Circ_RASGEF1B Promotes LPS-Induced Apoptosis and Inflammatory Response by Targeting MicroRNA-146a-5p/Pdk1 Axis in Septic Acute Kidney Injury Cell Model

Nephron ◽  
2021 ◽  
pp. 1-12
Author(s):  
Jianghong Cao ◽  
Dongwu Shi ◽  
Lili Zhu ◽  
Lu Song
Keyword(s):  
Acute Kidney Injury ◽  
Inflammatory Response ◽  
Cell Viability ◽  
Cell Model ◽  
Kidney Injury ◽  
Pyruvate Dehydrogenase Kinase ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Septic Acute Kidney Injury ◽  
Induced Apoptosis

<b><i>Background:</i></b> We intended to investigate the function of circular RNA RasGEF domain family member 1B (circ_RASGEF1B) in lipopolysaccharide (LPS)-induced septic acute kidney injury (AKI) cell model and its associated mechanism. <b><i>Methods:</i></b> TCMK-1 cells were exposed to 10 μg/mL LPS for 24 h to establish a septic AKI cell model. Mice were intraperitoneally injected with 10 mg/kg LPS to establish a septic AKI mice model. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were used to measure RNA and protein expression, respectively. Cell viability and apoptosis were assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. Cell inflammatory response was analyzed using enzyme-linked immunosorbent assay. Dual-luciferase reporter assay was conducted to confirm the predicted target relationship between microRNA-146a-5p (miR-146a-5p) and circ_RASGEF1B or pyruvate dehydrogenase kinase 1 (Pdk1). <b><i>Results:</i></b> The circ_RASGEF1B level was upregulated in LPS-induced TCMK-1 cells and septic AKI mice models. LPS exposure reduced cell viability and promoted cell apoptosis and inflammatory response partly by upregulating circ_RASGEF1B. Circ_RASGEF1B bound to miR-146a-5p and miR-146a-5p interference partly overturned circ_RASGEF1B silencing-mediated effects in LPS-induced TCMK-1 cells. Pdk1 was a target of miR-146a-5p, and Pdk1 accumulation partly counteracted miR-146a-5p-induced influences in TCMK-1 cells upon LPS stimulation. <b><i>Conclusion:</i></b> Circ_RASGEF1B promoted LPS-induced apoptosis and inflammatory response in renal tubular epithelial cells partly by upregulating Pdk1 via acting as miR-146a-5p sponge.

scholarly journals Knockdown of circ-FANCA alleviates LPS-induced HK2 cell injury via targeting miR-93-5p/OXSR1 axis in septic acute kidney injury

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Heyun Li ◽  
Xia Zhang ◽  
Peng Wang ◽  
Xiaoyan Zhou ◽  
Haiying Liang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Acute Kidney Injury ◽  
Cell Injury ◽  
Kidney Injury ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Septic Acute Kidney Injury ◽  
Hk2 Cells ◽  
And Oxidative Stress

Abstract Background Sepsis is life-threatening disease with systemic inflammation and can lead to various diseases, including septic acute kidney injury (AKI). Recently, diverse circular RNAs (circRNAs) are considered to be involved in the development of this disease. In this study, we aimed to elucidate the role of circ-FANCA and the potential action mechanism in sepsis-induced AKI. Methods HK2 cells were treated with lipopolysaccharide (LPS) to establish septic AKI cell model. The expression of circ-FANCA, microRNA-93-5p (miR-93-5p) and oxidative stress responsive 1 (OXSR1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. The inflammatory response was monitored according to the release of pro-inflammatory cytokines via enzyme-linked immunosorbent assay (ELISA). The activities of oxidative indicators were examined using the corresponding kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to validate the interaction between miR-93-5p and circ-FANCA or OXSR1. Protein analysis was conducted through western blot. Results Circ-FANCA was upregulated in septic AKI serum specimens and LPS-treated HK2 cells. Functionally, circ-FANCA knockdown facilitated cell proliferation and restrained apoptosis, inflammation and oxidative stress in LPS-triggered HK2 cells. Further mechanism analysis revealed that miR-93-5p was a target of circ-FANCA and circ-FANCA modulated LPS-induced cell damage by targeting miR-93-5p. Meanwhile, miR-93-5p overexpression repressed LPS-treated HK2 cell injury by sponging OXSR1. Furthermore, circ-FANCA regulated OXSR1 expression by sponging miR-93-5p. Besides, exosome-derived circ-FANCA was upregulated in LPS-induced HK2 cells, which was downregulated by GW4869. Conclusion Circ-FANCA knockdown attenuated LPS-induced HK2 cell injury by regulating OXSR1 expression via targeting miR-93-5p.

