LncRNA TMPO-AS1 facilitates cervical cancer cell tumorigenesis and ferroptosis resistance via interaction with LCN2

Abstract

Ferroptosis, characterized by iron accumulation and lipid peroxidation, has demonstrated anti-tumor properties in multiple malignancies. Long non-coding RNAs play a crucial role in the tumorigenesis and progression of cervical squamous cell cancer; however, the mechanisms underlying the actions of many lncRNAs in ferroptosis remain elusive. Here, the expression level of LICN-TMPO-AS1 in CESC was detected using quantitative real-time polymerase chain reaction. Loss- and gain-of-function experiments with TMPO-AS1 were performed using the CCK-8 assay, transwell assays, clone formation assays, and xenograft models. The relationship between TMPO-AS1, Lipocalin 2, and SFPQ were identified and validated by RNA pull-down/mass spectrometry, co-immunoprecipitation, RNA immunoprecipitation (RIP) assay and western blotting. We found that TMPO-AS1 expression was frequently upregulated in CESC tissues and cells and was strongly associated with poor prognosis. TMPO-AS1 decreased the lipid reactive oxygen species, intracellular Fe²⁺, and malondialdehyde content, leading to the inhibition of sulfasalazine- and erastin-induced ferroptosis. Overexpression of TMPO-AS1 weakened the anti-tumor sensitivity to sulfasalazine by inhibiting ferroptosis both in vitro and in vivo. Mechanistically, TMPO-AS1 bound LCN2 and activated LCN2 expression. Targeting LCN2 reduced iron accumulation and ROS generation in Siha cells. Furthermore, LCN2 regulated the expression of solute carrier family 7 member 11 by interacting with the splicing factor proline and glutamine-rich. Our study illustrates that TMPO-AS1 functions as a tumorigenic regulator and may be a promising therapeutic target for CESC patients with high TMPO-AS1 expression.

Keywords: Cervical squamous cell carcinoma; Ferroptosis; LCN2; Proliferation; TMPO-AS1.

Fig. 1

TMPO-AS1 is highly expressed in CESC and positively correlated with a poor CESC prognosis. A. Heatmap of differentially expressed ferroptosis-related-genes (FRGs) in CESC tissue and cervical cancer-adjacent tissue; B-C. Functional enrichment analysis and KEGG enrichment analyses of FRGs in the TCGA-GETX-CESC cohort; D. Forest map displays prognosis-related lncRNAs. Kaplan–Meier curves for four types of lncRNAs with poor prognosis; E. TMPO-AS1; F. NKILA; G. RNF216P1; and H. RUSC1-AS1 (p < 0.05); Differential expression analysis of I. RUSC1-AS1 and J. TMPO-AS1 in CESC and adjacent tissues; K.RUSC1-AS1 and L.TMPO-AS1 expression in CESC cell lines and the h8 cell line (an immortalized human normal cervix uteri cell line), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 2

TMPO-AS1 inhibits the sensitivity of ferroptosis in CESC cells. A-B. C11 BODIPY of Siha cells exposed to sulfasalazine, cystine (cys(-)) deprivation, erastin, or DMSO. Sh-TMPO-AS1 and OE-TMPO-AS1 developed from knockdown or overexpression of TMPO-AS1 in Siha cells; C-D. FerroOrange of Siha cells exposed to sulfasalazine, cys(-), erastin, or DMSO. The intracellular Fe²⁺ level was relative to the NC group; E. measurements of mitochondrial MDA in Siha cells transfected with vector control (vtr) or sh-TMPO-AS1 after exposure to sulfasalazine (1mM) and DMSO for 48 h; F. transmission electron microscopy observed mitochondrial ultrastructure change in sh-TMPO-AS1 and NC Siha cells under sulfasalazine (1mM) or DMSO treatment after 24 h. Scale bar, 5 μm and 2 μm; G. Protein expression levels of TFR1, ACSL4, SLC7A11, P53and GPX4 were validated via western bolt between the NC group and sh-TMPO-AS1 or OE-TMPO-AS1 Siha cells after treatment with sulfasalazine (1mM) or DMSO; H. RT-qPCR showed the knockdown efficiency of sh-TMPO-AS1 in the Siha cell line; I. RT-qPCR showed the overexpression efficiency of OE -TMPO-AS1 in the Siha cell line. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 3

Knockdown TMPO-AS1 inhibits CESC cell proliferation and migration through ferroptosis. A-C. Cell viability of Siha cells exposed to sulfasalazine, cys(-), erastin; D-E. colony formation assay was used to assess cell survival in sh-TMPO-AS1 and NC after exposure to sulfasalazine (0.25mM) for 2 weeks; F-G. transwell assay of sh-TMPO-AS1 and NC Siha after exposure to sulfasalazine (0.25mM) or DMSO for 5 days; H-I. Scratch assay of sh-TMPO-AS1 and NC Siha after exposure to sulfasalazine (0.25mM) or DMSO for 96 h. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 4

TMPO-AS1 inhibited CESC anti-cancer activity of sulfasalazine in vivo. A. Effect of overexpression of lncRNA TMPO-AS1 on the proliferation of CESC xenografts BALB/c nude mice treated with sulfasalazine or vehicle. Demonstrated by tumor volume B. and tumor weight C. Measurements of ALT (D) AST (E) and BUN (F) in mice treated with sulfasalazine or vehicle. (G) Measurements of mitochondrial MDA in tissues. Effects of sulfasalazine on immunohistochemistry (observed under 40× objective microscope, the scale bar represents 20 μm.) H-K. and western blot L. staining of GPX4, SLC7A11, ASCL4, TFR1, and Ki-67 in CESC xenografts. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 5

TMPO-AS1 performs its biological functions by interacting and regulating LCN2 in CESC. (A) subcellular fragmentation assays and RT-qPCR assay determine the distribution of TMPO-AS1; (B) RNA pull-down proteins by silver staining; (C) western blotting assay of TMPO-AS1-binding protein LCN2 in Siha cells; D-E. RIP assay using Flag-tagged LCN2 confirmed the interaction between TMPO-AS1 and LCN2 in SiHa cells. TMPO-AS1 enrichment in the Input, LCN2-Flag, and IgG groups was assessed by agarose gel electrophoresis and RT-qPCR.F. level of LCN2 mRNA in Siha after overexpressing TMPO-AS1; G. Interference efficiency of LCN2 in OE-TMPO-AS1 Siha cells assessed by RT-qPCR; H-I. FerroOrange of Siha cells exposed to sulfasalazine (1mM) or DMSO; J-K. C11 BODIPY of Siha cells exposed to sulfasalazine (1mM) or DMSO; L-M. transwell assay of Siha after exposure to sulfasalazine (0.25mM) or DMSO for 5 days; N-O. scratch assay of Siha after exposure to sulfasalazine (0.25mM) or DMSO for 6 days; P-Q. colony formation assay was used to assess cell survival after exposure to sulfasalazine (0.25mM) for 2 weeks; R. measurements of mitochondrial MDA in Siha cells after exposure to sulfasalazine (1mM) and DMSO for 48 h; S. protein expression levels of SLC7A11 and GPX4 between siLCN2-OE-TMPO-AS1 and NC groups treated with sulfasalazine (1mM). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 6

Identification of SFPQ as a binding partner of LCN2 in 293T Cells. (A) CoIP assay followed by western blotting to determine the interaction between LCN2 and SFPQ; (B) schematic of TMPO-AS1/LCN2/SFPQ circuit to modulate cervical cancer ferroptosis (Drawing with Fig DRAW 2.0 ID: SPTOUcccfb).