MiR-375 Mediates Chondrocyte Metabolism and Oxidative Stress in Osteoarthritis Mouse Models through the JAK2/STAT3 Signaling Pathway

2019 ◽  
Vol 208 (1-2) ◽  
pp. 13-24
Author(s):  
Li-xue Zou ◽  
Ling Yu ◽  
Xun-ming Zhao ◽  
Jun Liu ◽  
Hou-gen Lu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signaling Pathway ◽  
Mouse Models ◽  
Control Group ◽  
Tunel Staining ◽  
Stat3 Pathway ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway ◽  
Chondrocyte Metabolism ◽  
And Oxidative Stress

Objective: The aim of this work was to determine the effect of miR-375 on chondrocyte metabolism and oxidative stress in osteoarthritis (OA) mouse models through the JAK2/STAT3 signaling pathway. Methods: Chondrocytes were divided into control, IL-1β, IL-1β + miR-375 mimic, IL-1β + miR-375 inhibitor, IL-1β + miR-NC (negative control), and IL-1β + miR-375 inhibitor + siJAK2 groups. The chondrocyte proliferation was determined by MTT assay, the superoxide dismutase (SOD) and malondialdehyde (MDA) levels by corresponding kits, and the chondrocyte apoptosis by TUNEL staining. Furthermore, OA mouse models were divided into Sham, OA + miR-NC, and OA + miRNA-375 antagomir groups. The pathological changes were observed, and the expressions of miR-375 and the JAK2/STAT3 pathway were determined by qRT-PCR and Western blotting, respectively. Results: IL-1β-induced chondrocytes had significant increases in miR-375 and MDA, with decreased proliferation and SOD levels, as compared to the control group. Meanwhile, they also exhibited elevated apoptosis, with upregulations of ADAMTS-5 and MMP-13 and downregulations of COL2A1 and ACAN, as well as decreased p-JAK2/JAK2, p-STAT3/STAT3, and Bcl-2/Bax. However, these changes were significantly improved after transfection with miR-375 inhibitor, but transfection with miR-375 mimic resulted in severer exacerbation. Notably, the improvement of miR-375 inhibitor could be abolished by transfection with siJAK2. Furthermore, miR-375 antagomir significantly alleviated OA progression in OA mice in vivo. Conclusion: MiR-375 suppression enhanced the ability of chondrocyte to antagonize the oxidative stress and maintained the homeostasis of extracellular matrix metabolism to protect chondrocytes from OA via activation of the JAK2/STAT3 pathway, indicating that miR-375 is a potential molecular target for OA treatment.

scholarly journals Mechanism of miR-218-5p in autophagy, apoptosis and oxidative stress in rheumatoid arthritis synovial fibroblasts is mediated by KLF9 and JAK/STAT3 pathways

2021 ◽  
pp. jim-2020-001437
Author(s):  
Ming Chen ◽  
Minghui Li ◽  
Na Zhang ◽  
Wenwen Sun ◽  
Hui Wang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Oxidative Stress ◽  
Signaling Pathway ◽  
Synovial Tissue ◽  
Synovial Fibroblasts ◽  
Reverse Transcription Pcr ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway ◽  
Interfering Rna ◽  
And Oxidative Stress

This study was aimed to investigate the effects of miR-218-5p on the proliferation, apoptosis, autophagy, and oxidative stress of rheumatoid arthritis synovial fibroblasts (RASFs), and the related mechanisms. Quantitative reverse transcription–PCR showed that the expression of miR-218-5p in rheumatoid arthritis synovial tissue was significantly higher than that in healthy synovial tissue. Compared with healthy synovial fibroblasts, miR-218-5p expression was obviously upregulated in RASFs, while KLF9 protein expression was markedly downregulated. Mechanistically, miR-218-5p could directly bind to the 3′ untranslated region of KLF9 to inhibit the expression of KLF9. Additionally, transfection of miR-218-5p small interfering RNA (siRNA) inhibited the proliferation but promoted apoptosis and autophagy of RASFs. Simultaneously, miR-218-5p silencing reduced reactive oxygen species and malondialdehyde levels and increased superoxide dismutase and glutathione peroxidase activity to improve oxidative stress in RASFs. More importantly, the introduction of KLF9 siRNA reversed the effects of miR-218-5p siRNA transfection on RASF proliferation, apoptosis, autophagy, and oxidative stress. What is more, silencing miR-218-5p inhibited the activation of JAK2/STAT3 signaling pathway by targeting KLF9. Collectively, knockdown of miR-218-5p could regulate the proliferation, apoptosis, autophagy and oxidative stress of RASFs by increasing the expression of KLF9 and inhibiting the activation of the JAK2/STAT3 signaling pathway, which may provide a potential target for the mechanism research of RA.

