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MicroRNA 181a-2-3p Alleviates the Apoptosis of Renal Tubular Epithelial Cells via Targeting GJB2 in Sepsis-Induced Acute Kidney Injury.

ABSTRACT: 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 miRNA 181a-2-3p (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 quantitative real-time PCR (qRT-PCR). In addition, miR-181a-2-3p mimic alleviated the lipopolysaccharide (LPS)-induced inflammatory response, along with apoptosis of TCMK-1. Moreover, results from the GSE46955 data set 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 overexpression of GJB2 reversed the anti-inflammatory and antiapoptotic 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 upregulating GJB2 expression, which may provide a new therapeutic strategy for sepsis.

SUBMITTER: Yi HX

PROVIDER: S-EPMC8224234 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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MicroRNA 181a-2-3p Alleviates the Apoptosis of Renal Tubular Epithelial Cells via Targeting GJB2 in Sepsis-Induced Acute Kidney Injury.

Yi Hui-Xing HX Jiang Shou-Yin SY Yu Ling-Hua LH Chen Kan K Yang Zeng-Xiang ZX Wu Qin Q

Molecular and cellular biology 20210623 7

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 miRNA 181a-2-3p (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 i ...[more]

PMID: 33875577

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Project description:BackgroundAcute kidney injury (AKI) is a common complication in patients, especially elderly patients, who undergo cardiac surgery with cardiopulmonary bypass. Studies have indicated a protective role of autophagy in AKI. However, the mechanisms underlying the regulatory effect of autophagy in AKI among patients undergoing cardiac surgeries are poorly understood. In this study, we aimed to test the hypothesis that exosomal microRNAs (miRNAs) regulate autophagy in tubular epithelial cells after AKI.MethodsPlasma exosomal RNA was extracted from young and elderly AKI patients undergoing cardiac surgery, and the miRNAs expression during the perioperative period were analyzed using next-generation sequencing. The screened miRNAs and their target genes were subjected to gene oncology function and Kyoto Encyclopedia of Genes and Genome enrichment analyses. Renal tubular epithelial cell line (HK-2 cells) was cultured and hypoxia/reoxygenation (H/R) model was established, which is an in vitro renal ischemia/reperfusion (I/R) model. We used Western blot analysis, cell viability assay, transfection, luciferase assay to investigate the mechanisms underlying the observed increases in the levels of renal I/R injury-mediated exosomal miRNAs and their roles in regulating HK-2 cells autophagy.ResultsmiR-590-3p was highly enriched in the plasma exosomes of young AKI patients after cardiac surgery. Increased levels of miR-590-3p led to the increases in the expression of autophagy marker proteins, including Beclin-1 and microtubule associated protein 1 light chain 3 beta (LC3II), and prolonged the autophagic response in HK-2 cells after H/R treatment. These effects were achieved mainly via increases in the exosomal miR-590-3p levels, and the tumor necrosis factor receptor-associated factor 6 protein was shown to play a key role in I/R injury-mediated autophagy induction.ConclusionExosomes released from HK-2 cells after renal I/R injury regulate autophagy by transferring miR-590-3p in a paracrine manner, which suggests that increasing the miR-590-3p levels in HK-2 cell-derived exosomes may increase autophagy and protect against kidney injury after renal I/R injury.

Project description:BackgroundSystemic lupus erythematosus (SLE) is a common autoimmune disease that impacts various organs. Lupus nephritis (LN) significantly contributes to death in children with SLE. Toll-like receptor (TLR) adaptor interacting with SLC15A4 on the lysosome (TASL) acts as an innate immune adaptor for TLR and is implicated in the pathogenesis of SLE. A transcription factor known as signal transducer and activator of transcription 3 (STAT3), which is known to be linked to autoimmune diseases, is also involved in the development of SLE.MethodsBioinformatics and real-time quantitative PCR (qRT-PCR) was used to detect the expression of STAT3 and TASL in peripheral blood of SLE patients and their correlation. Bioinformatics analysis, qRT-PCR, luciferase assay and chromatin immunoprecipitation (ChIP) were used to verify the regulation of transcription factor STAT3 on TASL. The expression levels of STAT3, TASL and apoptosis-related genes in LPS-induced HK2 cells were detected by qRT-PCR and Western blot. TUNEL staining were used to detect the apoptosis of HK2 cells after LPS stimulation. ELISA and qRT-PCR were used to detect the levels of inflammatory cytokines in the cell culture supernatant. TASL knockdown in HK2 cells was used to detect the changes in apoptosis-related genes and inflammatory factors. The expression level of TASL in LPS-stimulated HK2 cells and its effect on cell apoptosis and inflammatory factors were observed by knocking down and overexpressing STAT3, respectively. It was also verified in a rescue experiment.ResultsThe expressions of STAT3 and TASL were higher in SLE than in healthy children, and the expression of STAT3 was positively correlated with TASL. Transcription factor STAT3 can directly and positively regulate the expression of TASL through the promoter region binding site. The expression of STAT3, TASL and inflammatory cytokines was elevated, and the change of apoptosis was up-regulated in LPS-stimulated HK2 cells. Inhibition of STAT3 alleviates LPS-stimulated apoptosis and inflammatory response in HK2 cells through transcriptional regulation of TASL.ConclusionsThese findings provide new insights into the transcriptional regulation of TASL and provide new evidence of a direct regulatory relationship between signaling nodes in the lupus signaling network.