scholarly journals NEAT1 aggravates sepsis-induced acute kidney injury by sponging miR-22-3p

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 333-342
Author(s):  
Yawei Feng ◽  
Jun Liu ◽  
Ranliang Wu ◽  
Peng Yang ◽  
Zhiqiang Ye ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Cell Injury ◽  
Kidney Injury ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay

AbstractBackground and aimAcute kidney injury (AKI) is a common complication of sepsis. Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) plays a vital role in various diseases, including AKI. This study aimed to investigate the function and mechanism of NEAT1 in sepsis-induced AKI.Materials and methodsA septic AKI model was established by treating HK-2 cells with lipopolysaccharide (LPS). The levels of NEAT1 and miR-22-3p were measured by quantitative real-time PCR. Cell apoptosis was assessed by flow cytometry. The levels of apoptosis-related protein and autophagy-related factors were examined by the western blot assay. An enzyme-linked immunosorbent assay was used to calculate the contents of inflammatory factors. The interaction between NEAT1 and miR-22-3p was validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay. The levels of nuclear factor (NF)-κB pathway-related proteins were evaluated by the western blot assay.ResultsNEAT1 was upregulated, while miR-22-3p was downregulated in patients with sepsis and in LPS-stimulated HK-2 cells. LPS treatment triggered cell apoptosis, autophagy, and inflammatory response in HK-2 cells. NEAT1 knockdown attenuated LPS-induced cell injury. NEAT1 modulated LPS-triggered cell injury by targeting miR-22-3p. Furthermore, NEAT1 regulated the NF-κB pathway by modulating miR-22-3p.ConclusionDepletion of NEAT1 alleviated sepsis-induced AKI via regulating the miR-22-3p/NF-κB pathway.

scholarly journals MiR-22-3p suppresses sepsis-induced acute kidney injury by targeting PTEN

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
Xudong Wang ◽  
Yali Wang ◽  
Mingjian Kong ◽  
Jianping Yang
Keyword(s):  
Acute Kidney Injury ◽  
Inflammatory Response ◽  
Periodic Acid ◽  
Kidney Injury ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
Tnf Α

Abstract Background: Septic acute kidney injury is considered as a severe and frequent complication that occurs during sepsis. The present study was performed to understand the role of miR-22-3p and its underlying mechanism in sepsis-induced acute kidney injury. Methods: Rats were injected with adenovirus carrying miR-22-3p or miR-NC in the caudal vein before cecal ligation. Meanwhile, HK-2 cells were transfected with the above adenovirus following LPS stimulation. We measured the markers of renal injury (blood urea nitrogen (BUN), serum creatinine (SCR)). Histological changes in kidney tissues were examined by hematoxylin and eosin (H&E), Masson staining, periodic acid Schiff staining and TUNEL staining. The levels of IL-1β, IL-6, TNF-α and NO were determined by ELISA assay. Using TargetScan prediction and luciferase reporter assay, we predicted and validated the association between PTEN and miR-22-3p. Results: Our data showed that miR-22-3p was significantly down-regulated in a rat model of sepsis-induced acute kidney injury, in vivo and LPS-induced sepsis model in HK-2 cells, in vitro. Overexpression of miR-22-3p remarkably suppressed the inflammatory response and apoptosis via down-regulating HMGB1, p-p65, TLR4 and pro-inflammatory factors (IL-1β, IL-6, TNF-α and NO), both in vivo and in vitro. Moreover, PTEN was identified as a target of miR-22-3p. Furthermore, PTEN knockdown augmented, while overexpression reversed the suppressive role of miR-22-3p in LPS-induced inflammatory response. Conclusions: Our results showed that miR-22-3p induced protective role in sepsis-induced acute kidney injury may rely on the repression of PTEN.

