doi: 10.1016/j.ymthe.2020.09.024.
Epub 2020 Sep 20.
A Nuclear Long Non-Coding RNA LINC00618 Accelerates Ferroptosis in a Manner Dependent upon Apoptosis
Affiliations
- PMID: 33002417
- PMCID: PMC7791008
- DOI: 10.1016/j.ymthe.2020.09.024
Item in Clipboard
A Nuclear Long Non-Coding RNA LINC00618 Accelerates Ferroptosis in a Manner Dependent upon Apoptosis
Mol Ther.
.
Abstract
Ferroptosis is primarily caused by intracellular iron catalytic activity and lipid peroxidation. The potential interplay between ferroptosis and apoptosis remains poorly understood. Here, we show that the expression of a nuclear long non-coding RNA (lncRNA), LINC00618, is reduced in human leukemia and strongly increased by vincristine (VCR) treatment. Furthermore, LINC00618 promotes apoptosis by increasing the levels of BCL2-Associated X (BAX) and cleavage of caspase-3. LINC00618 also accelerates ferroptosis by increasing the levels of lipid reactive oxygen species (ROS) and iron, two surrogate markers of ferroptosis, and decreasing the expression of solute carrier family 7 member 11 (SLC7A11). Interestingly, VCR-induced ferroptosis and apoptosis are promoted by LINC00618, and LINC00618 accelerates ferroptosis in a manner dependent upon apoptosis. LINC00618 attenuates the expression of lymphoid-specific helicase (LSH), and LSH enhances the transcription of SLC7A11 after the recruitment to the promoter regions of SLC7A11, further inhibiting ferroptosis. Knowledge of these mechanisms demonstrates that lncRNAs related to ferroptosis and apoptosis are critical to leukemogenesis and chemotherapy.
Keywords: LINC00618; apoptosis; ferroptosis; leukemia; lymphoid-specific helicase; solute carrier family 7 member 11; vincristine.
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
Figures
LINC00618 Is Induced by VCR and Highly Downregulated in Human Acute Myeloid Leukemia (AML) (A) The relative proliferation levels of K562, HL60, and MV4-11 cells checked by MTS assays with the indicated doses of VCR treatment. DMSO served as control. (B) The expression profiling of screened lncRNAs in AML from TCGA-LAML. (C) VCR induced the expression of LINC00618 for 0 h (0 H), 24 h (24 H), and 48 h (48 H) in MV4-11 cells and K562 cells. (D) LINC00618 expression is presented as box plot diagrams in AML patient samples. (E) RNA FISH analysis of LINC00618 localization in MV4-11 cells, K562 cells, and HL60 cells using Cy3-labeled probes. All scale bars, 25 μm long. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t test: ns, nonsignificant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Overexpression of LINC00618 Promotes Cell Apoptosis and Ferroptosis (A) qRT-PCR analyses of stably expressing vector and LINC00618 in MV4-11 cells. (B) qRT-PCR analyses of stably expressing vector and LINC00618 in K562 cells. (C) Statistical analysis of the relative cell viability in MV4-11 cells stably overexpressing LINC00618 and treated by VCR at 48 h. DMSO served as control. (D) Statistical analysis of cell viability in K562 cells stably overexpressing LINC00618 and treated by VCR at 48 h. DMSO served as control. (E) Western blot analyses of caspase-3 and BAX protein level in K562 and MV4-11 cells stably overexpressing LINC00618. (F) Electron microscopy images of mitochondria in LINC00618-overexpressed K562 cell lines. Scale bars,1 μm. (G) FACS and statistical analysis of ROS level in MV4-11 cells stably overexpressing LINC00618. (H) FACS and statistical analysis of relative lipid ROS level in MV4-11 cells stably overexpressing LINC00618. (I) FACS and statistical analysis of relative lipid ROS level in K562 cells stably overexpressing LINC00618. (J) Relative iron level checked by an Iron Assay Kit in MV4-11 cells stably overexpressing LINC00618. (K) Relative iron level checked by an Iron Assay Kit in K562 cells stably overexpressing LINC00618. