Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: miRNA mimics library screening for ARID1A synthetic lethal partners in CRC cells. A Western blot analysis showed loss of ARID1A expression in the two HCT116 ARID1A −/− clones. B Schematic illustration of the synthetic lethality miRNA mimics library screening. HCT116 ARID1A +/+ and ARID1A −/− #1 cell lines were screened with a miRNA mimics library. After incubation with the miRNA mimics for 72 h, cell viability was determined by AlamarBlue assay. C Results of the miRNA library screening. Cell viability of the miRNAs against HCT116 ARID1A +/+ and ARID1A −/− cells was plotted. D , E Synthetic lethality of miR-4653-3p in ARID1A −/− HCT116 cells. HCT116 ARID1A +/+ and ARID1A −/− cells were transfected with miR-4653-3p mimics for 72 h and nuclei were stained with Hoechst 33,342 ( D ). Scale bars, 100 μm. Nuclear density was measured with Image J software as a surrogate for cell viability ( E ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: Library Screening, Western Blot, Expressing, Clone Assay, Incubation, Alamar Blue Assay, Transfection, Staining, Software

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: In vitro and in vivo synthetic lethality of miR-4653-3p in HCT116 ARID1A −/− cells. A Synthetic lethality of miR-4653-3p in HCT116 ARID1A −/− cells. HCT116 ARID1A +/+ and ARID1A −/− cell lines were transfected with NC or miR-4653-3p mimics for 72 h and the cell viability was shown. Error bars represent s.d. from three independent experiments. ANOVA ** P < 0.01, *** P < 0.001. B-D Preferential induction of apoptosis in HCT116 ARID1A −/− cells by miR-4653-3p. Detection of apoptosis in HCT116 ARID1A +/+ and HCT116 ARID1A −/− cells transfected with NC or miR-4653-3p mimics for 72 h by flow cytometry ( B ) and the percentage of apoptotic cells were quantitated ( C ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001. Apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3) were detected by western blot ( D ). E , F Colony formation assay was performed in ARID1A isogenic cell pair to validate the synthetic lethality effects of miR-4653-3p. Cells were transfected with NC or miR-4653-3p mimics for 14 days and colonies were stained with crystal violet. Error bars represent s.d. from three independent experiments. Student’s t-test ** P < 0.01. G Western blot analysis showed overexpression of ARID1A in the two RKO ARID1A OE clones. H Synthetic lethality of miR-4653-3p in RKO ARID1A −/− cells. RKO ARID1A −/− and ARID1A OE cell lines were transfected with miR-4653-3p mimics for 72 h and the cell viability was shown. Error bars represent s.d. from three independent experiments. ANOVA *** P < 0.001. I Preferential induction of apoptosis in RKO ARID1A −/− cells by miR-4653-3p as indicated by apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3). J Schematic illustration of mouse tumor xenograft experiments with the HCT116 ARID1A isogenic cell pair. K , L Tumor growth curves in nude mice bearing HCT116 ARID1A +/+ ( K ) and ARID1A −/− ( L ) xenografts after treatment with NC or miR-4653-3p. M , N Tumor weight of HCT116 ARID1A +/+ ( M ) and ARID1A −/− ( N ) xenografts after treatment with NC or miR-4653-3p. Error bars represent s.d. * P < 0.05 between vehicle and RITA treatment groups ( n = 5). Student’s t-test

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: In Vitro, In Vivo, Transfection, Flow Cytometry, Western Blot, Colony Assay, Staining, Over Expression, Clone Assay

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: SLC25A51 is a downstream target of miR-4653-3p in CRC cells. A Schematic illustration of the RNA sequencing of HCT116 ARID1A +/+ and HCT116 ARID1A −/− cell lines after transfected with NC or miR-4653-3p. B Venn diagram revealed potential downstream target genes from RNA sequencing results and miRNA target prediction engine. C Heatmap showed expressions of candidate genes from RNA sequencing results. D Effects of siRNAs mediated deletion of candidate genes on cell viability of HCT116 ARID1A +/+ and ARID1A −/− cells. E RT-qPCR analysis of SLC25A51 mRNA level in HCT116 ARID1A +/+ and ARID1A −/− cells. * P < 0.05, one-sample t-test. F RT-qPCR analysis of SLC25A51 mRNA level in HCT116 cells after transfected with NC or miR-4653-3p mimics. ** P < 0.01, Student’s t-test. G , H Western blot analysis of SLC25A51 protein level in HCT116 ARID1A +/+ and ARID1A −/− cell lines ( G ) or RKO ARID1A −/− and ARID1A OE cell lines ( H ) after transfected with NC or miR-4653-3p mimics. I Bioinformatics analysis revealed the 3’UTR region of SLC25A51 contained binding sites of the sequence of miR-4653-3p. J Dual-luciferase reporter assay illustrated the binding of miR-4653-3p and SLC25A51 in HCT116 cells. Error bars represent s.d. from three independent experiments. Student’s t-test n.s. non-significant, *** P < 0.001

