Article Figures & Data
Figures
- Figure 1
The upregulation of DPP10-AS1 predicts poor prognosis in patients with lung cancer. (A) DPP10-AS1 expression in lung cancer tissues and corresponding noncancerous lung tissues was measured by qRT-PCR and normalized to β-actin. The horizontal lines in the box plots represent the medians. The boxes represent the interquartile ranges, and the whiskers represent percentiles 2.5 and 97.5. Statistical differences between groups were compared with the Wilcoxon signed-rank test (n = 94, P < 0.0001). (B) The ratio between the relative quantification of DPP10-AS1 expression in lung cancer tissues and paired adjacent noncancerous lung tissues of each case. Kaplan-Meier survival analyses of the correlation between DPP10-AS1 expression levels and recurrence-free survival (C) or overall survival (D) in 94 patients with lung cancer. The median expression level was used as a cutoff. Statistical significance was analyzed with the log-rank test. (E) DPP10-AS1 expression in a normal lung epithelial cell line (BEAS-2B) and in 5 lung cancer cell lines (NCI-H446, A549, LTEP-a-2, SPC-A1, and NCI-H1299). Data are shown as mean ± standard error from at least 3 independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.
- Figure 2
DPP10-AS1 promotes lung cancer cell proliferation in vitro and tumor growth in vitro. MTT assays were performed to determine the viability of lung cancer cells treated with siDPP10-AS1 in SPC-A1 (A) and NCI-H1299 (B) cells, and with pcDNA3.1-DPP10-AS1 in SPC-A1 (C) and NCI-H1299 (D) cells. Colony formation assays were used to detect the proliferation ability of lung cancer cells after transfection with siDPP10-AS1 in SPC-A1 (E) and NCI-H1299 (F) cells, and pcDNA3.1-DPP10-AS1 in SPC-A1 (G) and NCI-H1299 (H) cells. Lung cancer cells overexpressing pcDNA3.1-DPP10-AS1 were injected subcutaneously into nude mice to demonstrate xenograft tumor growth (I). Analysis of tumor volume (J) and tumor weight (K) in SPC-A1 cells with overexpression of pcDNA3.1-DPP10-AS1. Analysis of tumor volume (L) and tumor weight (M) in NCI-H1299 cells with overexpression of pcDNA3.1-DPP10-AS1. RT-qPCR analysis of lncRNA DPP10-AS1 (N) and DPP10 mRNA (O) expression in tumor tissues after injection of SPC-A1 cells. RT-qPCR analysis of lncRNA DPP10-AS1 (P) and DPP10 mRNA (Q) expression in tumor tissues after injection of NCI-H1299 cells. Western blot assays of DPP10 protein in tumor tissues overexpressing lncRNA DPP10-AS1 and empty plasmid (R). Colonies were counted and captured. The bar charts statistically compare the differences in colony formation in each experimental group compared with the corresponding control. Values are shown as the mean ± SD in 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
- Figure 3
DPP10-AS1 promotes cell cycle progression and inhibits apoptosis in lung cancer cells. SPC-A1 and NCI-H1299 lung cancer cells were treated with siDPP10-AS1 (A) or pcDNA3.1-DPP10-AS1 (B) and then were analyzed for cell cycle by flow cytometry. After the knockdown of DPP10-AS1 (C) or overexpression of DPP10-AS1 (D), lung cancer cell apoptosis was analyzed by flow cytometry with Annexin V/PI staining. The data are presented as the mean ± SD (n = 3), and *P < 0.05, **P < 0.01, ***P < 0.001.
- Figure 4
DPP10-AS1 positively regulates DPP10. (A) Genetic structure diagram of DPP10-AS1. (B) Effect of DPP10-AS1 knockdown on DPP10 mRNA. (C) Effect of DPP10-AS1 overexpression on DPP10 protein. (D) Effect of DPP10 overexpression on DPP10 mRNA. (E) Effect of DPP10-AS1 overexpression on DPP10 protein. (F) Effect of DPP10 overexpression on DPP10-AS1. (G) Effect of DPP10 knockdown on DPP10-AS1. Data are shown as mean ± SD from 3 independent experiments. **P < 0.01, ***P < 0.001.
