Long Noncoding RNA TCONS_00016406 Attenuates Lipopolysaccharide-Induced Acute Kidney Injury by Regulating the miR-687/PTEN Pathway

Abstract

Acute kidney injury (AKI) is a common and serious complication of sepsis accompanied by kidney dysfunction resulting from various etiologies and pathophysiological processes. Unfortunately, there is currently no ideal therapeutic strategy for AKI. Numerous studies have confirmed that long noncoding RNAs (lncRNAs) play important regulatory roles in the pathogenesis of sepsis-associated AKI. In this study, lncRNA TCONS_00016406 (termed lncRNA 6406), a novel lncRNA identified by using TargetScan, was significantly downregulated in the kidney tissues of mice with sepsis-associated AKI. This study aimed to explore the role of lncRNA 6406 in lipopolysaccharide (LPS)-induced AKI and its potential molecular mechanism. The models of sepsis-induced AKI (called LPS-induced AKI models) in mice and cell lines were established with male C57BL/6 mice and renal tubular epithelial (PTEC) cells, respectively. Twenty-four hours after LPS administration, kidneys and cell samples were collected after various treatments to examine the alterations in the lncRNA 6406 levels and to evaluate the effects on LPS-induced inflammation, oxidative stress, and apoptosis through real-time PCR (RT-PCR) analysis, western blotting, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The results revealed that lncRNA 6406 could significantly attenuate LPS-induced AKI, as shown by the alleviation of inflammation, the suppression of oxidative stress and the inhibition of apoptosis. Mechanistically, a luciferase reporter assay and additional research showed that lncRNA 6406 functioned as a ceRNA to sponge miRNA-687, thereby modulating LPS-stimulated AKI by targeting the miR-687/PTEN axis; thus, this study presents a novel therapeutic strategy or sepsis-associated AKI.

Keywords: PTEN; acute kidney injury; long noncoding RNA 6406; miR-687; sepsis.

Figure 1

Downregulation of long noncoding RNAs (lncRNA) 6406 was observed in the kidneys of mice in the lipopolysaccharide (LPS)-induced acute kidney injury (AKI) model. (A) HE staining was used to observe the outline of renal morphology, scale bar = 20 μm. (B) The relative expression of inflammatory cytokines in the kidneys of mice in the LPS-induced AKI model was detected by real-time PCR (RT-PCR) analysis (n = 6). (C,D) The LPS-induced oxidative stress in the kidneys of mice in the AKI model was determined by Dihydroethidium (DHE) staining and RT-PCR analysis (n = 6), scale bar = 20 μm. (E,F) The TUNEL-positive cells and Bax and Bcl-2 protein expression levels in the LPS-induced AKI model were determined to evaluate apoptosis (n = 6), scale bar = 10 μm. (G) The relative expression of lncRNA 6406 was detected in the AKI mouse kidneys (n = 6). (H) The expression of lncRNA 6406 was confirmed at different time points after LPS injection in the mice (n = 6). (I) The expression level of LncRNA 6406 was determined in different tissues (n = 6). (J) The subcellular localization of lncRNA 6406 was detected by extracting specific subcellular fractions from the PTEC cell line (n = 6). Each experiment was conducted independently three times. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; versus respective control.

Figure 2

The downregulation of LncRNA 6406 was determined in an in vitro cell model of LPS-induced AKI. (A,B) The viability of PTEC cells was evaluated after exposure to different concentrations of LPS (0, 1, 2, 5, and 10 μg/ml) for 24 h and after exposure to 2 μg/ml LPS for different time points (0, 6, 12, 24, and 48 h) (n = 6). (C) The relative expression was determined after exposure to LPS at different concentrations (n = 6). (D) The relative expression of inflammatory cytokines was detected in the LPS-stimulated AKI cell model. (E) Oxidative stress in the PTEC cells was determined after LPS stimulation by RT-PCR analysis (n = 6). (F,G) Apoptosis in the PTEC cells were evaluated via TUNEL assay and western blot analysis (n = 6), scale bar = 10 μm. Each experiment was conducted independently three times. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; versus respective control.

Figure 3

The function of lncRNA 6406 in LPS-stimulated AKI was determined. (A) The transfection efficacies of the lncRNA 6406 overexpressing vector (pcDNA-LncRNA 6406) or knockdown vector (Sh-LncRNA 6406) were verified in the PTEC cells via RT-PCR assays (n = 6). (B,C) LncRNA 6406 overexpression inhibited the LPS-induced upregulation of pro-inflammatory cytokines (including IL-1β, TNF-α, and IL-8) and oxidative stress-related proteins, while lncRNA 6406 knockdown showed the opposite effect on the PTEC cells (n = 6). (D,E) LncRNA 6406 attenuated LPS-induced apoptosis, and lncRNA 6406 inhibition further promoted LPS-induced apoptosis in vitro (n = 6), scale bar = 10 μm. Each experiment was conducted independently three times. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; versus respective control.

Figure 4

MiR-687 negatively regulated the function of lncRNA 6406. (A) The luciferase reporter assay indicated that miR-687 is a target of lncRNA 6406. (B) The transfection efficacy of the miR-687 mimic was determined by RT-PCR analysis (n = 6). (C,D) The miR-687 mimic inhibited the lncRNA 6406 overexpression-induced functional changes in the inflammatory response and oxidative stress (n = 6). (E,F) The miR-687 mimic promoted LPS-induced apoptosis and inhibited the effect of lncRNA 6406 on apoptosis in LPS-stimulated PTEC cells (n = 6), scale bar = 10 μm. Each experiment was conducted independently three times. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; versus respective control.

Figure 5

LncRNA 6406 exerted its effect on AKI by regulating PTEN. (A) RT-PCR analysis revealed the efficacy of the PTEN siRNA (n = 6). (B,C) The inhibition of PTEN inhibited the effect of lncRNA 6406 on inflammation and oxidative stress. (D,E) The inhibition of PTEN increased the LPS-induced apoptosis and attenuated the effect of lncRNA 6406 in PTEC cells (n = 6), scale bar = 10 μm. Each experiment was conducted independently three times. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; versus respective control.

Figure 6

LncRNA 6406 protected against LPS-induced AKI in vivo. (A–C) The relative expression of lncRNA 6406, miR-687 and PTEN was examined via qRT-PCR analysis (n = 6). (D,E) The difference in blood urea nitrogen (BUN) and serum creatinine (SCR) were detected via ELISA after 24 h of the LPS injection in mice. (F) RT-PCR analysis demonstrated that lncRNA 6406 attenuated the LPS-induced inflammatory response in the kidneys of the AKI mice (n = 6). (G) LncRNA 6406 improved the outline of renal morphology in LPS-induced AKI, scale bar = 20 μm. (H,I) RT-PCR analysis and DHE staining revealed that lncRNA 6406 improved the LPS-induced oxidative stress in the kidney (n = 6), scale bar = 20 μm. (J,K) LncRNA 6406 decreased the LPS-induced apoptosis in the kidney in vivo (n = 6), scale bar = 10 μm. Each experiment was conducted independently three times. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; versus respective control.