Acute Monocytic Leukemia Associated Antigen MLAA-34 up-Regulates JAK2/STAT3 Expression and JAK2/STAT3 Enhances MLAA-34 Activation in a Positive Feedback Loop

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1393-1393
Author(s):  
Bo Lei ◽  
Ju Bai ◽  
Wanggang Zhang ◽  
Aili He ◽  
Yinxia Chen ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Expression Vectors ◽  
Acute Monocytic Leukemia ◽  
U937 Cells ◽  
Rt Pcr ◽  
Monocytic Leukemia ◽  
Stat3 Pathway ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway

Abstract Backgroud: The MLAA-34 gene (GenBank no. AY288977.2) was first discovered in acute monocytic leukemia (M5) in an effort to identify monocytic leukemia-associated antigens by serologic analysis of a recombinant cDNA expression library (SEREX). Previous study showed that high MLAA-34 levels were independently associated with a poorer relapse-free survival and overall survival in AML patients. The MLAA-34 is located on 13q14.2 and has been confirmed to be a novel splice variant of CAB39L (calcium binding protein 39-like). Both mRNA and protein levels of MLAA-34 were found to be higher in U937 cells and M5 patients. The study confirmed that MLAA-34 plays a role in the antiapoptosis of U937 cells, and has the function of oncogenes. Objective: This study is to explore the relationship of MLAA-34 and JAK2/STAT3 signaling pathway so as to further clarify antiapoptotic mechanisms of MLAA-34. Method: Potential binding sites for STAT3 was identified by computer-assisted analysis of the core promoter of MLAA-34 gene. We analyzed the role of STAT3 in the regulation of MLAA-34 gene expression in U937 cells by site-specific mutation, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). The expression of MLAA-34 was detected by RT-PCR and western blot after over-expressing and interfering STAT3. Through the different concentrations of AG490 (JAK2 inhibitors) and different concentrations of IL- 6 (JAK2 activator) study JAK2/STAT3 signaling pathway upregulated MLAA-34 expression. The gene chip and co-IP were applied to study the MLAA-34-induced JAK2/STAT3 activation. Peripheral blood mononuclear cells and bone marrow (BM) cells of M5 patients were obtained. In M5 patients, mRNA and protein expression of JAK-2, STAT3, p-STAT3 and MLAA-34 were determined by quantitative RT-PCR and western blot respectively to verify the forward feedback regulation pathway. Result: The reporter assay showed that the activity of reporter gene was downregulated after the mutation of STAT3 binding sites. ChIP assay and EMSA showed that STAT3 can directly bind to MLAA-34 gene promoter. The expression vectors of MLAA-34 as well as siRNA eukaryotic expression vectors respectively targeting STAT3 were successfully constructed. RT-PCR and western blot results showed that STAT3 can increase the level of MLAA-34. Research of administration of different concentrations of AG490 and different concentrations of IL-6 showed that JAK2/STAT3 signaling pathway could upregulate MLAA-34 expression. AML-M5 NOD / SCID mice leukemia model was successfully constructed,.Konckdown of MLAA-34 gene can promote apoptosis of leukemia cells, inhibit tumor growth and prolong survival time. Total RNA from shRNA-MLAA-34/U937 and Vec/U937 cells were analyzed by Human Genome U133 chip. Heat-map showed reduced expression of JAK2/STAT3 pathway genes. The result was verified by qPCR and western blot. CO-IP showed that MLAA-34 could form a complex with endogenous JAK2 under the enhanced role of IL-6. Thereby activating JAK2 may directly or indirectly dependent on the presence of MLAA-34. Furthermore, we detected JAK-2, STAT3, P-STAT3 and MLAA-34 gene and protein level in M5 patients, the results showed that they have a positive correlation. Conclusion: JAK2/STAT3 pathway up-regulates MLAA-34 transcription, and MLAA-34 enhances JAK2/STAT3 pathway activation. The forward feedback regulation may have profound therapeutic implications for M5 and could help invent novel approaches in treatment for acute monocytic leukemia. Disclosures No relevant conflicts of interest to declare.

