Upregulation of lncRNA NONRATG019935.2 suppresses the p53-mediated apoptosis of renal tubular epithelial cells in septic acute kidney injury

Abstract

Although increasing evidence has confirmed that the apoptosis of renal tubular epithelial cells (RTECs) is a crucial contributor to the onset and development of septic acute kidney injury (AKI), the pathological mechanism by which RTEC apoptosis is upregulated during septic AKI is not entirely clear. In this study, a rat model of septic AKI was induced by a cecal ligation puncture procedure or lipopolysaccharide (LPS) injection. Four differentially expressed long noncoding RNAs (DE-Lncs) in the rat model of septic AKI were determined using RNA-sequencing and verified by qRT-PCR. Among the four DE-Lncs, the expression level of lncRNA NONRATG019935.2 (9935) exhibited the most significant reduction in both septic AKI rats and LPS-treated NRK-52E cells (a rat RTEC line). The overexpression of 9935 suppressed cell apoptosis and p53 protein level in LPS-treated NRK-52E cells, and retarded septic AKI development in the rat model of septic AKI. Mechanistically, 9935 decreased the human antigen R (HuR)-mediated Tp53 mRNA stability by limiting the combination of HuR and the 3'UTR region of Tp53 mRNA in RTECs. The overexpression of HuR abrogated the inhibitory effect of pcDNA-9935 on the LPS-induced apoptosis of NRK-52E and rat primary RTECs. In conclusion, 9935 exerts its role in septic AKI by suppressing the p53-mediated apoptosis of RTECs, and this essential role of 9935 relies on its destructive effect on HuR-mediated Tp53 mRNA stability.

Fig. 1. Identification of differentially expressed lncRNAs (DE-Lncs) in cecal ligation puncture (CLP)-engendered acute kidney injury (AKI).

Clustered heat map of the DE-Lncs in renal cortical of A CLP 12 h (n = 3) and B CLP 24 h (n = 3). Rows represent lncRNAs and columns represent tissue types. The color scale runs from blue (low intensity) to white (medium intensity), to red (strong intensity). C There were 69 common DE-Lncs between CLP 12 and 24 h group. D The heat map of the 16 known lncRNAs among 69 common DE-Lncs. E qRT-PCR analysis of the expression levels of 16 known lncRNAs in the renal cortical of rats in each group (n = 5). *P < 0.05, **P < 0.01 vs. sham group.

Fig. 2. Effect of lncRNA NONRATG019935.2 (9935) on LPS-induced renal tubular epithelial cell (RTEC) apoptosis in vitro.

The expression levels of NONRATG019917.2 (9917), NONRATG001687.2 (1687), 9935, and NONRATG009918.2 (9918) in the A renal cortical of control (n = 5), LPS 12 h (n = 5), and LPS 24 h (n = 5) rats; and B rat RTEC line NRK-52E treated with or without 10 µg/ml LPS (12 or 24 h). *P < 0.05, **P < 0.01 vs. control. C The correlations between 9917/9935/1687 and apoptosis-related genes were analyzed by Pearson correlation analysis. Bak1 B-cell lymphoma 2-antagonist/killer 1, Ctsd cathepsin D, Tnfrsf1a tumor necrosis factor receptor superfamily member 1A, Atf4 activating transcription factor 4, Traf2 Tnf receptor-associated factor 2, Apaf1 apoptotic peptidase activating factor 1, Bid BH3 interacting domain death agonist, Fas Fas cell surface death receptor, Nfkbia nuclear factor-kappa B inhibitor alpha, Tp53 tumor protein p53. D The cleaved caspase 3 (c-caspase 3) protein level was measured in LPS-treated NRK-52E cells transfected with pcDNA-9935/9917/1687 overexpressing vector (OVE) or corresponding negative control (pcDNA-NC) using western blot. Caspase 3 was served as a control. *P < 0.05, **P < 0.01 vs. control; ##P < 0.01 vs. LPS + pcDNA-NC. E–G NRK-52E cells were transfected with/without pcDNA-9935 and then treated with or without LPS. E Cell apoptosis was measured by flow cytometry. **P < 0.01 vs. control; #P < 0.05 vs. LPS. F The mRNAs levels of Tp53, Bcl-2 binding component 3 (Puma), Bid, and Apaf1 were measured by qRT-PCR. *P < 0.05 vs. LPS. G The protein levels of phospho-p53 (p-p53), p53, and c-caspase 3 were measured using western blot with β-actin as an internal control. **P < 0.01 vs. control, ##P < 0.01 vs. LPS. H, I NRK-52E cells were divided into four groups: si-NC + LPS, si-NC + LPS + pcDNA-9935, si-p53+LPS, si-p53+LPS + pcDNA-9935. H The protein levels of p-p53, p53, and c-caspase 3. **P < 0.01 vs. LPS + si-NC. I TUNEL staining was performed to determine cell apoptosis. Triangles: TUNEL-positive cells (green). The nuclei were counterstained with DAPI. **P < 0.01 vs. LPS + si-NC.

Fig. 3. Effect of lncRNA 9935 on sepsis-induced kidney injury in vivo.

