Figures and data
Human RUNX2 isoform II is overexpressed and associated with poor prognosis in OSCC. (A) Schematic diagram of the isoforms and alternative promoters of the human RUNX2 gene. Boxes and lines represent exons or introns in the pre-mRNA, respectively. P1 and P2 represent promoters. Isoform II is transcribed from P1, while isoform I is transcribed from P2. Exon 5 and 7 are alternative exons. (B-D) The expression levels of total RUNX2 and isoforms in TCGA OSCC patients. (B) The normalized expression levels of total RUNX2, obtained from an online program, TSVdb, in normal (32 cases) or OSCC tissues (309 cases). (C, D) The PSI (percent-splice-in) values of exon 1.1 (isoform II) (C) and exon 2.1 (isoform I) (D) (total 288 cases with PSI values of exon 1.1 and exon 2.1) in normal (27 cases) and OSCC tissues (288 cases) were obtained from an online program, TCGA SpliceSeq. The PSI values represent the relative expression levels of individual isoform. (E) Comparison of exon 1.1 (isoform II) PSI between patients in stage I, II and III (132 cases) and those in stage IV (156 cases) of OSCC TCGA patients. (F) OSCC TCGA patients (total 287 cases with survival data) with low expression (254 cases) or high expression (33 cases) of exon 1.1 (isoform II) in OSCC. Low exon 1.1 (isoform II) PSI was defined as less than mean + 1.44SD (standard deviation). (G) The representative RT-PCR results of isoform II and isoform I in our OSCC or normal samples. GAPDH served as a loading control. (H-I) The scatter dot plot summarized the ratio of isoform II versus isoform I (isoform II/isoform I) (H) or the relative expression levels of isoform II (isoform II/GAPDH) (I) in our clinical OSCC (11 cases) and normal samples (11 cases). * P < 0.05, ** P < 0.01.
RUNX2 isoform II is required for the proliferation in vitro and tumorigenesis in vivo. (A) CAL 27 or SCC-9 cells were stably transfected by isoform II-expression, isoform I-expression or vector control lentivirus. CAL 27 cells were seeded into 24-well plates at day 0 and counted on day 1, 2 and 3. SCC-9 cells were seeded into 24-well plates at day 0 and counted on day 2 and 4. Data are means ± SD, n = 3. (B) Overexpression of RUNX2 isoform II or isoform I was confirmed by Western blot. Actin served as a loading control. (C) Proliferation curves of CAL 27 and SCC-9 cells treated with anti-isoform II siRNAs (si-II-1 or si-II-2) or negative control siRNA (NC). Data are means ± SD, n = 3. (D) Knockdown efficiency of isoform II was analyzed by RT-PCR. GAPDH served as a loading control. (E) Effects of isoform II-knockdown on the clonogenic ability in CAL 27 and SCC-9. The histograms on the right summarized the numbers of colonies (at least 50 cells/colony). Data are means ± SD, n = 3. (F) CAL 27 and SCC-9 cells were treated with si-II-1, si-II-2 or NC siRNA. The cellular apoptosis was analyzed by flow cytometry. The histograms on the right summarized the cellular apoptosis. Data are means ± SD, n = 4 for CAL 27, n = 3 for SCC-9. (G-I) CAL 27 cells with stable isoform II shRNA (shisoform II) or nonspecific shRNA (shNC) were injected into both sides of the dorsum of BALB/c nude mice. (G, H) Tumors were dissected out and weighed on day 21. (I) Tumor volumes were measured on different days. (J) Knockdown efficiency of isoform II was analyzed by RT-PCR. 18S rRNA served as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001.