Effect of miR-155 on Aplastic Anemia Rats Through Regulating Signal Transducer and Activator of Transcription 3 Signaling Pathway

2020 ◽  
Vol 10 (3) ◽  
pp. 397-401
Author(s):  
Qingxian Yan ◽  
Zhengjun Hou ◽  
Shuting Ye ◽  
Meiyun Su
Keyword(s):  
Aplastic Anemia ◽  
Signaling Pathway ◽  
Blood Cells ◽  
Mrna Level ◽  
White Blood Cells ◽  
Control Group ◽  
Stat3 Signaling ◽  
Group I ◽  
Stat3 Signaling Pathway ◽  
Experimental Group

Objective: To assess miR-155’s effect on aplastic anemia (AA) rats. Methods: In the present study, the healthy rats (control group) and AA rats including AA rats with miR-155 overexpression (experimental group I) and those with miR-155 deficiency (experimental group II), were selected. The levels of miR-155, STAT3 (a key gene in STAT3 signaling pathway) and phosphorylated STAT3 (p-STAT3) in control group and experimental group were detected via qPCR and Western blotting. Moreover, the number of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs) and hemoglobin (HGB) level were also measured. Results: The level of miR-155 in AA rats was significantly declined compared with that in healthy rats (p < 0.05). STAT3 mRNA level was significantly declined in AA rats with miR-155 overexpression compared with that in AA rats with miR-155 deficiency (p < 0.05). STAT3 and p-STAT3 protein expression in AA rats with miR-155 overexpression were significantly lower than those in AA rats with miR-155 deficiency (p < 0.05). Besides, it was found that the number of WBC, RBC, PLT and HGB level were significantly elevated in AA rats with miR-155 overexpression compared to those in AA rats with miR-155 deficiency (p < 0.05). Conclusion: miR-155 can improve the AA symptoms of rats through inhibiting the STAT3 signaling pathway.

Hydrodynamic tail vein injection of SOCS3 eukaryotic expression vector in vivo promoted liver lipid metabolism and hepatocyte apoptosis in mouse

2014 ◽  
Vol 92 (2) ◽  
pp. 119-125 ◽  
Author(s):  
Zhenjiang Liu ◽  
Lu Gan ◽  
Xiaobo Yang ◽  
Zhenzhen Zhang ◽  
Chao Sun
Keyword(s):  
Lipid Metabolism ◽  
Signaling Pathway ◽  
Cytokine Signaling ◽  
Tunel Staining ◽  
Regulation Mechanism ◽  
Hepatocyte Apoptosis ◽  
Tail Vein ◽  
Stat3 Signaling ◽  
Tail Vein Injection ◽  
Stat3 Signaling Pathway

Suppressor of cytokine signaling 3 (SOCS3), a signal transduction cytokine, is involved in lipid metabolism as well as in cell proliferation, differentiation, apoptosis, and so on. To explore the effects of SOCS3 on apoptosis and lipid metabolism in liver, we used a simple effective method named hydrodynamic tail vein injection to overexpress SOCS3. Then orbital blood was obtained for the assessment of blood lipid after injection. Lipid metabolism related genes were detected by Western blot after the determination of serum lipids. Meanwhile, liver cell apoptosis was observed by Hoechst and TUNEL staining and the expression of apoptosis related proteins Bax, Bcl-2, and Caspase3 were detected as well as the JAK2/STAT3 signaling pathway. In addition, we also demonstrated the effect of SOCS3 in prime hepatocyte by overexpression or interference of SOCS3 along with SD1008, which is a specific inhibitor of the JAK2/STAT3 signaling pathway. Taken together, all the results indicated that SOCS3 promoted lipid synthesis in mice liver and promoted hepatocyte apoptosis by inhibiting the activation of the JAK2/STAT3 signaling pathway, however the detailed regulation mechanism had not yet been fully understood and needs further study.

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