The rat model of septic AKI was constructed by a CLP procedure or LPS injection. Two weeks before the modeling, lentivirus (LV)-9935 or its negative control (LV-NC) was injected into rats via the tail vein. n = 6 in each group. A, B Representative images of hematoxylin-eosin (H&E) staining and TUNEL staining performed on kidney sections of rats. C, D The quantitation of TUNEL staining was expressed as the mean number of TUNEL-positive cells in ten high-power fields (HPF). The serum levels of creatinine (Cre) and urea were measured by assay kits. E, F Representative images of immunohistochemical staining for p53 and c-caspase 3 performed on kidney sections of rats. G qRT-PCR analysis of the 9935 expression level in the renal tubules of rats. *P < 0.05, **P < 0.01.

Fig. 4. LncRNA 9935 regulated the stability of Tp53.

A Fluorescence in situ hybridization (FISH) was executed to confirm the location of 9935 (red) in NRK-52E cells and rat primary RTECs. Nuclei were stained blue with DAPI. B NRK-52E cells were transfected with pcDNA-NC or pcDNA-9935 and then treated with LPS and cycloheximide (CHX; 5 μg/ml). The protein level of p53 was measured at the indicated time points after CHX administration. C, D NRK-52E cells were divided into four groups: si-NC, si-9935, pcDNA-NC+LPS, and pcDNA-9935+LPS. C The promoter activity of Tp53 was detected by luciferase gene reporter assay. D Each group of cells was treated with actinomycin D (AtcD; 5 μg/ml) and the Tp53 mRNA level was determined by qRT-PCR at the indicated time points. **P < 0.01 vs. pcDNA-NC or si-NC.

Fig. 5. LncRNA 9935 reduced the Tp53 mRNA stability through human antigen R (HuR).

A The dual-luciferase reporter assay was performed on si-9935 (or si-NC)-transfected NRK-52E cells using pGL3 reporters that contain various regions of the Tp53 transcript (full length, 5′UTR, 5’UTR + Open Reading Frame (ORF), 3′UTR, 3′UTR + ORF). *P < 0.05 vs. si-NC. B Expression vectors containing p53 ORF in combination with either the 3′UTR, the 5′UTR, or both (named as full length) were generated and labeled with histone (His). Western bolt analysis for histone was performed on NRK-52E cells co-transfected with si-9935/pcDNA-9935 and indicated vectors. Empty vector was served as the negative control. **P < 0.01 vs. control-tp53 (Full length), ##P < 0.01 vs. control-tp53 (3′UTR + ORF). C Upper: NRK-52E cells were transfected with an antisense biotin-labeled DNA oligomer against 9935 or its negative control (NC, a biotin-labeled sense DNA oligomer probe), followed by the pull-down experiments. The pull-downed complexes were analyzed by western blot with anti-HuR and anti-β-actin antibodies. Below: Lysates of NRK-52E cells were incubated with biotin-labeled 9935 or its antisense RNA, followed by the pull-down experiments. The pull-downed complexes were analyzed by western blot with anti-HuR and anti-β-actin antibodies. **P < 0.01. D RNA immunoprecipitation (RIP) assay was performed to examine the combination of HuR and 9935/Tp53 mRNA in NRK-52E cells. **P < 0.01 vs. Anti-IgG. E–H NRK-52E cells were transfected with indicated vectors, followed by LPS treatment or normal culture. E Tp53 mRNA stability (*P < 0.05, **P < 0.01 vs. pcDNA-NC/si-NC; #P < 0.05, ##P < 0.01 vs. pcDNA-9935/si-9935), F Tp53 mRNA level (*P < 0.05, **P < 0.01, n.s. no significant difference), and G protein levels of p53 and HuR were detected. *P < 0.05, **P < 0.01 vs. pcDNA-NC or si-NC, #P < 0.05, ##P < 0.01 vs. pcDNA-9935. H RIP assay was performed to detect the influence of 9935 on the combination of HuR and Tp53 mRNA. **P < 0.01. I RNA pull-down assay was performed to detect the influence of 9935 on the combination of Tp53 3′UTR and HuR. **P < 0.01 vs. biotin-tp53 3′UTR.

Fig. 6. LncRNA 9935 overexpression suppressed LPS-induced RTEC apoptosis through HuR/p53 axis.

A HuR protein level was measured in the rat model of septic AKI induced by CLP or LPS using western blot. The correlation between the HuR protein level and Tp53 mRNA was assessed. B, C The influence of 9935 and HuR on p53 expression was measured in rat primary RTECs. *P < 0.05,**P < 0.01 vs. LPS + pcDNA-NC or si-NC; ##P < 0.01 vs. LPS + pcDNA-9935 or si-9935. D RIP was performed to detect the influence of 9935 on the combination of HuR and Tp53 mRNA in RTECs. *P < 0.05. E–H The influence of 9935 and HuR on LPS-induced cell apoptosis was performed on primary RTECs and NRK-52E cells using flow cytometry. The representative flow scatters plots and quantitative results were shown. **P < 0.01 vs. LPS + pcDNA-NC; #P < 0.05 vs. LPS + pcDNA-9935.

Fig. 7. Mechanism figure.

9935 is downregulated during septic AKI progression and its overexpression restrains the combination of HuR and 3′UTR region of Tp53, thereby lessening the p53 expression and suppressing the apoptosis of RTECs.