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: RNA Sequencing, Transfection, Quantitative RT-PCR, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: Synthetic lethality of ARID1A and miR-4653-3p is mediated by SLC25A51 and reduced SLC25A51 expression suppressed SIRT3 activity. A Western blot analysis of SLC25A51 expression in HCT116 cells by siRNA silencing. B , C Representative images ( B ) and cell viability ( C ) of HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with SLC25A51 siRNA for 72 h. The cell images were taken with Olympus microscope. Scale bars, 100 μm. Cell viability was measured with Image J software. Error bars represent s.d. from three independent experiments. ANOVA * P < 0.05, ** P < 0.01, *** P < 0.001. D HCT116 ARID1A +/+ and ARID1A −/− cell lines were transfected with SLC25A51 siRNA for 72 h, and the expressions of apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3) were detected by western blot. E Western blot analysis of SLC25A51 in HCT116 cells by overexpressing SLC25A51. F Cell viability of HCT116 ARID1A +/+ and ARID1A −/− cell lines with or without miR-4653-3p mimics and SLC25A51 plasmid transfection was shown. Error bars represent s.d. from three independent experiments. Student’s t-test * P < 0.05. G Mitochondrial NAD + levels in HCT116 cells transfected with SLC25A51 siRNA. Error bars represent s.d. from three independent experiments. Student’s t-test ** P < 0.01. H SIRT3 activity in HCT116 cells transfected with SLC25A51 siRNA. Error bars represent s.d. from three independent experiments. Student’s t-test * P < 0.05. I , J HCT116 cells were transfected with or without SLC25A51 siRNA and SIRT3 overexpression plasmids, and the acetylation levels of SOD2 were analyzed by western blot ( I ). Band intensity quantification for acetylation of SOD2 was performed using ImageJ software, normalized to the total levels of SOD2 ( J ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001. K Cell viability of HCT116 ARID1A +/+ and ARID1A −/− cell lines after treated with 3-TYP for 72 h was shown. Error bars represent s.d. from three independent experiments. ANOVA *** P < 0.001

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Microscopy, Software, Plasmid Preparation, Over Expression

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: miR-4653-3p selectively induces DNA damage in ARID1A-deficient CRC cells. A GO analysis for significantly altered genes regulated by ARID1A by RNA sequencing. B KEGG pathway analysis for significantly altered genes regulated by ARID1A by RNA sequencing. C , D Western blot analysis of γ-H2AX level in HCT116 ARID1A +/+ and ARID1A −/− cell lines ( C ) or RKO ARID1A −/− and RKO ARID1A OE cell lines ( D ) after transfected with NC or miR-4653-3p. E , F Representative images of comet assay ( E ) and quantification of the tail moment ( F ) of HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with NC or miR-4653-3p. Scale bars, 100 μm. Analyses of the percentage tail DNA in the comet assay ( n = 50). Error bars represent s.d. Student’s t-test *** P < 0.001. G , H Representative images of comet assay ( G ) and quantification of the tail moment ( H ) of RKO ARID1A −/− and ARID1A OE cell lines after transfected with NC or miR-4653-3p. Scale bars, 100 μm. Analyses of the percentage tail DNA in the comet assay ( n = 50). Error bars represent s.d. Student’s t-test *** P < 0.001. I Representative fluorescent staining images of γ-H2AX in HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with NC or miR-4653-3p. Scale bars, 20 μm

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: RNA Sequencing, Western Blot, Transfection, Single Cell Gel Electrophoresis, Staining

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: Effects of ARID1A and miR-4653-3p on DNA damage repair. A , B Western blot analysis of DNA damage repair protein levels (ATM, pATM, ATR, p-ATR, CHK1, p-Chk1, CHK2, p-Chk2) in HCT116 ( A ) and LOVO ( B ) cells after transfected with NC or miR-4653-3p. C Western blot analysis of DNA damage repair protein levels in HCT116 ARID1A +/+ and ARID1A −/− cell lines. D The level of DNA damage repair proteins was detected by western blot in HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with NC or miR-4653-3p. E The level of DNA damage repair proteins was detected by western blot in HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with or without miR-4653-3p and SIRT3

