Figure 1.
Expression of HOTAIR in cisplatin-resistant A549/DDP cells is significantly upregulated compared with that in parental A549 cells.
(A) Morphologies of A549 and A549/DDP cells. Cells were grown to 70% confluency and then photographe under 40× magnification. (B) The IC50 value of cisplatin to A549/DDP cells was significantly higher than that to A549 cells. (C) Flow cytometric analysis of cell cycle distribution in A549 and A549/DDP cells. (D) The colony formation of A549 and A549/DDP cells treated with various concentrations of cisplatin (0.5, 1.0, 1.5 and 2.0 μg/L). (E) qRT-PCR analysis of HOTAIR expression in A549/DDP and A549 cells. (F) A549 cells were cultured in the presence of various concentrations of cisplatin (0.0, 0.5, 1.0, 1.5 or 2.0 μg/L) for 24h. qRT-PCR assay was performed to detect HOTAIR expression. GAPDH was used as an internal control. Results represent the average of three independent experiments (mean±SD). N.S indicates P>0.05 and * or ** indicates P<0.05 or <0.01, respectively.
Figure 2.
siRNA/HOTAIR significantly increases the in vitro sensitivity of A549/DDP to cisplatin.
(A) 48h after A549/DDP cells transfected with siRNA/control, siRNA/HOTAIR1 or siRNA/HOTAIR2, qRT-PCR detection of HOTAIR expression in those cells. GAPDH was used as an internal control. (B) MTT analysis of the IC50 values of cisplatin to siRNA/HOTAIR1 or siRNA/control-transfected A549/DDP cells. (C) Flow cytometry analysis of apoptosis in siRNA/control or siRNA/HOTAIR1-transfected A549/DDP cells combined with various concentrations of cisplatin (0.0, 1.0 or 2.0 μg/L). (D) Flow cytometry analysis of cell cycle distribution in siRNA/control or siRNA/HOTAIR1-transfected A549/DDP cells combined with various concentrations of cisplatin (0.0, 1.0 or 2.0 μg/L). Results represent the average of three independent experiments (mean±SD). N.S indicates P>0.05 and * or ** indicates P<0.05 or <0.01, respectively.
Figure 3.
pcDNA/HOTAIR significantly promotes the resistance of parental A549 cells to cisplatin.
(A) qRT-PCR detection of HOTAIR expression in A549 cells stably transfected with pcDNA/control or pcDNA/HOTAIR. GAPDH was used as an internal control. (B) MTT analysis of the IC50 values of cisplatin to A549/control or A549/HOTAIR cells. (C) Flow cytometry analysis of apoptosis in A549/control or A549/HOTAIR cells combined with various concentrations of cisplatin (0.0, 1.0 or 1.5 μg/L). (D) Flow cytometry analysis of cell cycle distribution in A549/control or A549/HOTAIR cells combined with various concentrations of cisplatin (0.0, 1.0 or 1.5 μg/L). Results represent the average of three independent experiments (mean±SD). N.S indicates P>0.05 and * or ** indicates P<0.05 or <0.01, respectively.
Figure 4.
pcDNA/p21 or siRNA/p21 could mimic the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the sensitivity of LAD cells to cisplatin.
(A) Western blot analysis of the effect of HOTAIR on p21 protein expression in pcDNA/HOTAIR (or pcDNA/control) or siRNA/HOTAIR1 (or siRNA/control)-transfected A549 or A549/DDP cells. GAPDH was used as an internal control. (B) MTT analysis of the IC50 values of cisplatin to pcDNA/control or pcDNA/p21-transfected A549/DDP cells and siRNA/control or siRNA/p21-transfected A549 cells. (C) Flow cytometry analysis of apoptosis in pcDNA/control or pcDNA/p21-transfected A549/DDP cells combined with various concentrations of cisplatin (0.0, 1.0 or 2.0 μg/L). (D) Flow cytometry analysis of apoptosis in siRNA/control or siRNA/p21-transfected A549 cells combined with various concentrations of cisplatin (0.0, 1.0 or 1.5 μg/L). (E) Flow cytometry analysis of cell cycle distribution in pcDNA/control or pcDNA/p21-transfected A549/DDP cells combined with various concentrations of cisplatin (0.0, 1.0 or 2.0 μg/L). (F) Flow cytometry analysis of cell cycle distribution in siRNA/control or siRNA/p21-transfected A549 cells combined with various concentrations of cisplatin (0.0, 1.0 or 1.5 μg/L). Results represent the average of three independent experiments (mean±SD). N.S indicates P>0.05 and * or ** indicates P<0.05 or <0.01, respectively.