RUNX2 isoform II suppresses ferroptosis. (A, B) CAL 27 and SCC-9 cells were treated with anti-isoform II siRNAs (si-II-1 and si-II-2) or negative control siRNA (NC). (A) The levels of total ROS were detected with DCFH-DA using flow cytometry. The histograms below summarized the levels of mean fluorescent intensity (MFI). Data are means ± SD, n = 4. (B) The lipid peroxidation of cells was analyzed with C11 BODIPY 581/591 reagent using flow cytometry. The histograms below summarized the levels of MFI. Data are means ± SD, n = 4 for CAL 27, n = 3 for SCC-9. (C, D) CAL 27 or SCC-9 cells were stably transfected by isoform II-expression or vector control lentivirus. The levels of ROS (C) or lipid peroxidation (D) were detected by flow cytometry. The histograms below summarized the levels of MFI. Data are means ± SD, n = 4. (E) Transmission electron microscopy images of CAL 27 cells transfected with si-II-1, si-II-2 or NC. RSL3 (a ferroptosis activator) served as a positive control. (F) CAL 27 cells transfected with anti-isoform II siRNAs were also treated with ferrostatin-1 (Fer-1, 10 μM), a ferroptosis inhibitor. Negative control siRNA and DMSO were used as controls. Cells were divided into six groups: NC+DMSO, si-II-1+DMSO, si-II-2+DMSO, NC+Fer-1, si-II-1+Fer-1 and si-II-2+Fer-1. To display clear diagrams, the proliferation curves of si-II-1-treated cells or si-II-2-treated cells were shown separately. Data are means ± SD, n = 3. (G) CAL 27 cells transfected with anti-isoform II siRNAs were also treated with Z-VAD (20 μM, an apoptosis inhibitor) or necrostatin-1 (Nec-1, 20 μM, a necroptosis inhibitor). Negative control siRNA and DMSO were used as controls. Cells were divided into nine groups: NC+DMSO, si-II-1+DMSO, si-II-2+DMSO, NC+Z-VAD, si-II-1+Z-VAD, si-II-2+Z-VAD, NC+Nec-1, si-II-1+Nec-1, si-II-2+Nec-1. To display clear diagrams, the proliferation curves of si-II-1-transfected cells or si-II-2-transfected cells were shown separately. Data are means ± SD, n = 3. (H, I) The total ROS levels (H) or lipid peroxidation (I) of cells simultaneously transfected with anti-isoform II siRNAs or NC and treated with Fer-1 or DMSO were detected with DCFH-DA (H) or BODIPY 581/591 reagent (I) by flow cytometry. The histogram on the right summarized the levels of MFI. Data are means ± SD, n = 3. (J, K) The total ROS levels (J) or lipid peroxidation (K) of isoform II-overexpressed cells treated with RSL3 (2 μM, a ferroptosis activator) or DMSO were detected with DCFH-DA (J) or BODIPY 581/591 reagent (K) by flow cytometry. The histogram on the right summarized the levels of MFI. Data are means ± SD, n = 4 for J, n = 5 for K. (L) Representative images of immunohistochemical staining of 4-HNE in tumors with or without isoform II-knockdown (shisoform II vs shNC) in Figure 2G. The histogram below summarized the H score of 4-HNE staining in tumors. * P < 0.05, ** P < 0.01, *** P < 0.001.
RUNX2 isoform II promotes the expression of PRDX2. (A) Screening analysis of the expression of enzymatic antioxidant genes upon isoform II-knockdown (si-II-1 and si-II-2) in CAL 27 cells via RT-PCR. (B, C) Effects of RUNX2 isoform II-knockdown on PRDX2 expression levels were analyzed by RT-PCR (B) or Western blot (C) in CAL 27 or SCC-9. 18S rRNA (B) or actin (C) served as loading controls. Data are means ± SD, n = 3. (D) Representative images of immunohistochemical staining of PRDX2 in tumors with or without isoform II-knockdown (shisoform II vs shNC) in Figure 2G. The histogram below summarized the expression levels of PRDX2 in tumors. (E, F) Effects of RUNX2 isoform II or isoform I overexpression on PRDX2 expression levels were analyzed by RT-PCR (E) or Western blot (F) in CAL 27 or SCC-9. 18S rRNA (E) or actin (F) served as loading controls. Data are means ± SD, n = 6 or 3 for CAL 27, n = 3 for SCC-9. (G) RUNX2 binding motifs on PRDX2 promoter were obtained from JASPAR. (H) Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) assay was performed in CAL 27 with or without FLAG-tagged RUNX2 isoform II overexpression (isoform II vs vector) by using anti-FLAG or control IgG antibody. Data are means ± SD, n = 3. (I) The immunoprecipitated protein levels of FLAG-tagged RUNX2 isoform II in the ChIP assays were determined by Western blot. (J-M) CAL 27 cells were co-transfected with PRDX2-expression, empty control lentivirus, and anti-isoform II siRNA (si-II), negative control siRNA (NC). Transfected cells were divided into four groups: Vector+NC, Vector+si-II, PRDX2+NC and PRDX2+si-II. (J, K) The total ROS levels (J) or lipid peroxidation (K) of transfected cells were detected with DCFH-DA (J) or BODIPY 581/591 reagent (K) by flow cytometry. The histograms below summarized the levels of MFI. Data are means ± SD, n = 3. (L) Overexpression of PRDX2 was confirmed by Western blot. Actin served as a loading control. (M) Knockdown efficiency of isoform II was analyzed by RT-PCR. 18S rRNA served as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001.
HOXA10 is required for RUNX2 isoform II expression and cell proliferation in OSCC. (A) Effects of HOXA10 knockdown (siHOX-1 and siHOX-2) on isoform II expression levels were analyzed by RT-PCR in CAL 27 or SCC-9. 18S rRNA served as a loading control. Data are means ± SD, n = 3. (B-D) CAL 27 or SCC-9 cells were treated with siHOX-1, siHOX-2 or NC siRNA. (B) Proliferation curves of CAL 27 or SCC-9 cells treated with HOXA10 siRNAs or NC siRNA. Data are means ± SD, n = 3. (C) Effects of HOXA10 knockdown on the clonogenic ability in CAL 27 and SCC-9. The histograms on the right summarized the numbers of colonies (at least 50 cells/colony). Data are means ± SD, n = 3. (D) The cellular apoptosis was analyzed by flow cytometry. The histograms on the right summarized the cellular apoptosis. Data are means ± SD, n = 4 for CAL 27, n = 3 for SCC-9. (E) The expression of HOXA10 in TCGA OSCC patients (288 cases) is positively correlated with RUNX2 exon 1.1 (isoform II) PSI (Spearman’s rank correlation coefficient, r=0.21, P < 0.001). * P < 0.05, ** P < 0.01, *** P < 0.001.
