N4-acetylcytidine modification of LINC02802 promotes non-small cell lung cancer progression by modulating mitochondrial NAD+/NADH ratio

Abstract

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cancer progression through their interaction with microRNAs and modulation of gene expression. However, their role in mitochondrial metabolism, particularly in non-small cell lung cancer (NSCLC), remains poorly defined. In this study, we found that LINC02802 was significantly upregulated in NSCLC tissues and associated with poor prognosis. Mechanistically, LINC02802 acts as a competing endogenous RNA (ceRNA) for miR-1976, thereby relieving the suppression of solute carrier family 25 member 51(SLC25A51). Elevated SLC25A51 enhances mitochondrial NAD⁺ import, leading to an increased NAD⁺/NADH ratio and promoting oxidative TCA cycle flux. Functionally, this shift supports tumor cell proliferation and migration. Rescue experiments confirmed that the oncogenic effect of LINC02802 is dependent on the miR-1976/SLC25A51 axis. Interestingly, either silencing LINC02802 with antisense oligonucleotides (ASOs) or treating cells with fludarabine phosphate, an SLC25A51 inhibitor, successfully reversed cisplatin resistance in lung cancer cells. Our findings reveal a novel lncRNA-microRNA-metabolic axis wherein LINC02802 facilitates NSCLC progression by reprogramming mitochondrial metabolism via miR-1976-mediated upregulation of SLC25A51. Targeting this axis may offer therapeutic potential for metabolic intervention in NSCLC.

Keywords: LINC02802; NAD+/NADH ratio; NSCLC; SLC25A51; miR-1976; mitochondrial metabolism.

Figure 1

LINC02802 is highly expressed in lung cancer. (A) Integration of TCGA and GEO datasets to identify LINC02802 as a potential lung cancer-associated lncRNA. (B) Combined TCGA and GTEx analysis of LINC02802 expression across multiple cancer types. (C-D) Analysis of LINC02802 expression in lung adenocarcinoma (LUAD) based on TCGA data. (n=52). (E-F) Validation of LINC02802 expression in lung cancer tissues using in situ hybridization (ISH) and qPCR, ((E) n=35, (F) n=10). (G) qPCR analysis of LINC02802 expression in lung cancer cell lines compared to normal lung epithelial cells. (H-L) Clinical relevance of LINC02802 expression in LUAD patients analyzed using the TCGA database. (M-N) Prognostic significance of LINC02802 expression in LUAD assessed by Kaplan-Meier survival analysis using TCGA data. (O) ROC curve analysis evaluating the diagnostic performance of LINC02802 in LUAD. (P-Q) Analysis of LINC02802 subcellular localization and coding potential using CPC and related databases. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Knockdown of LINC02802 inhibits lung cancer cell proliferation and migration. (A-B) qPCR analysis confirming LINC02802 knockdown efficiency in lung cancer cell lines. (C-D) CCK-8 assays assessing the effect of LINC02802 knockdown on cell proliferation. (E-F) Colony formation assays evaluating the impact of LINC02802 knockdown on clonogenic potential. (G) Wound healing assay assessing the effects of LINC02802 knockdown on migration and invasion. (H-J) LINC02802 knockdown reduces tumor formation in xenograft mouse models. Representative images of tumors (H), tumor volumes (I), and tumor weights (J) are shown. (n = 5 mice per group). Data are presented as mean ± SD (bar plots). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 3

The LINC02802/miR-1976 axis regulates proliferation and migration in lung adenocarcinoma. (A) Bioinformatics analysis combined with RNA pull-down assays to identify miRNAs interacting with LINC02802. (B) RNA pull-down using a LINC02802-specific probe to validate its binding with miR-1976. (C) Dual-luciferase reporter assays confirming direct interaction between LINC02802 and miR-1976. (D-E) TCGA-based analysis of miR-1976 expression and its correlation with LINC02802 in LUAD. (F) ROC curve evaluating the diagnostic potential of miR-1976 in LUAD. (G) Kaplan-Meier survival analysis of miR-1976 in LUAD. (H-I) CCK-8 and colony formation assays demonstrating the regulatory effect of the LINC02802/miR-1976 axis on cell proliferation. (J-K) Transwell assays evaluating the impact of the LINC02802/miR-1976 axis on cell migration and invasion. (L-N) miR-1976 mimics suppress tumor formation in vivo by H1299 cells, Representative images (L), tumor volume (M), and tumor weight (N) are shown. (n = 3 mice per group) Data are presented as mean ± SD (bar plots). sh#1=LINC02802 shRNA#1, sh#2=LINC02802 shRNA#2, Ove=LINC02802 Overexpression. NC= negative control, *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 4