Project description:Rho-associated protein kinase 2 (ROCK2) is an important regulator of the inflammatory response and has been reported to serve a role in sepsis. The present study aimed to investigate whether ROCK2 served a role in sepsis-associated acute kidney injury (S-AKI). HK-2 cells were stimulated with lipopolysaccharide (LPS) to simulate S-AKI in vitro. Subsequently, the change in ROCK2 expression levels were determined. ROCK2 in LPS-induced HK-2 cells was knocked down using short hairpin RNA-ROCK2, in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB. Cell viability, cytotoxicity, inflammation and apoptosis were assessed using MTT, lactate dehydrogenase (LDH) release, reverse transcription-quantitative PCR, ELISA, TUNEL and western blotting assays. The protein expression levels of proteins involved in the NF-κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway were also assessed using western blotting. The results demonstrated that ROCK2 was upregulated in HK-2 cells upon LPS treatment. LPS also reduced cell viability, promoted LDH activity and increased TNF-α, IL-6 and IL-1β mRNA expression levels and concentrations. Apoptosis was also induced by LPS as indicated by an increase in the proportion of TUNEL-positive cells, decreased Bcl-2 protein expression levels and increased cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase protein expression levels. However, ROCK2 knockdown in LPS-induced HK-2 cells reversed cell viability damage and inhibited LDH activity, the generation of pro-inflammatory cytokines and apoptosis caused by LPS. Furthermore, ROCK2 knockdown inhibited the LPS-induced expression of phosphorylated-NF-κB p65, NLRP3, apoptosis-associated speck-like protein containing a CARD and caspase-1 p20. PMA treatment reversed all the aforementioned effects of ROCK2 knockdown on LPS-treated HK-2 cells. Therefore, ROCK2 knockdown may alleviate LPS-induced HK-2 cell injury via the inactivation of the NF-κB/NLRP3 signaling pathway.

Project description:Renal ischemia is the initial stage of kidney damage, leading to mitochondrial metabolism disorders and cell necrosis. In this study, we aimed to investigate the biological functions and potential mechanisms of miR-21 in protecting renal tubular epithelial cells from oxidative stress and apoptosis following oxygen glucose deprivation (OGD). Following an OGD injury, miR-21 levels increased in HK-2 renal tubular epithelial cells. Overexpression of miR-21 decreased the protein expressions of cleaved caspase-3, BAX, P53, cell apoptosis and increased Bcl-2 expression in HK-2 cells with OGD injury. In vivo studies found that miR-21 agomir reduced renal tissue apoptosis, while miR-21 antagomir increased it. In addition, overexpression of miR-21 reduced levels of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in HK-2 cells with OGD injury. However, miR-21 inhibition exhibited the opposite effect. A dual-luciferase reporter assay demonstrated that miR-21 directly regulates Toll-like receptor 4 (TLR4) by targeting the 3'-UTR of TLR4 mRNA. Overexpression of miR-21 led to decreased TLR4 protein expression, and TLR4 knockdown was shown to greatly increase AKT activity in HK-2 cells by in vitro kinase assay. Additionally, TLR4 knockdown promoted AKT phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression, while TLR4 overexpression inhibited these processes. Furthermore, AKT activation abolished the effect of TLR4 on HIF-1α, while AKT inhibition decreased the expression of TLR4 on HIF-1α in TLR4 knockdown HK-2 cells. Further study revealed that HIF-1α inhibition abolished the protective effect of miR-21 overexpression on ROS, LDH levels and cell apoptosis in HK-2 cells after OGD injury, which is indicated by increased levels of ROS and LDH, as well as increased cell apoptosis after HIF-1α inhibition in miR-21-treated HK-2 cells. In conclusion, miR-21 defends OGD-induced HK-2 cell injury via the TLR4/AKT/HIF-1α axis.

Dataset Information

MicroRNA 181a-2-3p Alleviates the Apoptosis of Renal Tubular Epithelial Cells via Targeting GJB2 in Sepsis-Induced Acute Kidney Injury.

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MicroRNA 181a-2-3p Alleviates the Apoptosis of Renal Tubular Epithelial Cells via Targeting GJB2 in Sepsis-Induced Acute Kidney Injury.

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