- Figure 5
DPP10-AS1 and DPP10 are coordinately upregulated in lung cancer. (A) Difference in expression levels of DPP10 mRNA between lung cancer tissues and adjacent cancerous tissues. β-actin served as the internal control for normalization. The statistical difference was analyzed with Wilcoxon signed-rank test. (B) The ratio between the relative quantification of DPP10 mRNA expression in lung cancer tissues vs. paired adjacent noncancerous lung tissues for each case. qRT-PCR (C) and Western blot (D) analysis of the relative expression of DPP10 mRNA and protein level in lung cell lines. The correlation between DPP10-AS1 and DPP10 mRNA in lung cancer tissues (E) and cell lines (F). Data were subjected to Pearson correlation analysis. (G, H) According to the expression of DPP10-AS1, the qRT-PCR data from 94 pairs of clinical samples were classified as DPP10-AS1 high and DPP10-AS1 low. The relative expression of DPP10 mRNA and DPP10-AS1 is compared in box plots. (I, J) According to the expression of DPP10, the qRT-PCR data from 94 pairs of clinical samples were classified as DPP10 high and DPP10 low. The relative expression of DPP10-AS1 and DPP10 is compared in box plots. *P < 0.05, **P < 0.01, ****P < 0.0001.
- Figure 6
DPP10-AS1 promotes lung cancer cell growth and proliferation through upregulating DPP10 mRNA expression. (A) Rescue effect of DPP10 overexpression on DPP10-AS1 knockdown-mediated cell growth inhibition in SPC-A1 and NCI-H1299 cells, determined with MTT assays. (B) Rescue effect of DPP10 knockdown on DPP10-AS1 overexpression-mediated cell growth promotion, determined with MTT assays. (C) Colony formation assays were performed to investigate the effects of DPP10-AS1 knockdown and DPP10 overexpression on SPC-A1 and NCI-H1299 cell proliferation. (D) The effects of DPP10-AS1 overexpression and DPP10 knockdown on SPC-A1 and NCI-H1299 cell proliferation were detected with colony formation assays. The number of colonies was counted, and statistical analysis is shown at right. (E) Rescue effects of DPP10 overexpression or depletion on DPP10-AS1-mediated DPP10 mRNA expression changes. Data are shown as mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
- Figure 7
DPP10-AS1 promotes cell cycle progression and inhibits apoptosis by upregulating DPP10 mRNA. (A) Effects of DPP10-AS1 knockdown and DPP10 overexpression on the SPC-A1 and NCI-H1299 cell cycle, as assessed by flow cytometry. (B) Effects of DPP10-AS1 overexpression and DPP10 knockdown on the SPC-A1 and NCI-H1299 cell cycle, as assessed by flow cytometry. (C) Effects of DPP10-AS1 knockdown and DPP10 overexpression on SPC-A1 and NCI-H1299 cell apoptosis, as assessed by flow cytometry with Annexin V/PI staining. (D) Effects of DPP10-AS1 overexpression and DPP10 knockdown on SPC-A1 and NCI-H1299 cell apoptosis, as assessed by flow cytometry with Annexin V/PI staining. Data are shown as mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
- Figure 8
DPP10-AS1 does not increase DPP10 mRNA stability, but both are regulated by epigenetic methylation. (A) qRT-PCR analysis of DPP10-AS1 in the nucleus and cytoplasm, as determined with cytoplasmic and nuclear extract isolation assays. (B) qRT-PCR analysis of DPP10 mRNA levels in SPC-A1 and NCI-H1299 cells cotransfected with siDPP10-AS1, as determined with RNase protection assays. (C) qRT-PCR analysis of DPP10 mRNA levels in SPC-A1 and NCI-H1299 cells cotransfected with pcDNA3.1-DPP10-AS1, as determined with RNase protection assay. (D) qRT-PCR analysis of DPP10-AS1 levels in SPC-A1 and NCI-H1299 cells treated with different doses of the DNA methyltransferase inhibitor 5-azacytidine. (E) qRT-PCR analysis of DPP10 mRNA levels in SPC-A1 and NCI-H1299 cells treated with different doses of the DNA methyltransferase inhibitor 5-azacytidine. (F) Hypomethylation of DPP10 in patients with lung cancer (LUAD) compared with healthy controls, according to the MethHC database. (G) DNA methylation analysis of CpG islands of DPP10 and DPP10-AS1 genes in lung cancer tissues. (H) The effect of DPP10-AS1 overexpression on DPP10 methylation in SPC-A1 cells. (I) The working model in which the lncRNA DPP10-AS1 promotes lung carcinoma malignant processes through epigenetic activation of DPP10. *P < 0.05, **P < 0.01, ***P < 0.001.
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