miR-30e-5p regulates autophagy and apoptosis by targeting Beclin1 involved in contrast-induced acute kidney injury

2021 ◽  
Vol 28 ◽  
Author(s):  
Xiaoqin Liu ◽  
Qingzhao Li ◽  
Lixin Sun ◽  
Limei Chen ◽  
Yue Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Kidney Injury ◽  
Cell Apoptosis ◽  
Cell Injury ◽  
Cell Model ◽  
Kidney Injury ◽  
Cell Vitality ◽  
A Cell ◽  
Renal Tubular ◽  
Induced Apoptosis

Aims: This study aims to verify if miR-30e-5p targets Beclin1 (BECN1), a key regulator of autophagy, and investigate the function of miR-30e-5p and Beclin1 through mediating autophagy and apoptosis in contrast-induced acute kidney injury (CI-AKI). Methods: Human renal tubular epithelial HK-2 cells were treated with Urografin to construct a cell model of CI-AKI. Real-time reverse transcription–polymerase chain reaction was used to detect gene expression. The dual-luciferase reporting assay and endogenous validation were used to verify targeting and regulating function. The expressions of protein were detected using Western blot. Cell proliferation was detected using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was detected using terminal-deoxynucleoitidyl transferase mediated nick end labeling assay, and autophagy was detected using transmission electron microscopy. Results: HK-2 cells exposed to Urografin for 2 h induced a significant increase in miR-30e-5p. miR-30e-5p had a targeting effect on Beclin1. Moreover, Urografin exposure can enhance cell apoptosis by increasing caspase 3 gene expression and inhibiting autophagy, which was induced by decreased Beclin1 expression regulated by miR-30e-5p, thereby resulting in renal cell injury. Downregulation of miR-30e-5p or upregulation of Beclin1 restored cell vitality by promoting autophagy and suppressing apoptosis in Urografin-treated cells. Conclusions: Urografin increased the expression of miR-30e-5p in HK-2 cells and thus decreased Beclin1 levels to inhibit autophagy, but induced apoptosis, which may be the mechanism for CI-AKI.

scholarly journals Hyperoside Protects HK-2 Cells Against High Glucose-Induced Apoptosis and Inflammation via the miR-499a-5p/NRIP1 Pathway

2021 ◽  
Vol 27 ◽  
Author(s):  
Jingbo Zhou ◽  
Shu Zhang ◽  
Xinyi Sun ◽  
Yan Lou ◽  
Jiangyi Yu
Keyword(s):  
Inflammatory Response ◽  
High Glucose ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Protective Effects ◽  
Human Papillomavirus 16 ◽  
Induced Apoptosis ◽  
Apoptosis And Inflammation ◽  
Dose Dependent

Hyperoside, a flavonol glycoside, is derived from plants of the genera Hypericum and Crataegus. Recent studies have indicated the anti-apoptotic and anti-inflammatory roles of hyperoside. The present study was designed to measure the effects of hyperoside on high glucose (HG)-treated HK-2 cells. HK-2 is a human papillomavirus 16 transformed cell line and can be used as a model for normal tubular cell. Cell apoptosis was examined by TUNEL assays and flow cytometry analysis. Inflammatory response was detected by Enzyme linked immunosorbent assay kits. Western blotting was applied to detect protein levels of apoptosis-related genes and inflammatory cytokines. Mechanistical assays including luciferase reporter and RNA pull down assays were applied to detect the binding relationship between molecules. We identified that hyperoside protected HK-2 cells against HG-induced apoptosis and inflammation. Moreover, miR-499a-5p was upregulated by hyperoside in a dose dependent manner. MiR-499a-5p inhibition rescued the suppressive effects of hyperoside on apoptosis and inflammation of HG-treated HK-2 cells. Furthermore, miR-499a-5p targeted NRIP1 to inhibit its mRNA expression, and further suppressed its translation. NRIP1 was downregulated by hyperoside in a dose dependent manner. Finally, rescue assays indicated that miR-499a-5p inhibition rescued the protective effects of hyperoside on apoptosis and inflammatory response of HK-2 cells by NRIP1. In conclusion, our findings revealed that hyperoside alleviates HG-induced apoptosis and inflammatory response of HK-2 cells by the miR-499a-5p/NRIP1 axis.