SLC25A51 is a downstream target of the LINC02802/miR-1976 axis. (A-B) Volcano plot and heatmap showing differentially expressed genes identified by transcriptome sequencing after LINC02802 knockdown. (C) RNA-seq and bioinformatic analysis to predict miR-1976 downstream target genes. (D) Dual-luciferase reporter assays confirming the binding of miR-1976 to the 3′UTR of SLC25A51. (E) TCGA-based expression analysis of SLC25A51 in LUAD. (F) Correlation analysis between LINC02802 and SLC25A51 expression in LUAD. (G-I) Expression changes of SLC25A51 following LINC02802 knockdown or miR-1976 overexpression. (J) Expression analysis of SLC25A51 after overexpression of LINC02802 and miR-1976. Data are presented as mean ± SD (bar plots). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 5

Knockdown of SLC25A51 suppresses NSCLC progression. (A) qPCR analysis of SLC25A51 expression in lung cancer cell lines compared to normal lung epithelial cells. (B) qPCR analysis confirming SLC25A51 knockdown efficiency in lung cancer cell lines. (C) CCK-8 assays assessing the effect of SLC25A51 knockdown on cell proliferation. (D-F) Transwell and wound healing assay assessing the effects of SLC25A51 knockdown on migration and invasion. (G) Flow cytometry analysis of apoptosis in lung cancer cells following SLC25A51 knockdown. (H) Changes in MitoSOX fluorescence intensity and NAD⁺/NADH ratio in SLC25A51 knockdown cell lines. (I-K) fludarabine phosphate effectively inhibited NSCLC tumor growth, combined treatment with fludarabine phosphate and cisplatin (DDP) resulted in a more pronounced suppression of tumor growth, as evidenced by reduced tumor volume and size, Representative images of xenograft tumors (J), tumor volume measurements (F), and tumor weights (K) are shown. (n = 4 mice per group) Data are presented as mean ± SD (bar plots). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 6

LINC02802 regulates mitochondrial metabolism by modulating SLC25A51 expression. (A-B) Changes in MitoSOX fluorescence intensity and NAD⁺/NADH ratio in LINC02802 knockdown cell lines. (C) Western blot analysis of mitochondrial electron transport chain complex proteins after LINC02802 knockdown. (D-G) Alterations in tricarboxylic acid (TCA) cycle intermediates in LINC02802 knockdown cell lines. (H) Assessment of mitochondrial respiratory capacity in LINC02802 knockdown cell lines. Data are presented as mean ± SD (bar plots). sh#1=LINC02802 shRNA#1, sh#2=LINC02802 shRNA#2, Ove=LINC02802 Overexpression. NC= negative control,*P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 7

LINC02802 promotes malignant phenotypes of lung cancer cells by upregulating SLC25A51 expression. (A-B) Clonogenic and CCK-8 assays demonstrating the role of the LINC02802/SLC25A51 axis in promoting lung cancer cell proliferation. (C-D) Transwell assays showing that the LINC02802/SLC25A51 axis enhances the migratory capacity of lung cancer cells. (G-J) Wound healing assays further validating the involvement of the LINC02802/SLC25A51 axis in lung cancer cell migration. sh#1=LINC02802 shRNA#1, sh#2=LINC02802 shRNA#2, Ove=LINC02802 Overexpression. Data are presented as mean ± SD (bar plots). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 8

Targeting LINC02802 reverses cisplatin resistance in lung cancer cells. (A) qPCR analysis confirming the knockdown efficiency of LINC02802-specific antisense oligonucleotides (ASOs) in lung cancer cells. (B) CCK-8 assay showing the inhibitory effect of LINC02802-specific ASOs on cell proliferation. (C) Colony formation assay indicating reduced colony-forming ability following ASO treatment. (D) Transwell assay demonstrating decreased migration and invasion of lung cancer cells upon LINC02802 knockdown. (E-G) LINC02802 knockdown suppresses tumor growth in vivo. Representative images of xenograft tumors (E), tumor volume measurements (F), and tumor weights (G) are shown. (n = 5 mice per group) Data are presented as mean ± SD (bar plots). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Figure 9

NAT10 mediates Ac4C modification of LINC02802, contributing to its stability. (A) TCGA-based correlation analysis of NAT10 and LINC02802 expression in LUAD tissues. (B-C) qPCR analysis showing decreased LINC02802 expression following NAT10 knockdown. (D) qPCR results of LINC02802 levels after treatment with a NAT10 inhibitor. (E-F) RNA pull-down and RIP-qPCR assays validating the interaction between NAT10 and LINC02802. (G) Ac4C-RIP-qPCR assays confirming the presence of Ac4C modification on LINC02802 transcripts. (H-I) RNA stability assays showing a reduced half-life of LINC02802 following NAT10 knockdown. Data are presented as mean ± SD (bar plots). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.