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: Western Blot, Transfection

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: Proposed working model of the synthetic lethality between ARID1A and miR-4653-3p

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: In vitro and in vivo synthetic lethality of miR-4653-3p in HCT116 ARID1A −/− cells. A Synthetic lethality of miR-4653-3p in HCT116 ARID1A −/− cells. HCT116 ARID1A +/+ and ARID1A −/− cell lines were transfected with NC or miR-4653-3p mimics for 72 h and the cell viability was shown. Error bars represent s.d. from three independent experiments. ANOVA ** P < 0.01, *** P < 0.001. B-D Preferential induction of apoptosis in HCT116 ARID1A −/− cells by miR-4653-3p. Detection of apoptosis in HCT116 ARID1A +/+ and HCT116 ARID1A −/− cells transfected with NC or miR-4653-3p mimics for 72 h by flow cytometry ( B ) and the percentage of apoptotic cells were quantitated ( C ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001. Apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3) were detected by western blot ( D ). E , F Colony formation assay was performed in ARID1A isogenic cell pair to validate the synthetic lethality effects of miR-4653-3p. Cells were transfected with NC or miR-4653-3p mimics for 14 days and colonies were stained with crystal violet. Error bars represent s.d. from three independent experiments. Student’s t-test ** P < 0.01. G Western blot analysis showed overexpression of ARID1A in the two RKO ARID1A OE clones. H Synthetic lethality of miR-4653-3p in RKO ARID1A −/− cells. RKO ARID1A −/− and ARID1A OE cell lines were transfected with miR-4653-3p mimics for 72 h and the cell viability was shown. Error bars represent s.d. from three independent experiments. ANOVA *** P < 0.001. I Preferential induction of apoptosis in RKO ARID1A −/− cells by miR-4653-3p as indicated by apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3). J Schematic illustration of mouse tumor xenograft experiments with the HCT116 ARID1A isogenic cell pair. K , L Tumor growth curves in nude mice bearing HCT116 ARID1A +/+ ( K ) and ARID1A −/− ( L ) xenografts after treatment with NC or miR-4653-3p. M , N Tumor weight of HCT116 ARID1A +/+ ( M ) and ARID1A −/− ( N ) xenografts after treatment with NC or miR-4653-3p. Error bars represent s.d. * P < 0.05 between vehicle and RITA treatment groups ( n = 5). Student’s t-test

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: In Vitro, In Vivo, Transfection, Flow Cytometry, Western Blot, Colony Assay, Staining, Over Expression, Clone Assay

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: Synthetic lethality of ARID1A and miR-4653-3p is mediated by SLC25A51 and reduced SLC25A51 expression suppressed SIRT3 activity. A Western blot analysis of SLC25A51 expression in HCT116 cells by siRNA silencing. B , C Representative images ( B ) and cell viability ( C ) of HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with SLC25A51 siRNA for 72 h. The cell images were taken with Olympus microscope. Scale bars, 100 μm. Cell viability was measured with Image J software. Error bars represent s.d. from three independent experiments. ANOVA * P < 0.05, ** P < 0.01, *** P < 0.001. D HCT116 ARID1A +/+ and ARID1A −/− cell lines were transfected with SLC25A51 siRNA for 72 h, and the expressions of apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3) were detected by western blot. E Western blot analysis of SLC25A51 in HCT116 cells by overexpressing SLC25A51. F Cell viability of HCT116 ARID1A +/+ and ARID1A −/− cell lines with or without miR-4653-3p mimics and SLC25A51 plasmid transfection was shown. Error bars represent s.d. from three independent experiments. Student’s t-test * P < 0.05. G Mitochondrial NAD + levels in HCT116 cells transfected with SLC25A51 siRNA. Error bars represent s.d. from three independent experiments. Student’s t-test ** P < 0.01. H SIRT3 activity in HCT116 cells transfected with SLC25A51 siRNA. Error bars represent s.d. from three independent experiments. Student’s t-test * P < 0.05. I , J HCT116 cells were transfected with or without SLC25A51 siRNA and SIRT3 overexpression plasmids, and the acetylation levels of SOD2 were analyzed by western blot ( I ). Band intensity quantification for acetylation of SOD2 was performed using ImageJ software, normalized to the total levels of SOD2 ( J ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001. K Cell viability of HCT116 ARID1A +/+ and ARID1A −/− cell lines after treated with 3-TYP for 72 h was shown. Error bars represent s.d. from three independent experiments. ANOVA *** P < 0.001

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Microscopy, Software, Plasmid Preparation, Over Expression