Fig 1.
ChREBP expression in human LUAD cell lines.
(A) Expression levels of ChREBP (MLXIPL) in 66 human LUAD cell lines, derived from RNA-seq datasets available in the EMBL-EBI Expression Atlas, are presented in transcripts per million (TPM). (B-F) Expression of ChREBP in NCI-H1975, NCI-H1650, NCI-H2228 and HepG2 cells assessed using RT-qPCR assays. (B) Expression level of seven candidate HKGs. Cycle threshold (Ct) values are displayed. (C) Average expression stability values of seven HKGs determined by geNorm are shown. Expression levels of total ChREBP (D), ChREBP-α (E), and ChREBP-β (F) were normalized against the three most stable HKGs, RPS13, QARS, and RNA18S. Data represents the results of three independent experiments. Asterisks (*) indicate a significance level of p < 0.05 compared to the expression levels in the HepG2 cell line.
Fig 2.
Impact of dominant negative ChREBP (dnChREBP) overexpression on colony formation ability and migration in NCI-H1975 LUAD cells.
(A) Schematic representation of doxycycline-inducible vectors used for overexpression of dnChREBP. LTR, long terminal repeat; TRE, tetracycline-responsive promoter element; UBC, human ubiquitin C promoter; rtTA3, reverse tetracycline-transactivator 3; IRES, internal ribosomal entry site; PuroR, puromycin resistance gene; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; SIN LTR, self-inactivating long terminal repeat. (B) Amino acid alignment of human ChREBP-α and ChREBP-β. bHLH, basic Helix-Loop-Helix-Leucine; ZIP, leucine zipper. (C) Outline of the process used to generate dnChREBP-overexpressing NCI-H1975 cells. (D-E) Colony formation assay. Macroscopic visualization of the crystal violet-stained colonies is shown (D). Violin plot showing the intensity distribution of stained colonies formed in dnChREBP-overexpressing NCI-H1975 cells compared to control cells (E). A significant difference (p-value < 0.01) is indicated. (F-G) Transwell migration assay. Representative images of migrated control and dnChREBP cells at 20x magnification is shown (F). The bar graph shows the percentage of migrated cells, with data representing the mean ± SD from three independent experiments (G).
Fig 3.
Transcriptome analysis of NCI-H1975 LUAD cells overexpressing dnChREBP.
(A) Brief diagram outlining the key steps of RNA sequencing analysis. (B) Heatmap displaying sample-to-sample distances calculated from the variance-stabilizing transformation of count data for overall gene expression. (C) Volcano plot illustrating differentially expressed genes (DEGs); red dots represent upregulated genes, blue dots indicate downregulated genes, and grey dots denote unchanged genes (adjusted p-value < 0.05). (D) Chromosomal localization of DEGs. The DEGs are plotted at their chromosomal positions.
Fig 4.
Enrichment analysis of DEGs associated with dnChREBP overexpression in NCI-H1975 cells.
(A) Gene ontology (GO) analysis displaying the enriched biological processes related to DEGs, indicating fold enrichment and significance levels (−log10 adjusted p-value). (B) Hallmark gene sets highlighting significant pathways associated with the expression changes. The gene ratio and adjusted p-values are shown. (C) Interaction network of hub genes, illustrating their interconnections and potential regulatory relationships. (D) Venn diagram illustrating the overlap of genes associated with the EMT Hallmark, the extracellular space GO term, and the identified hub genes.
Fig 5.
Alteration of expression levels of selected genes in dnChREBP-overexpressing NCI-H1975 cells.
(A-D) RNA-seq results. Bar graphs depict the Log2 FC in genes enriched in the extracellular space GO term (A), genes enriched in the Hallmark EMT gene set (B), hub genes (C), and ChREBP target genes (D). (E) Gene expression analysis using RT-qPCR was conducted on selected genes to validate the RNA-seq findings. Gene expression levels were normalized against the three most stable HKGs, RPL13a, ACTB, and B2M. Asterisks (*) denote statistically significant differences (p < 0.05) compared to the expression levels in control cells.
Fig 6.
Prognostic significance of ChREBP and its associated genes in LUAD.
Kaplan–Meier plot demonstrates the median overall survival (OS) of LUAD patients. Only genes with statistical significance (p < 0.05) and a false discovery rate (FDR) of 1% are shown. (A) ChREBP-α (MLXIPL). (B-E) Top 10 downregulated genes by dnChREBP in NCI-H1975 cells: KLHL4 (B), SHROOM4 (C), TMEM47 D) and ARMCX2B (E). (F-J) Top 10 upregulated genes by dnChREBP in NCI-H1975 cells: IRX2 (F), CD99P1 (G), PELI2 (H), NFASC (I), and TFPT (J). (K-O) Hub genes: SPP1 (K), PECAM1 (L), FLG (M), CCN2 (N), IL1A (O).