MicroRNA (miR)-429 Promotes Inflammatory Injury by Targeting Kruppel-like Factor 4 (KLF4) in Neonatal Pneumonia

2020 ◽  
Vol 17 (1) ◽  
pp. 102-109
Author(s):  
Lan Zhang ◽  
HuanLi Yan ◽  
Huiping Wang ◽  
Li Wang ◽  
Boling Bai ◽  
...  
Keyword(s):  
Cell Viability ◽  
Peripheral Blood ◽  
Cell Model ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Common Disease ◽  
Human Lung Fibroblast ◽  
Inflammatory Injury ◽  
Tnf Α

Background: Neonatal pneumonia is a common disease in the neonatal period with a high incidence and death. This study aimed to investigate the molecular mechanism and effect of microRNA (miR)-429 in neonatal pneumonia. Methods: The peripheral blood was collected from neonatal pneumonia and healthy patients, respectively. Human lung fibroblast WI-38 cells were treated with lipopolysaccharide (LPS) to establish neonatal pneumonia cell model. Then, the miR-429 expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the relationship between miR- 429 and kruppel-like factor 4 (KLF4) was confirmed by dual luciferase reporter assay. Cell viability, the level of interleukin 6 (IL-6), IL-1β and tumor necrosis factor α (TNF-α) and apoptosis were measured by Cell Counting Kit-8 (CCK-8), enzyme linked immunosorbent assay (ELISA) and flow cytometry. Meanwhile, apoptosis and nuclear factor kappa-B (NF-κB) pathway related proteins expression were analyzed by western blot. Results: MiR-429 expression level was increased in neonatal peripheral blood and LPS-stimulated WI-38 cells. Then, miR-429 overexpression increased apoptosis, the level of IL-6, IL-1β, TNF-α, Bax and cleaved caspase-3, while reduced cell viability in LPS-stimulated WI-38 cells. Besides, KLF4 was identified as the target gene of miR-429, and reversed the changes caused by miR-429 overexpression. Finally, miR-429 suppressor down-regulated p-NF-κB level in LPS-stimulated cells and KLF4 knockdown reversed these reductions. Conclusion: MiR-429 promotes inflammatory injury, apoptosis and activates the NF-κB signaling pathway by targeting KLF4 in neonatal pneumonia, and then these results provide evidence for clinical diagnosis and treatment for neonatal pneumonia.

scholarly journals miR-181a-2-3p alleviates the apoptosis of renal tubular epithelial cell via targeting GJB2 in sepsis-induced acute kidney injury

2021 ◽  
Author(s):  
Hui-xing Yi ◽  
Shou-yin Jiang ◽  
Ling-hua Yu ◽  
Kan Chen ◽  
Zeng-xiang Yang ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Inflammatory Response ◽  
Mouse Models ◽  
Cecal Ligation ◽  
Kidney Injury ◽  
Underlying Mechanism ◽  
Tubular Epithelial Cell ◽  
Luciferase Reporter ◽  
Rna Immunoprecipitation ◽  
Renal Tubular

Acute kidney injury (AKI) is the most common complication of sepsis. microRNAs (miRNAs) play important roles in the sepsis-induced AKI. This paper aimed to explore the role of miR-181a-2-3p in the sepsis-induced AKI and the underlying mechanism. Our results revealed that miR-181a-2-3p showed low expression levels in patients with sepsis and mouse models undergoing cecal ligation and puncture (CLP). The addition of miR-181a-2-3p antagonists aggravated the sepsis-induced kidney injuries and inflammatory response in CLP mouse models, as suggested by hematoxylin and eosin (H&E) staining and qRT-PCR. Besides, miR-181a-2-3p mimic alleviated the lipopolysaccharide (LPS)-induced inflammatory response, along with apoptosis of TCMK-1. Moreover, results from the GSE46955 dataset indicated that GJB2 was highly expressed in septic patients, but lowly expressed after recovery. Further, the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were carried out, which confirmed that GJB2 was a target of miR-181a-2-3p, and over-expression of GJB2 reversed the anti-inflammatory and anti-apoptotic effects of miR-181a-2-3p mimic on the LPS-induced sepsis cell models. In conclusion, miR-181a-2-3p alleviates the inflammatory response and cell apoptosis of septic patients and animal models by up-regulating GJB2 expression, which may provide a new therapeutic strategy for sepsis.

