological processes in RA. However, knowledge of ZFAS1 in RA is limited, the potential work pathway of ZFAS1 needs to be further investigated. Methods: Levels of ZFAS1, microRNA (miR)-2682-5p, and ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) were estimated using quantitative real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to explore the ability of cell proliferation in fibroblast-like synoviocytes (FLS-RA). Cell apoptosis was measured via flow cytometry. Also, levels of ADAMTS9, apoptosis-related proteins, cleaved-caspase-3 (active large subunit), and autophagy-related proteins were identified adopting Western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the productions of inflammatory cytokines. Beside, the interrelation between miR-2682-5p and ZFAS1 or ADAMTS9 was verified utilizing dual-luciferase reporter assay. Results: High levels of ZFAS1 and ADAMTS9, and a low level of miR-2682-5p were observed in RA synovial tissues and FLS-RA. Knockdown of ZFAS1 led to the curbs of cell proliferation, inflammation, autophagy, and boost apoptosis in FLS-RA, while these effects were abolished via regaining miR-2682-5p inhibition. Additionally, the influence of miR-2682-5p on cell phenotypes and inflammatory response were eliminated by ADAMTS9 up-regulation in FLS-RA. Mechanically, ZFAS1 exerted its role through miR-2682-5p/ADAMTS9 axis in RA. Conclusion: ZFAS1/miR-2682-5p/ADAMTS9 axis could modulate the cell behaviors, inflammatory response in FLS-RA, might provide a potential therapeutic target for RA treatment.

Sign in / Sign up

Export Citation Format

Share Document