PMC

HDAC3 knockdown restores deficient invasiveness of KDM2A-depleted NSCLC cells. A and B, the effect of HDAC3 knockdown on the invasiveness of KDM2A-depleted H1975 (A) and H1792 (B) cells. Representative images of invaded cells are shown (left panels), and cells were counted (right panels). Data are presented as the mean ± S.E. (error bars). ***, p < 0.001.

HDAC3 occupies the regions spanning the transcription start sites in the CDK6, NEK7, NANOS1, and RAPH1 genes. A–D, analysis of chromatin occupancy of HDAC3 at the CDK6 (A), NEK7 (B), NANOS1 (C), and RAPH1 (D) genes in H1975 and H1792 cells by quantitative ChIP (qChIP). A diagrammatic representation of individual genes is also shown. Arrows indicate the PCR-amplified regions (a and b). TSS, transcription start site. Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

HDAC3 knockdown rescues proliferation defect of KDM2A-depleted NSCLC cells. A and B, the effect of HDAC3 knockdown on the proliferation of KDM2A-depleted H1975 (A) and H1792 (B) cells. C, the effect of single and double knockdown of KDM2A and HDAC3 on S phase cell percentages. The percentages of sub-G₁, G₁, S, and G₂/M phase cells were analyzed. D, BrdU incorporation assay. d, days. Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

CDK6 knockdown inhibits mainly cell proliferation, whereas NANOS1 knockdown impedes largely cell invasiveness. A–D, the effect of CDK6 knockdown on the proliferation and invasiveness of H1792 cells. CDK6 mRNA levels were analyzed in siControl-treated and CDK6-depleted (siCDK6-1 or -3) cells using quantitative RT-PCR (A). Cell proliferation (B), BrdU incorporation (C), and invasion (D) assays were performed. E–H, the effect of NANOS1 knockdown on the proliferation and invasiveness of H1792 cells. NANOS1 mRNA levels were analyzed in siControl-treated and NANOS1-depleted (siNANOS1-1 or -3) cells using quantitative RT-PCR (E). Cell proliferation (F), BrdU incorporation (G), and invasion (H) assays were performed. d, days. Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

HDAC3 knockdown significantly increases expression of the CDK6 and NANOS1 genes in KDM2A-depleted NSCLC cells. A and B, analysis of HDAC3 and KDM2A mRNA levels in siControl-treated, KDM2A-depleted (siKDM2A-3), and KDM2A/HDAC3-depleted (siKDM2A-3 + siHDAC3-9) cells by quantitative RT-PCR. H1975 (A) and H1792 (B) cells were treated with siControl, siKDM2A-3, or a mixture of siKDM2A-3 and siHDAC3-9. C–F, the effect of HDAC3 knockdown on CDK6 and NANOS1 mRNA levels in KDM2A-depleted cells. Expression levels of the CDK6 (C and D) and NANOS1 (E and F) genes in H1975 (C and E) and H1792 (D and F) cells were measured by quantitative RT-PCR. Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

HDAC3 represses expression of the cell cycle-associated genes CDK6 and NEK7 and the invasion-related genes NANOS1 and RAPH1. A and B, analysis of HDAC3 knockdown efficacy in H1975 and H1792 cells. Cells were treated with two siRNAs against HDAC3 (siHDAC3-3 and siHDAC3-9). HDAC3 mRNA and protein levels were measured by quantitative RT-PCR (A) and Western blot analysis (B), respectively. C and D, the effect of HDAC3 knockdown on CDK6, NEK7, NANOS1, RAPH1, and GPR157 mRNA levels in H1975 (C) and H1792 (D) cells. Expression levels of individual genes were analyzed by quantitative RT-PCR. Data are presented as the mean ± S.E. (error bars). **, p < 0.01; ***, p < 0.001.