scholarly journals SIRT1 attenuates sepsis-induced acute kidney injury via Beclin1 deacetylation-mediated autophagy activation

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiya Deng ◽  
Maomao Sun ◽  
Jie Wu ◽  
Haihong Fang ◽  
Shumin Cai ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Protective Effect ◽  
Cell Model ◽  
Kidney Injury ◽  
Underlying Mechanism ◽  
Autophagy Induction ◽  
Promising Therapeutic Approach ◽  
Related Proteins ◽  
Caecal Ligation And Puncture

AbstractOur previous studies showed that silent mating-type information regulation 2 homologue-1 (SIRT1, a deacetylase) upregulation could attenuate sepsis-induced acute kidney injury (SAKI). Upregulated SIRT1 can deacetylate certain autophagy-related proteins (Beclin1, Atg5, Atg7 and LC3) in vitro. However, it remains unclear whether the beneficial effect of SIRT1 is related to autophagy induction and the underlying mechanism of this effect is also unknown. In the present study, caecal ligation and puncture (CLP)-induced mice, and an LPS-challenged HK-2 cell line were established to mimic a SAKI animal model and a SAKI cell model, respectively. Our results demonstrated that SIRT1 activation promoted autophagy and attenuated SAKI. SIRT1 deacetylated only Beclin1 but not the other autophagy-related proteins in SAKI. SIRT1-induced autophagy and its protective effect against SAKI were mediated by the deacetylation of Beclin1 at K430 and K437. Moreover, two SIRT1 activators, resveratrol and polydatin, attenuated SAKI in CLP-induced septic mice. Our study was the first to demonstrate the important role of SIRT1-induced Beclin1 deacetylation in autophagy and its protective effect against SAKI. These findings suggest that pharmacologic induction of autophagy via SIRT1-mediated Beclin1 deacetylation may be a promising therapeutic approach for future SAKI treatment.

scholarly journals Hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2 Cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian Gong ◽  
Zhi-ming Shen ◽  
Zhe Sheng ◽  
Shi Jiang ◽  
Sheng-lin Ge
Keyword(s):  
Cardiac Surgery ◽  
Inflammatory Response ◽  
Target Gene ◽  
Kidney Injury ◽  
Human Kidney ◽  
Luciferase Reporter ◽  
The Relationship ◽  
Hk2 Cells ◽  
Dual Luciferase Reporter Assay ◽  
Hypoxia Reoxygenation

AbstractThe occurrence of cardiac surgery-associated acute kidney injury (CSA-AKI) increases hospital stay and mortality. MicroRNAs has a crucial role in AKI. This objective of the current study is to explore the function of hsa-miR-494-3p in inflammatory response in human kidney tubular epithelial (HK2) cells with hypoxia/reoxygenation. According to KDIGO standard, patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI (n = 10) and non-AKI patients (n = 8). HK2 were raised in the normal and hypoxia/reoxygenation circumstances and mainly treated by overexpression ofmiR-494-3p and HtrA3. The relationship between miR-494-3p and HtrA3 was determined by dual-luciferase reporter assay. Our result showed that Hsa-miR-494-3p was elevated in the serum of patients with CSA-AKI, and also induced in hypoxic reoxygenated HK2 cells. Hsa-miR-494-3p also increased a hypoxia-reoxygenation induced inflammatory response in HK2 cells. Moreover, as a target gene of miR-494-3p, overexpression of HtrA3 downregulated the hypoxia-reoxygenation induced inflammatory response in HK2 cells. Overexpression of hsa-miR-494-3p-induced inflammatory response was inhibited by overexpression of HtrA3. Collectively, we identified that hsa-miR-494-3p, a miRNA induced in both circulation of AKI patients and hypoxia-reoxygenation-treated HK2 cells, enhanced renal inflammation by targeting HtrA3, which may suggest a possible role as a new therapeutic target for CSA-AKI.

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