Ferroptosis‑related long non‑coding RNAs and the roles of LASTR in stomach adenocarcinoma

Abstract

Ferroptosis is a form of programmed cell death that participates in diverse physiological processes. Increasing evidence suggests that long noncoding RNAs (lncRNAs) regulate ferroptosis in tumors, including stomach adenocarcinoma (STAD). In the present study, RNA‑sequencing data from The Cancer Genome Atlas database and ferroptosis‑related markers from the FerrDb data resource were analyzed to select differentially expressed lncRNAs. Univariate and multivariate Cox regression analyses were performed on these differentially expressed lncRNAs to screen 12 lncRNAs linked with overall survival (OS) and 13 associated with progression‑free survival (PFS). Subsequently, two signatures for predicting OS and PFS were established based on these lncRNAs. Kaplan‑Meier analyses indicated that the high‑risk group of patients with STAD had relatively poor prognosis. The areas under the receiver operating characteristic curves of the two signatures indicated their excellent efficacy in predicting STAD prognosis. In addition, the effect of the lncRNA LASTR on proliferation and migration in gastric cancer was confirmed and the relationship between LASTR and ferroptosis was initially explored through experiments. These results provide potential novel targets for tumor treatment and promote personalized medicine.

Keywords: LASTR; TCGA; ferroptosis; lncRNA; prognosis signature; stomach adenocarcinoma.

Figure 1.

Expression and enrichment analyses of differentially expressed lncRNAs in individuals with stomach adenocarcinoma. (A) Heatmap of ferroptosis-related markers. (B) Heatmap of ferroptosis-related lncRNAs. (C) Bubble chart illustrating the top 10 most significant terms in the Gene Ontology functional analysis, consisting of biological process, cellular component and molecular function; the x-axis refers to the ratio of lncRNAs abundant in the matching function. (D) Bubble chart illustrating the top 30 most significant Kyoto Encyclopedia of Genes and Genomes pathway terms of ferroptosis-related lncRNAs; the x-axis refers to the ratio of lncRNAs abundant in the matching function. lncRNA, long noncoding RNA; N, normal tissue; T, tumor tissue.

Figure 2.

Establishment of 12-differentially expressed ferroptosis-related lncRNA-based OS signature. (A) Univariate Cox regression analysis established 27 ferroptosis-related lncRNAs significantly associated with OS. (B) Time-based ROC curves of the OS signature at 1, 3 and 5 years. (C) Kaplan-Meier survival plots illustrate OS differences between low-risk and high-risk groups. (D) Levels of expression of lncRNAs in the high-risk and low-risk groups according to the OS signature. (E) OS scatter plots for individuals with stomach adenocarcinoma. (F) Risk score distribution of patients according to OS signature. OS, overall survival; lncRNA, long noncoding RNA; AUC, area under the ROC curve; ROC, receiver operating characteristic.

Figure 3.

Establishment of 13-differentially expressed ferroptosis-related lncRNA-based PFS signature. (A) Univariate Cox regression uncovered 40 ferroptosis-related lncRNAs that were significantly associated with PFS. (B) Time-based ROC curves of the PFS signature at 1, 3 and 5 years. (C) Kaplan-Meier survival plots illustrate PFS differences between the low-risk and high-risk groups. (D) Levels of lncRNA expression in the high-risk and low-risk groups according to the PFS signature. (E) PFS scatter plots for individuals with stomach adenocarcinoma. (F) Risk score distribution of patients according to PFS signature. PFS, progression-free survival; lncRNA, long noncoding RNA; AUC, area under the ROC curve; ROC, receiver operating characteristic.

Figure 4.

Development of nomogram combining the DEFRL-based signature with independent predictive clinical variables to estimate OS in individuals with stomach adenocarcinoma. (A) Univariate regression of DEFRL-based prognostic signature along with clinical factors. (B) Multivariate regression of the significant characteristics in the univariate regression. (C) Decision Curve Analysis of the risk factors with OS. (D) Nomogram of OS integrating the OS signature with the two clinical variables of patients. (E) Heatmap illustrating ferroptosis-related lncRNAs by OS signature and clinicopathological manifestations. (F) Regulatory network of ferroptosis-related markers and OS-linked ferroptosis-related lncRNAs. *P<0.05, **P<0.01 and ***P<0.001. OS, overall survival; lncRNA, long noncoding RNA; DEFRL, differentially expressed ferroptosis-related lncRNAs; Pr, probability.

Figure 5.

Development of nomogram combining DEFR-based signature with independent predictive clinical variables to predict PFS in individuals with stomach adenocarcinoma. (A) Univariate regression of the DEFRL-based predictive signature along with clinical factors. (B) Multivariate regression of the significant characteristics in the univariate Cox analyses. (C) DCA of the risk factors with PFS. (D) Nomogram of PFS integrating the PFS signature with the two clinical variables of patients. (E) Heatmap illustrating the ferroptosis-related lncRNA PFS signature and clinicopathological manifestations. (F) Modulatory network of ferroptosis-related markers and PFS-linked ferroptosis-related lncRNAs. *P<0.05, **P<0.01, and ***P<0.001. PFS, progression-free survival; lncRNA, long noncoding RNA; DEFRL, differentially expressed ferroptosis-related lncRNAs; Pr, probability.

Figure 6.

High expression of LASTR predicts poor prognosis in individuals with stomach adenocarcinoma. (A) LASTR expression in gastric cancer tissues and neighboring non-malignant tissues from The Cancer Genome Atlas database. (B) Kaplan-Meier survival plots illustrating the differences in OS between low-level and high-level LASTR. (C) Kaplan-Meier survival plots illustrating PFS differences between low-level and high-level LASTR. STAD, stomach adenocarcinoma; LASTR, lncRNA associated with spliceosome associated factor 3, U4/U6 recycling protein regulation of splicing.

Figure 7.

Impact of LASTR knockdown on the growth of gastric cancer cells. (A and B) LASTR RNA expression in (A) AGS or (B) MKN7 cells transfected with siLASTR and siNC was confirmed by reverse transcription-quantitative PCR. (C and D) Comparison of the proliferative potential between the siLASTR and siNC groups of (C) AGS or (D) MKN7 cells by an EdU incorporation assay. (E and F) Comparison of the clonogenic potential between the siLASTR and siNC groups of (E) AGS or (F) MKN7 cells by a colony formation assay. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. LASTR, lncRNA associated with spliceosome associated factor 3, U4/U6 recycling protein regulation of splicing; siLASTR, small interfering RNA targeting LASTR; NC, negative control; RFP, red fluorescent protein; EdU, 5-ethynyl-2′-deoxyuridine.

Figure 8.

Impact of LASTR knockdown on the migration and the ferroptosis of gastric cancer cells. (A and B) Wound-healing assays comparing the migration distance between the siLASTR and siNC groups of (A) AGS or (B) MKN7 cells. (C and D) Transwell assay comparing the number of migrated cells between the siLASTR and siNC groups of (C) AGS or (D) MKN7 cells. (E and F) Comparison of the protein expression of GPX4 between the siLASTR and siNC groups of (E) AGS or (E) MKN7 cells by western blot assay. *P<0.05 and **P<0.01. LASTR, lncRNA associated with spliceosome associated factor 3, U4/U6 recycling protein regulation of splicing; siLASTR, small interfering RNA targeting LASTR; NC, negative control; GPX4, phospholipid hydroperoxide glutathione peroxidase 4.