KDM2A represses HDAC3 expression by demethylating H3K36me2 at the HDAC3 promoter. A, the effect of KDM2A knockdown on HDAC3 mRNA levels in H1975 and H1792 cells. Cells were transfected with two different siKDM2As (siKDM2A-3 and -4). B and C, analysis of HDAC3 mRNA levels in KDM2A-depleted H1975 (B) and H1792 (C) cells after ectopic expression of GFP, wild-type KDM2A, and the catalytic mutant mKDM2A. HDAC3 mRNA levels were measured by quantitative RT-PCR. D, schematic representation of the promoter region of the HDAC3 gene. Arrows indicate the PCR-amplified region. TSS, transcription start site. E, analysis of KDM2A occupancy at HDAC3 gene in H1975 and H1792 cells by quantitative ChIP (qChIP). F and G, analysis of occupied levels of KDM2A, H3K36me2, H3K9me3, and H3 at the HDAC3 promoter region in H1975 (F) and H1792 (G) cells by quantitative ChIP. Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

HDAC3 knockdown increases acetylation levels in histones H3 and H4 at the promoters of the CDK6 and NANOS1 genes. A–D, analysis of chromatin levels of HDAC3, H3K9ac, H3K14ac, H4ac, and H3 at the promoter regions of the CDK6 (A and B) and NANOS1 genes (C and D) in H1975 (A and C) and H1792 (B and D) cells. Chromatin levels of proteins and histone marks were measured by quantitative ChIP. Anti-H3 was used as a ChIP control. E, the effect of KDM2A or HDAC3 knockdown on total cellular levels of H3K36me2, H3K9 acetylation, and H4 acetylation. Histone marks were examined by Western blot analysis. H3 and β-actin were used as internal loading controls. Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01.

Transcriptional expression of cell cycle-associated genes and invasion-related genes is positively regulated by KDM2A in the KDM2A-overexpressing NSCLC cell lines H1975 and H1792. A and B, analysis of CDK6, NEK7, NANOS1, RAPH1, DOCK4, and ZNF652 mRNA levels in H1975 (A) and H1792 (B) cells after KDM2A knockdown. Expression levels were analyzed by quantitative RT-PCR. C–F, rescue experiments for CDK6 (C and D) and NANOS1 (E and F) expression in KDM2A-depleted H1975 (C and E) and H1792 (D and F) cells. After ectopic expression of GFP, wild-type KDM2A, and its catalytic mutant mKDM2A in KDM2A-depleted H1975 and H1792 cells, expression levels were measured by quantitative RT-PCR. The siControl-treated cells were used as controls. G and H, analysis of KDM2A occupancy at CDK6, NEK7, NANOS1, RAPH1, DOCK4, and ZNF652 genes in H1975 (G) and H1792 (H) cells by quantitative ChIP (qChIP). Data are presented as the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

HDAC3 knockdown greatly recovers impaired tumorigenicity of KDM2A-depleted NSCLC cells in a mouse xenograft model. A and B, analysis of KDM2A (A) and HDAC3 (B) mRNA levels in control (shLuciferase), stably KDM2A-depleted (shKDM2A-1), and KDM2A/HDAC3-depleted H1792 cells. Stably KDM2A-depleted H1792 cells (shKDM2A-1) were transfected with siHDAC3-9 to generate double (KDM2A/HDAC3) knockdown cells. C and D, the effect of HDAC3 knockdown on tumor development of KDM2A-depleted cells in a subcutaneous xenograft model. Three different groups of cells (shControl, shKDM2A-1, and shKDM2A-1 + siHDAC3-9) were subcutaneously injected into five mice per group. Representative tumors (10 weeks after subcutaneous injection) are shown in dotted circles and indicated by black arrows (C). Tumor volumes were monitored and plotted for 10 weeks (D). E, representative images of H&E staining of xenograft tumor samples (shControl group and shKDM2A-1 + siHDAC3-9 group) and normal skin tissues (shKDM2A-1 group) obtained from the injection sites (10 weeks after subcutaneous injection). Scale bars are indicated (upper panels, 200 μm; lower panels, 100 μm). F, representative images of immunohistochemical staining of NANOS1 and CDK6 in xenograft tumor samples. Immunohistochemical staining was performed only using tumor tissues from the shControl group and shKDM2A-1 + siHDAC3-9 group. Normal mouse skin tissues from the shKDM2A-1 group were not used for staining because they were not a proper control. CDK6 and NANOS1 levels were quantified using the Chromavision Automated Cellular Imaging System (ACIS-III) from Dako and compared between the shControl group and shKDM2A-1 + siHDAC3-9 group: CDK6, 1 versus 0.73; NANOS, 1 versus 0.54. G, in this hypothetical model, KDM2A transcriptionally represses the HDAC3 gene. Subsequently, cell cycle-associated genes (e.g. CDK6) and invasiveness-related genes (e.g. NANOS1) are released from HDAC3-mediated repression. Data are presented as the mean ± S.E. (error bars). **, p < 0.01; ***, p < 0.001.