CircDTL Functions as an Oncogene and Regulates Both Apoptosis and Ferroptosis in Non-small Cell Lung Cancer Cells

Abstract

Background: Circular RNAs (circRNA) play an essential role in the tumorigenesis of non-small cell lung cancer (NSCLC). CircDTL is a novel identified circRNA with little information regarding its biological role. However, the role of circDTL in NSCLC has not been investigated yet. Method: In this study, the levels of circDTL in tissues and cells were measured by RT-PCR. Cell viability was measured by the CCK-8 assay. Cell migration and invasion were evaluated using the wound healing assay and transwell assay, respectively. Cell death was measured by the cell death ELISA kit. The levels of Fe²⁺, ROS, MDA and GSH were measured using the commercial kits. The interactions between miR-1287-5p and circDTL/3'UTR GPX4 were verified by dual-luciferase activity assay. The effects of circDTL on tumor growth were evaluated in vivo. Results: CircDTL was found to be upregulated and acted as an oncogene in NSCLC cells. Knockdown of circDTL promoted both apoptosis and ferroptosis of NSCLC cells. It was identified that circDTL exerts its oncogenic effects via the circDTL/miR-1287-5p/GPX4 axis and GPX4 inhibits both ferroptosis and apoptosis. Finally, this study showed that silencing of circDTL promoted the sensitivity of NSCLC cells to chemotherapeutic agents and inhibited the growth of tumors in vivo. Conclusion: CircDTL acts as an oncogene and exerts its effects via the miR-1287-5p/GPX4 axis in NSCLC, providing a potential therapeutic target for NSCLC cancer therapy.

Keywords: apoptosis; circDTL; ferroptosis; miRNA; non-small cell lung cancer.

FIGURE 1

circDTL was upregulated and acted as an oncogene in NSCLC. (A) Levels of circDTL in 84 pairs of NSCLC and adjacent tissues. (B) Levels of circDTL in normal cell line BEAS-2B and NSCLC cell lines. (C) NSCLC cells were transfected with shNC or shRNA against circDTL (sh#1, sh#2) for 24 h, levels of circDTL were measured. (D) NSCLC cells were transfected as indicated and cell viability was measured at different time points. (E) NSCLC cells were transfected as indicated, cell migration was assayed. (F) Cell invasion was measured. Data were presented as mean ± SD. Experiments were performed at least three times. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 2

Silencing of circDTL induced apoptosis and ferroptosis of NSCLC cells. (A) NSCLC cells were transfected as indicated with or without different inhibitors for 24 h, cellular death was assayed. (B) NSCLC cells were transfected as indicated 24 h, caspase-3 activities were measured. (C) Apoptosis-related proteins were measured by western blots. (D) NSCLC cells were transfected as indicated and treated with or without ferroptosis inhibitors (Fer-1, Lip-1) for 24 h, ROS levels were measured. (E) MDA levels. (F) GSH levels. (G) Fe²⁺ levels. Data were presented as mean ± SD. Experiments were performed at least three times. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 3

circDTL negatively regulates the expression of miR-1287-5p in NSCLC cells. (A) Cellular location of GAPDH, U6, and circDTL was assayed in NSCLC cells. (B) RNA pull-down assay was used for the detection of circDTL. (C) RNA pull-down was performed to examine different miRNAs that bind with circDTL. (D) Predictive binding sites between circDTL and miR-1287-5p (left), the interaction between miR-1287-5p and circDTL was evaluated by dual-luciferase reporter assay (right). (E) NSCLC cells were transfected with shNC or shRNAs against circDTL (sh#1, sh#2) for 24 h, levels of miR-1287-5p were measured. (F) The expression of miR-1287-5p in 84 pairs of NSCLC and adjacent normal tissues. Data were presented as mean ± SD. Experiments were performed at least three times. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 4

miR-1287-5p targets GPX4 to regulate both apoptosis and ferroptosis of NSCLC cells. (A) Predictive binding sites between miR-1287-5p and 3′-UTR of GPX4 (left), the interaction between miR-1287-5p and 3′-UTR of GPX4 was evaluated by dual-luciferase reporter assay (right). (B) NSCLC cells were transfected as indicated for 24 h, mRNA levels of GPX4 were measured by RT-PCR. (C) Protein levels of GPX4 were measured. (D) mRNA levels of GPX4 were measured in NSCLC and adjacent normal tissues. (E) RNA pull-down was conducted to evaluate the GPX4 mRNA that binds with miR-1287-5p. (F) NSCLC cells were transfected with shNC or shRNAs against circDTL (sh#1, sh#2), protein levels of GPX4 were measured. (G) mRNA levels of GPX4 were measured. (H) Protein levels of GPX4 were measured. (I) Cell viabilities were measured at different time points. (J) cell migration distance was calculated. (K) Several invaded cells were measured. (L) Cell death was measured. (M) the release of mitochondrial proteins was measured. (N) Levels of ROS, MDA, GSH, and Fe² + were measured. Data were presented as mean ± SD. Experiments were performed at least three times. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 5

Silencing of circDTL increased the chemosensitivity of NSCLC cells and inhibited the growth of NSCLC in vivo. (A) NSCLC cells were exposed to different chemotherapy agents with or without z.VAD for 24 h and cellular death was measured. (B) NSCLC cells were exposed to RSL3 or Erastin with or without Fer-1/Lip-1 for 24 h, cellular deaths were measured. (C) NSCLC cells stably transfected with shNC or shcircDTL and inoculated into nude mice, tumor volumes were measured at different time points. (D) Xenografts were subjected to western blotting analysis. Data were presented as mean ± SD. Experiments were performed at least three times. ^∗P < 0.05; ^∗∗P < 0.01.