Journal: Advanced Science

Article Title: Novel Evolutionarily Conserved Oncogene COA4 is Driven by KRAS Mutant and Promotes Cancer Metastasis Through Dual Mitochondrial Metabolism‐Dependent and ‐Independent Mechanisms

doi: 10.1002/advs.202507533

Figure Lengend Snippet: E2F1 directly binds to COA4 promoter and promotes its expression under KRAS mutation. A,B) Western blot (A) and RT‐qPCR (B) analyses of COA4 expression in A549 cells following E2F1 overexpression or knockdown. ( n = 3; ** p < 0.01; **** p < 0.0001) C) Luciferase reporter assay confirming that E2F1 directly regulates COA4 transcription in 293T and A549 cells. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) D) Luciferase reporter assay demonstrating that KRAS enhances E2F1‐mediated transcriptional activation of the COA4 promoter in 293T cells. ( n = 3; **** p < 0.0001) E) ChIP‐qPCR analysis identifying the specific E2F1 binding region within the COA4 promoter in A549 cells. ( n = 3; *** p < 0.001) F) AlphaFold 3 molecular docking of the COA4 promoter with E2F1, supporting their direct interaction. G) Chromatin immunoprecipitation followed by qPCR (ChIP‐qPCR) was performed to evaluate E2F1 binding to the COA4 promoter in normal tissues and KRAS‐mutant tumor tissues. Data are presented as mean ± SD ( n = 6, *** p < 0.001) H) Spearman's rank correlation analysis of COA4 and E2F1 expression in LUAD using the GEPIA2 database. I. IHC analysis of E2F1 expression in LUAD tumor versus normal tissues ( n = 12 per group; **** p < 0.0001) J) ATP quantification in E2F1 ‐overexpressing A549 cells following COA4 knockdown.( n = 3; ** p < 0.01) K) Complex IV enzyme activity in E2F1 ‐overexpressing A549 cells after COA4 knockdown. ( n = 3; ** p < 0.01) L,M) Western blot (L) and RT‐qPCR (M) analyses of COA4 expression in KRAS G12C ‐overexpressing A549 cells following E2F1 knockdown. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) N) Western blot analysis of COA4 expression in A549 cells treated with the PI3K agonist 740Y‐P(15 µM) for 48 h (left), and in 740Y‐P‐treated A549 cells following E2F1 knockdown (right).( n = 3) O) Western blot analysis of COA4 expression in A549 and H23 cells treated with the PI3K inhibitor LY294002 following E2F1 overexpression. ( n = 3) P) RT‐qPCR analysis of COA4 mRNA levels in A549 cells treated with LY294002 following E2F1 overexpression (left), and in A549 cells treated with 740Y‐P following E2F1 knockdown (right). ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) Q) Transwell assays assessing migration of COA4 ‐overexpressing A549 and H23 cells following E2F1 knockdown (top), and of COA4‐knockdown cells following E2F1 overexpression (bottom). Scale bar: 275 µm (n = 3; ** p < 0.01; **** p < 0.0001) R) Transwell assays analyzing migration of A549 and H23 cells treated with 740Y‐P following E2F1 knockdown (left), and with LY294002 following E2F1 overexpression (right). Scale bar: 275 µm ( n = 3) S) Transwell assays evaluating migration and invasion in A549 and H23 cells following KRAS G12D overexpression, E2F1 knockdown, and COA4 overexpression. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) T) Analysis of E2F1 expression in LUAD tissues: left, comparing KRAS wild‐type versus KRAS mutant samples using TCGA data; right, comparing LUAD tissues with high versus low KRAS expression using combined datasets ( GSE30219 , GSE37745 , GSE118370 , and GSE140797 ). U) Survival analysis based on COA4 and E2F1 expression levels using the GSE30219 database. V) Combined analysis of COA4 and E2F1 expression with survival status and overall survival time, performed using the linkET package on the GSE30219 dataset. The data are given as mean ± SD and compared by Student's t test (A–E, G, I–T), Spearman's rank correlation analysis (H). The Log‐rank (Mantel–Cox) test was used for survival analyses (U,V). Significance levels are indicated as follows: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Primary antibodies: ACTIN (1:5000, Proteintech, 81115‐1‐RR); Coa4 (1:2500, Invitrogen, PA5‐59076); CDC42 (1:1000, Proteintech, 10155‐1‐AP); E2F1 (1:1000, Proteintech, 12171‐1‐AP); MTCO1 (1:500, ABclonal, A17889); MTCO2 (1:3000, Proteintech, 55070‐1‐AP); MTCO3 (1:2000, Proteintech, 55082‐1‐AP); KRAS (1:10000, Proteintech, 12063‐1‐AP); FAK (1:5000, ABclonal, A11131); p‐FAK‐Y397 (1:300, ABclonal, AP0302).

Techniques: Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR, Over Expression, Knockdown, Luciferase, Reporter Assay, Activation Assay, ChIP-qPCR, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Migration

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 2. Changes in mRNA expression of BCL2, E2F1, E2F3, RB1 and P53 in K562 cells after cisplatin treatment. BCL2, E2F1, E2F3, RB1 and P53 were detected by (A) RT-PCR, (B) real-time PCR and (C) ELISA.

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 4. Correlative expression of miRNAs and oncogenes using antisense oligos (ASO). (A) RT-PCR, (B) real-time PCR. Correlative expression of (C) E2F1 and its targeted miR-17-5p, (D) E2F3 and its targeted miRNAs and (E) Bcl-2 and its targeted miRNAs (miR-16, 34a-c) using ELISA is shown. *Significant difference (p<0.05).

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 5. Expression of E2F1, E2F3, BCL2, RB1 and P53 genes regulated by miRNAs. Detection of the expression of (A) E2F1, (B) E2F3, (C) BCL2, (D) RB1 and (E) P53. *Significant difference (p<0.05).

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing