Article Figures & Data
Figures
- Figure 1.
Relative ANRIL expression in NSCLC tissues and its clinical significance. A, relative expression of ANRIL in NSCLC tissues (n = 68) compared with corresponding nontumor tissues (n = 68). ANRIL expression was examined by qPCR and normalized to GAPDH expression. Results are presented as the fold change in tumor tissues relative to normal tissues. B, ANRIL expression was classified into two groups. C and D, Kaplan–Meier DFS and OS curves according to ANRIL expression levels.
- Figure 2.
Effects of knockdown of ANRIL on NSCLC cell viability in vitro. A, ANRIL expression levels of NSCLC cell lines (PC9, SPC-A1, NCI-H1975, H1299, and H358 and H520) compared with that in normal human bronchial epithelial cells (16HBE). B, SPC-A1, H1299, and PC9 cells were transfected with si-ANRIL. C, MTT assays were used to determine the cell viability for si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. Values represent the mean ± SD from three independent experiments. D, colony-forming assays were conducted to determine the proliferation of si-ANRIL–transfected SPC-A1, H1299, and PC9 cells. Flow-cytometry assays were performed to analysis the cell-cycle progression and apoptosis when NSCLC cells transfected with si-ANRIL; *, P < 0.05 and **, P < 0.01.
- Figure 3.
Effects of knockdown of ANRIL on NSCLC cell cycle and apoptosis in vitro. A, the bar chart represents the percentage of cells in G0–G1, S, or G2–M phase, as indicated. B, apoptosis was determined by flow cytometry; upper left, necrotic cells; upper right, terminal apoptotic cells; lower right, early apoptotic cells. C, apoptosis was determined by Tunel staining. All experiments were performed in biologic triplicates with three technical replicates; **, P < 0.01.
- Figure 4.
Effects of downregulation of ANRIL on tumor growth in vivo. A, the tumor volume was calculated once every 3 days after injection of PC9 cells stably transfected with sh-ANRIL or empty vector. Points, mean (n = 7); bars, SD. B, tumor weights are represented as means of tumor weights ± SD. C, qPCR analysis of ANRIL expression in tumor tissues formed from PC9/sh-ANRIL, PC9/empty vector. D, tumors developed from sh-ANRIL–transfected PC9 cells showed lower Ki67 protein levels than tumors developed by control cells. Top, H&E staining; bottom, immunostaining; *, P < 0.05 and **, P < 0.01.
- Figure 5.
ANRIL could silence KLF2 and P21 expression. A, the levels of p15INK4B and p16 mRNA were determined by qPCR in SPC-A1 and PC9 cells transfected with si-ANRIL and results are expressed relative to the corresponding values for control cells. B and C, the levels of p21 and KLF2 mRNA and protein levels were determined by qPCR and Western blot when PC9 cells were transfected with si-EZH2 or si-SUZ12. D, ANRIL expression levels in cell cytoplasm or nucleus of NSCLC cell lines SPC-A1, PC9, and H520 were detected by qPCR. *, P < 0.05 and **, P < 0.01; N.S., not significant.
- Figure 6.
ANRIL could directly bind PRC2 and silence KLF2 and P21 transcription. A, RIP with rabbit monoclonal anti-EZH2, preimmune IgG, or 10% input from PC9 cell extracts. RNA levels in immunoprecipitates were determined by qPCR. Expression levels of ANRIL RNA are presented as fold enrichment in EZH2 relative to IgG immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates were used as positive control. B and C, ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells, and IgG as a negative control; ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9 cells treated with ANRIL siRNA (48 hours) or scrambled siRNA. D, analysis of the relationship between ANRIL expression and KLF2 mRNA level (ΔCt value) in 40 NSCLC tissues. The mean values and SDs were calculated from triplicates of a representative experiment.
- Figure 7.
Overexpression of KLF2 expression inhibits PC9 cells proliferation. PC9 cells were transfected with pCDNA–KLF2 or cotransfected with si-ANRIL and si-KLF2. A, the protein level of KLF2 in PC9 cells transfected with pCDNA–KLF2 was detected by Western blot analysis. B, MTT assays and colony-forming assays were used to determine the cell viability for pCDNA–KLF2-transfected PC9 cells. Values represent the mean ± SD from three independent experiments. C, apoptosis was determined by flow cytometry. Upper left, necrotic cells; upper right, terminal apoptotic cells; lower right, early apoptotic cells. D, the protein level of KLF2 in PC9 cells cotransfected with si-ANRIL and si-KLF2 was detected by Western blot analysis. E, MTT assays and colony-forming assays were used to determine the cell viability for si-ANRIL and si-KLF2 cotransfected PC9 cells. Values represent the mean ± SD from three independent experiments; *, P < 0.05 and **, P < 0.01.
Tables
- Table 1.
Correlation between ANRIL expression and clinicopathologic characteristics of NSCLC patients
ANRIL Characteristics High, number of cases (34) Low, number of cases (34) χ2 test (P value) Age, y 0.627 ≤65 17 19 >65 17 15 Gender 0.625 Male 18 20 Female 16 14 Histologic subtype 0.324 SCC 22 18 Adenocarcinoma 12 16 TNM stage 0.007a Ia + Ib 4 15 IIa + IIb 14 12 IIIa 16 7 Tumor size 0.001a ≤5 cm 13 26 >5 cm 21 8 Lymph node metastasis 0.051 Negative 11 19 Positive 23 15 Smoking history 0.793 Smokers 23 24 Never smokers 11 10 -
↵aOverall P < 0.05.
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Supplementary Data
Files in this Data Supplement:
- Supplementary Fig. S1 Legend - Supplementary Fig. S1 Legend
- Supplementary figure 1 - Supplementary figure 1. The effect of ANRIL on cells proliferation and migration
- Supplementary table 1 - Supplementary table 1. Univariate and multivariate analysis of progression-free survival in NSCLC patients (n=68)
- Supplementary table 2 - Supplementary table 2. Univariate and multivariate analysis of overall survival in NSCLC patients (n=68)
- Supplementary table 3 - Supplementary table 3. Sequence of primers and siRNA
- Supplementary table 4 - Supplementary table 4. The raw data of ANRIL expression level in all patients
Volume 14, Issue 1
