METTL3/m6A/miRNA-873-5p Attenuated Oxidative Stress and Apoptosis in Colistin-Induced Kidney Injury by Modulating Keap1/Nrf2 Pathway

Abstract

Nephrotoxicity of colistin is the major factor limiting its clinical application. However, the exact mechanism of colistin-induced nephrotoxicity is still elusive. N⁶-Methyladenosine (m⁶A) modification has been implicated in many biological processes, however, its role in colistin-induced nephrotoxicity needs to be elucidated. Mouse renal tubular epithelial cells (mRTECs) were treated with 200 μM colistin with or without METTL3 overexpression. Cells injury, m⁶A assay, oxidative stress and apoptosis were examined. Levels of m⁶A are decreased after colistin treatment in mRTECs. METTL3 is the major factor involved in abnormal m⁶A modification. METTL3 overexpression plays a protective role against colistin-induced oxidative stress and apoptosis. Moreover, METTL3 interacts with the microprocessor protein DGCR8 and positively modulates miR-873-5p mature process in an m⁶A-dependent manner. Further experiments show that miR-873-5p could regulate Keap1-Nrf2 pathway against colistin-induced oxidative stress and apoptosis. These studies revealed an important role of METTL3/m⁶A in colistin-induced nephrotoxicity and provide a new insight on m⁶A modification in drug induced toxicity.

Keywords: apoptosis; colistin; m6A modification; miRNA; oxidative stress.

FIGURE 1

Colistin-induced nephrotoxicity in vitro. (A) Assessment of cell viability of mRTECs following a series dose of colistin treatment at different time point by MTT assay. (B) LDH assay. (C) Oxidative stress markers detection. (D) Cyt-c content and (E) DNA fragment measured by ELISA Kit. All the data were represented by the mean ± SD from at least three independent experiments. Values that are significantly different from the values for the control group are indicated by asterisks as follows: ^∗∗p < 0.01.

FIGURE 2

Aberrant m⁶A modification involved in colistin-induced renal injury. (A) Effect of colistin on m⁶A contents of total RNAs in mTRECs. (B) mRNA levels of m⁶A modification-associated genes and immunoblotting of METTL3 after colistin treatment. (C) Effect of METTL3 overexpression on m⁶A contents of total RNAs. Values that are significantly different from the values for the control group are indicated by asterisks as follows: ^∗∗p < 0.01. Values that are significantly different from the values for the colistin group are indicated as follows: ^##p < 0.01.

FIGURE 3

METTL3 overexpression reduces oxidative stress and apoptosis after colistin treatment. (A) Activity of Caspase-3 and Caspase-9 in each group. (B) Apoptosis of mRTECs was analyzed by flow cytometry following annexin V-FITV/PI staining. Q1, necrosis cells; Q2, later apoptotic cells; Q3, live cells; Q4, early apoptotic cells. (C) METTL3 overexpression attenuates colistin-induced ROS production. (D) Oxidative stress markers in each group. (E) Cyt-c content and DNA fragment measurement. (F) Western blot and qPCR analysis of Keap1, Nrf2, and HO-1 expression. The values that are significantly different from the values relative to control group are indicated by asterisks as follows: ^∗∗p < 0.01. Values that are significantly different from the values of colistin-treated group are indicated as follows: ^##p < 0.01.

FIGURE 4

METTL3-dependent m⁶A methylation regulates the processing of mmu-miR-873-5p by DGCR8. (A) Co-IP of the METTL3-interacting protein DGCR8. mREECs were crosslinked before the immunoprecipitation. IgG antibody was used as control. (B) MiR-126 and pri-miR126 were quantified by qPCR in all groups. (C) IP of DGCR8-associated RNA from control or METTL3-overexpressing cells followed by qPCR to detect pri-mmu-miR-873-5p binding toDGCR8. Immunoprecipitation of m⁶A modified RNA in control or METTL3-overexpressing cells followed by qPCR to assess the pri-mmu-miR-873-5p m⁶A modification levels. (D) Bioinformatics analysis of predicted interactions of mmu-miR-873-5p with its binding sites in Keap1. Luciferase activity was measured by dual-luciferase reporter assay. Luciferase activity was normalized to Renilla luciferase activity. (E) Western blot and qPCR analysis of Keap1, Nrf2, and HO-1 expression. The values that are significantly different from the values relative to control group are indicated by asterisks as follows: ^∗∗p < 0.01. Values that are significantly different from the values of colistin-treated group are indicated as follows: ^##p < 0.01.

FIGURE 5

Overexpressed miR-873-5p suppresses oxidative stress and apoptosis. (A) Apoptosis of mRTECs was analyzed by flow cytometry following annexin V-FITV/PI staining. Q1, necrosis cells; Q2, later apoptotic cells; Q3, live cells; Q4, early apoptotic cells. (B) MiR-873-5p overexpression attenuates colistin-induced ROS production. (C) Oxidative stress markers in each group. (D) Activity of Caspase-3 and Caspase-9 in each group. (E) Cyt-c content and DNA fragment measurement. The values that are significantly different from the values for the control group are indicated by asterisks as follows: ^∗∗p < 0.01. Values that are significantly different from the values of colistin-treated group are indicated as follows: ^##p < 0.01.

FIGURE 6

Schematic diagram of the proposed mechanisms of colistin-induced oxidative stress and apoptosis in the present study.