HOXA10 promotes the expression of PRDX2 and inhibits ferroptosis in OSCC. (A, B) Effects of HOXA10 knockdown (siHOX-1 and siHOX-2) on PRDX2 expression levels were analyzed by RT-PCR (A) or Western blot (B) in CAL 27 or SCC-9. 18S rRNA (A) or actin (B) served as loading controls. Data are means ± SD, n = 4 or 3 for CAL 27, n = 3 for SCC-9. (C, D) Effects of HOXA10 knockdown on ROS levels (C) or lipid peroxidation (D) were detected with DCFH-DA (C) or BODIPY 581/591 reagent (D) by flow cytometry in CAL 27 or SCC-9. The histograms below summarized the levels of MFI. Data are means ± SD, n = 4 for CAL 27, n = 3 or 4 for SCC-9. (E) CAL 27 cells transfected with anti-HOXA10 siRNAs were also treated with Fer-1 (10 μM), a ferroptosis inhibitor. Negative control siRNA and DMSO were used as controls. Cells were divided into six groups: NC+DMSO, siHOX-1+DMSO, siHOX-2+DMSO, NC+Fer-1, siHOX-1+Fer-1 and siHOX-2+Fer-1. To display clear diagrams, the proliferation curves of siHOX-1-treated cells or siHOX-2-treated cells were shown separately. Data are means ± SD, n = 3. (F) CAL 27 cells transfected with anti-HOXA10 siRNAs were also treated with Z-VAD (20 μM, an apoptosis inhibitor) or Nec-1 (20 μM, a necroptosis inhibitor). Negative control siRNA and DMSO were used as controls. Cells were divided into nine groups: NC+DMSO, siHOX-1+DMSO, siHOX-2+DMSO, NC+Z-VAD, siHOX-1+Z-VAD, siHOX-2+Z-VAD, NC+Nec-1, siHOX-1+Nec-1, siHOX-2+Nec-1. To display clear diagrams, the proliferation curves of siHOX-1-transfected cells or siHOX-2-transfected cells were shown separately. Data are means ± SD, n = 3. (G, H) The total ROS levels (H) or lipid peroxidation (I) of cells simultaneously transfected with anti-HOXA10 siRNAs or NC and treated with Fer-1 or DMSO were detected with DCFH-DA (G) or BODIPY 581/591 reagent (H) by flow cytometry. The histogram on the right summarized the levels of MFI. Data are means ± SD, n = 3 for G, n=4 for H. * P < 0.05, ** P < 0.01, *** P < 0.001.
Ferroptosis induced by HOXA10-knockdown can be rescued by isoform II overexpression and PRDX2 overexpression. (A-F) CAL 27 cells were co-transfected with isoform II-expression lentivirus, empty control lentivirus, and HOXA10 siRNA (siHOX), negative control siRNA (NC). Transfected cells were divided into four groups: Vector+NC, Vector+siHOX, Isoform II+NC and Isoform II+siHOX. (A) Cell number was counted on day 2 and day 4. Data are means ± SD, n = 5. (B) The cellular apoptosis of transfected cells was analyzed by flow cytometry. The histogram on the right summarized the cell apoptosis. Data are means ± SD, n = 4. (C, D) The total ROS levels (C) or lipid peroxidation (D) of transfected cells were detected with DCFH-DA (C) or BODIPY 581/591 reagent (D) by flow cytometry. The histograms below summarized the levels of MFI. Data are means ± SD, n = 4 or 3. (E, F) Effect of HOXA10-knockdown in isoform II-overexpressed cells on PRDX2 expression levels was analyzed by RT-PCR (E) or Western blot (F). 18S rRNA (E) or actin (F) served as loading controls. Data are means ± SD, n = 3 or 4. (G, H) CAL 27 cells were co-transfected with PRDX2-expression lentivirus, empty control lentivirus, and siHOX, NC. Transfected cells were divided into four groups: Vector+NC, Vector+siHOX, PRDX2+NC and PRDX2+siHOX. The total ROS levels (G) or lipid peroxidation (H) of transfected cells were detected with DCFH-DA (G) or BODIPY 581/591 reagent (H) by flow cytometry. The histograms on the right summarized the levels of MFI. Data are means ± SD, n = 3. (I) The model of a new ferroptosis-related pathway-HOXA10/RUNX2 isoform II/PRDX2 in this study. * P < 0.05, ** P <0.01, *** P < 0.001.