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t test: ns, nonsignificant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Knockdown of LINC00618 Inhibits Cell Apoptosis and Ferroptosis (A) qRT-PCR analyses of stably knockdown vector and LINC00618 in HL60 cells. (B) Statistical analysis of cell viability in HL60 cells stably overexpressing LINC00618 and treated by VCR at 48 h. DMSO served as control. (C) Western blot analyses of caspase-3 and BAX protein levels in HL60 cells after the knockdown of LINC00618. (D and E) FACS (D) and statistical analysis (E) showed that LINC00618 promoted early apoptosis in HL60 cells after the knockdown of LINC00618. (F) FACS and statistical analysis of relative ROS level in HL60 cells after the knockdown of LINC00618. (G) FACS and statistical analysis of relative lipid ROS level in HL60 cells after the knockdown of LINC00618. (H) Relative iron level checked by an Iron Assay Kit in HL60 cells after the knockdown of LINC00618. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t test: ns, nonsignificant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
LINC00618 Accelerates Ferroptosis in Dependence of Apoptosis (A) Flow cytometry and statistical analysis of apoptosis level in K562 cells stably overexpressing LINC00618 with VCR, VAD, and erastin treatment. (B) Flow cytometry and statistical analysis of relative lipid ROS level in K562 cells stably overexpressing LINC00618 with VCR, VAD, and erastin treatment. (C) Flow cytometry and statistical analysis of apoptosis levels in HL60 cells after the knockdown of LINC00618 with VCR, VAD, and erastin treatment. (D) Flow cytometry and statistical analysis of relative lipid ROS levels in HL60 cells after the knockdown of LINC00618 with VCR, VAD, and erastin treatment. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t test: ns, nonsignificant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
SLC7A11 Is Regulated by LINC00618 through LSH (A) qPCR-based heatmap indicating the changes of ferroptosis-related genes in K562 cells stably overexpressing LINC00618. (B) Western blot analyses of LSH, SLC7A11, and ferroptosis-related proteins in K562 and MV4-11 cells stably overexpressing LINC00618. (C) Western blot analyses of LSH, SLC7A11, and ferroptosis-related proteins in HL60 cells after the knockdown of LINC00618. (D) Protein expression level of LSH and SLC7A11 in four leukemia cells. (E) The correlation between LSH and SLC7A11 mRNA level was measured in 38 AML tissues. (F) The correlation between LSH and SLC7A11 mRNA level was analyzed in TCGA-LAML samples. (G) qPCR analyses of SLC7A11 in CCRF-CEM cells after the knockdown of LSH. (H) Western blot analyses of SLC7A11 in CCRF-CEM cells after the knockdown of LSH. (I) Western blot analyses of LSH and SLC7A11 in K562 cells stably overexpressing LINC00618 with LSH or SLC7A11 overexpression. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t test: ns, nonsignificant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
LINC00618 Recruits LSH to the Promoter Regions of SLC7A11 (A) RIP assays indicated that LINC00618 can be sequestered by LSH in K562 and MV4-11 cells. (B) ChIP-qPCR assays indicate that LSH is recruited to the promoter regions of SLC7A11 in HL60 cells after the knockdown of LINC00618 with SLC7A11 ChIP primer #1. (C) ChIRP enriches for LINC00618. GAPDH served as negative controls. (D) LINC00618 ChIRP-qPCR detects LSH and SLC7A11. (E) Dot blot analyses of LINC00618 ChIRP retrieves LSH and SLC7A11. (F) Working model for LINC00618 in cell apoptosis and ferroptosis. Nuclear LINC00618 amplification reduces LSH expression. Decreased LSH inhibits SLC7A11 transcription and expression through occupying the promoter, leading to cell ferroptosis. Moreover, LINC00618 overexpression increases the expression levels of BAX and cleaved caspase-3 and in turn, triggers apoptosis. Lastly, LINC00618 accelerates ferroptosis independent of apoptosis. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t test: ns, nonsignificant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.