Long non-coding RNA MEG3 promotes cisplatin-induced nephrotoxicity through regulating AKT/TSC/mTOR-mediated autophagy

Abstract

Cis-Diamminedichloroplatinum (II) (DDP)-induced nephrotoxicity (DDPIN) may cause irreversible renal injury associated with high morbidity and mortality. Current standard therapies have not achieved satisfactory clinical outcomes due to unclear molecular and cellular mechanisms. Therefore, exploring potential therapies on DDPIN represents an urgent medical need. Present study characterized the role of lncRNA maternally expressed gene 3 (lnc-MEG3) in the pathogenesis of DDPIN. In both in vitro and in murine models of DDP-induced nephrotoxicity, lnc-MEG3 exacerbated DDPIN by negatively regulating miRNA-126 subsequently causing a decreased AKT/TSC/mTOR-mediated autophagy. By silencing lnc-MEG3 or incorporating miRNA-126 mimetics, the proliferation and migration of DDP-treated cells were restored. In vivo, we identified Paeonol to alleviate DDPIN by the inhibition of lnc-MEG3. Taken together, lnc-MEG3 represents a novel therapeutic target for DDPIN and Paeonol may serve as a promising treatment by inhibiting lnc-MEG3 and its related signaling pathways.

Keywords: DDPIN; autophagy; lncRNA MEG3; miRNA-126.

Figure 1

lnc-MEG3 is upregulated significantly and lnc-MEG3 silencing alleviates DDP-induced inflammation and apoptosis. (A) lnc-MEG3 expressions were visualized by R package “pheatmap” based on the RNA sequence from GEO database. Relative mRNA levels of lnc-MEG3 in DDP-treated HK-2 cells (B), the kidneys of DDPIN mice (C) on RT-PCR analysis. (D) FISH assays of lnc-MEG3 location at the cytoplasm fractionation of proximal tubule epithelial cells. Relative mRNA levels of lnc-MEG3 silenced HK-2 cells on RT-PCR analysis (E). Flow cytometry (F) and data analysis (G) of apoptosis in DDPIN or/and lnc-MEG3 silenced HK-2 cells. (H) RT-PCR analysis of IL-1, IL-6, IL-10, IL-18, MCP-1 and TNF-α. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DDP-treated control (n=3).

Figure 2

AKT/TSC/mTOR-mediated autophagy is impaired in DDPIN. (A and B) Western blot analysis of p-AKT, AKT, TSC2, TSC1, TBC1D7, p- mTOR, mTOR, Atg5, Beclin-1 and LC3 in the kidneys of DDPIN mice. (C) Representative images showing LC3 staining in the kidneys of DDPIN mice. (D-F) Co-immunofluorescence staining for TSC2 and tubular segment-specific markers in the kidney of DDPIN. The following segment-specific tubular markers were used: proximal tubule, aquaporin-1 (AQP1); distal tubule, calbindin D28k; and collecting duct, aquaporin-3 (AQP3). (G-H) The data analysis of LC3 and TSC2. *P < 0.05, **P < 0.01, ***P < 0.001 versus control (n=3).

Figure 3

lnc-MEG3 silencing reverse the impaired AKT/TSC/mTOR-mediated autophagy induced by DDP. (A and B) Western blot analysis of p-AKT, AKT, TSC2, TSC1, TBC1D7, p-mTOR, mTOR, Atg5, Beclin-1 and LC3 in the DDP-treated and/or lnc-MEG3 silenced HK-2 cells. (C) Immunofluoresence of LC3 staining in the DDP-treated HK-2 cells under the condition of lnc-MEG3 silence. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DDP-treated control (n=3).

Figure 4

miRNA-126 is negatively regulated by lnc-MEG3 and ablates DDP-induced autophagy depression. (A) The targeting relationship between lncRNA MEG3 and miRNA-126. Relative mRNA level of miRNA-126 in HK-2 cells (B) and the kidney of DDPIN mice (C). The luciferase activity in the MEG3-WT and MEG3-MUT compared to control group, and the elements responsible for miR126 expression are between -1871 and -1889 as shown in (D) and (E). Negative correlation between lnc-MEG3 and miRNA-126 in vivo (F) and in vitro (G). Relative mRNA level of miRNA-126 under the condition of lnc-MEG3 silencing or miRNA-126 mimics in DDP-treated HK-2 cells, respectively (H and I). (J and K) Western blot analysis of p-AKT, AKT, TSC2, TSC1, TBC1D7, p-mTOR, mTOR, Atg5, Beclin-1 and LC3 in the DDP-treated and/or miRNA-126 mimics HK-2 cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DDP-treated control (n=3).

Figure 5

lnc-MEG3 silencing and miRNA-126 mimics are beneficial to the proliferation and migration of the DDP-treated HK-2 cells. (A and B) CCK8 assay, (C-F) Colony formation assay, Transwell assay of HK-2 cell (G-J) in the condition of lnc-MEG3 silence or miRNA-126 mimics. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DDP-treated control (n=3).

Figure 6

Pae functions as an effective protection in DDPIN. HE staining, (A and B) TUNEL staining, (C and D), Immunohistochemical staining of neutrophils and CD68+ macrophages (E and F) and RT-PCR analysis (G) of IL-1, IL-6, IL-10, IL-18, MCP-1 and TNF-α in DDP or/and Pae-treated (15 mg/kg and 30 mg/kg) mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DDP-treated control (n=3).

Figure 7

Pae protects against DDPIN through regulating lnc-MEG3 /miRNA-126/mTOR-mediated autophagy. Relative mRNA level of lnc-MEG3 (A) and (B) miRNA-126 in DDP or/and Pae-treated (15 mg/kg and 30 mg/kg) mice. (C and D) Western blot analysis of p-AKT, AKT, TSC2, TSC1, TBC1D7, p-mTOR, mTOR, Atg5, Beclin-1 and LC3 in the DDP or/and Pae-treated (15 mg/kg and 30 mg/kg) mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DDP-treated control (n=3).