Figure 5.
siRNA/p21 or pcDNA/21 reverses the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the chemosensitivity of LAD cells to cisplatin.
(A) 48h after A549/DDP cells transfected with siRNA/control, siRNA/HOTAIR1 alone or combination with siRNA/p21, Western blot detection of p21 protein expression in those cells. GAPDH was used as an internal control. (B) MTT analysis of the IC50 values of cisplatin to A549/DDP cells transfected with siRNA/control, siRNA/HOTAIR1 alone or combination with siRNA/p21. (C) 48h after A549 cells transfected with pcDNA/control, pcDNA/HOTAIR alone or combination with pcDNA/p21, Western blot detection of p21 protein expression in those cells. GAPDH was used as an internal control. (D) MTT analysis of the IC50 values of cisplatin to A549/DDP cells transfected with pcDNA/control, pcDNA/HOTAIR alone or combination with pcDNA/p21. Results represent the average of three independent experiments (mean±SD). N.S indicates P>0.05 and * or ** indicates P<0.05 or <0.01, respectively.
Figure 6.
Effects of HOTAIR expression on the in vivo sensitivity of A549/DDP cells to cisplatin.
(A) Tumor volume was calculated twice weekly following injection of A549/DDP cells transfected with siRNA/control or siRNA/HOTAIR after cisplatin treatment. Points, mean (n=3); bars indicate SD. (B) After 28 days, tumor weights are represented as means of tumor weights ± SD. (C) qRT-PCR detection of HOTAIR expression in tumor tissues formed from siRNA/control or siRNA/HOTAIR-transfected A549/DDP cells combined with cisplatin treatment. GAPDH was used as an internal control. (D) Western blot detection of p21 protein expression in tumor tissues formed from siRNA/control or siRNA/HOTAIR-transfected A549/DDP cells combined with cisplatin treatment. GAPDH was used as an internal control. (E) Tumors developed from siRNA/HOTAIR-transfected A549/DDP cells showed higher positive rate of p21 protein and lower positive rate of PCNA protein levels than tumors developed from siRNA/control-A549/DDP cells. Upper: H&E staining; Intermediate and lower: immunostaining, Origninal magnification, 100× or 400×. (F) Detection of apoptosis in tumors developed from siRNA/HOTAIR or siRNA/control-transfected A549/DDP cells combined with cisplatin treatment. Results represent the average of three independent experiments (mean±SD). * or ** indicates P<0.05 or <0.01, respectively.
Figure 7.
The inverse correlation between HOTAIR and p21 expression in LAD tissue samples.
(A) qRT-PCR detection of HOTAIR expression in cisplatin-sensitive (n=21) or insensitve (n=20) LAD tissues. Abundance of HOTAIR was normalized to GAPDH. (B) qRT-PCR detection of p21 mRNA expression in cisplatin-sensitive (n=21) or insensitve (n=20) LAD tissues. Abundance of p21 mRNA was normalized to GAPDH. (C) Immunostaining of p21 protein expression cisplatin-sensitive (n=20) or insensitve (n=21) LAD tissues. Origninal magnification, 100×(upper), 400×(low). (D) The inversely correlated expression of HOTAIR and p21 mRNA among those LAD tissue samples (n=41) as indicated by linear regression analysis. Spearman rank test r and P values (2-tailed) were shown. (E) Kaplan-Meier survival analysis of the association between PFS of patients and HOTAIR expression according to the level of HOTAIR expression. The P-value was determined with the log-rank test. ** indicates P<0.01.