FIGURE 4 from Targeting MALAT1 Augments Sensitivity to PARP Inhibition by Impairing Homologous Recombination in Prostate Cancer
MALAT1 knockdown restrains cell-cycle progression and instigates apoptosis in prostate cancer cells. A, Flow cytometry analysis for accessing the cell-cycle distribution by propidium iodide (PI) DNA staining assay in MALAT1-silenced prostate cancer cells. The percentage of cells in each phase was calculated using FlowJo software. B, Representative images depicting EdU incorporation in the same cells as in A. Nuclei were stained with Hoechst 33342. Scale bar, 20 µm. Right, bar graph showing quantification of EdU uptake in the indicated cells. C, qRT-PCR analysis showing expression of genes associated with G1 and S-phase of the cell cycle in MALAT1-silenced 22RV1 cells. The expression level for each gene was normalized to GAPDH. D, Immunoblot showing the change in expression of E2F1 in the same cells as in A. β-Actin was used as an internal control. E, Line graph showing cell proliferation assay using the same cells as in A, at the indicated time points. F, Flow cytometry–based apoptosis assay using Annexin V-PE and 7-AAD staining in the same cells as in A. The percentage of apoptotic cells was calculated using FlowJo software. G, Immunoblots showing a change in the expression of apoptosis markers in the same cells as in A. β-Actin was used as an internal control. H, Schematic depicting that MALAT1 is a novel regulator of HR and plays an important role in the maintenance of genome stability in prostate cancer. MALAT1 depletion induces HR deficiency by decreasing the expression of several DDR genes and results in DSB accumulation which in turn induces cell-cycle arrest and instigates apoptosis. Experiments were performed with n = 3 biologically independent samples; the data represents mean ± SEM. For B and C, one-way ANOVA with Dunnett multiple comparisons posthoc test was applied, while for E, two-way ANOVA with Tukey multiple comparisons test was applied.