<i>MEG3</i> overexpression impairs A549/DDP cell proliferation <i>in vitro</i>.

<p>(Fig 3A) MTT assay of A549/DDP cell proliferation with or without cisplatin. (Fig 3B) Colony formation analysis of cell proliferation in combination with increasing concentrations of cisplatin (0.0, 1.0, and 2.0 μg/ml). (Fig 3C) Flow cytometry analysis of cell cycle distribution in combination with increasing concentrations of cisplatin. *<i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

<i>MEG3</i> expression levels are positively correlated with the responses of patients to cisplatin-based chemotherapy.

<p>(Fig 8A) qRT-PCR analysis of <i>MEG3</i> expression levels in cisplatin-sensitive (n = 20) and cisplatin-insensitive (n = 21) LAD tissues. (Fig 8B) Kaplan–Meier survival analysis of the association between PFS of LAD patients and <i>MEG3</i> expression (log rank <i>P</i> < 0.001). (Fig 8C) Cisplatin-sensitive tissues exhibited higher p53 and lower Bcl-xl protein expression than cisplatin-insensitive tissues. Original magnification, ×200 or ×400. ** <i>P</i> < 0.01.</p

Differentially expressed lncRNAs in A549/DDP cells compared with A549 cells as determined by microarray.

<p>Differentially expressed lncRNAs in A549/DDP cells compared with A549 cells as determined by microarray.</p

Knockdown of <i>MEG3</i> promotes A549 cell proliferation <i>in vitro</i>.

<p>(Fig 5A) MTT assay of the proliferation of si-NC- and si-<i>MEG3</i>-transfected A549 cells. (Fig 5B) Flow cytometry analysis of cell cycle distribution in combination with increasing concentrations of cisplatin (0.0, 1.0, and 1.5 μg/ml). *<i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

The level of <i>MEG3</i> expression level in LAD cells.

<p>(Fig 1A) qRT-PCR analysis of <i>MEG3</i> expression levels in A549 and A549/DDP cells. (Fig 1B) MTT assay of the IC₅₀ values of A549 and A549/DDP cells to cisplatin. (Fig 1C) qRT-PCR analysis of <i>MEG3</i> expression levels following the transfection of A549/DDP cells with empty vector or pCDNA-<i>MEG3</i>. (Fig 1D) MTT assay of the IC₅₀ values of empty vector- and pCDNA-<i>MEG3</i>-transfected A549/DDP cells to cisplatin. *<i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

<i>MEG3</i> improves the <i>in vivo</i> sensitivity of A549/DDP cells to cisplatin.

<p>(Fig 7A, 7B) Four weeks after initial cisplatin administration, the tumor volume and average weight of mice receiving pCDNA-<i>MEG3</i>- or empty vector-transfected A549/DDP cells were recorded. (Fig 7C) Tumor volume was calculated twice weekly after cisplatin treatment. (Fig 7D) qRT-PCR analysis of <i>MEG3</i> expression levels in tumor tissues formed from pCDNA-<i>MEG3</i>- or empty vector-transfected A549/DDP cells. (Fig 7E) Tumors developed from pCDNA-<i>MEG3</i>-transfected A549/DDP cells showed lower PCNA protein levels, higher p53 protein expression, and lower Bcl-xl protein expression than those developed from control cells. Upper: H&E staining; second, third row and lower: immunostaining. Original magnification, ×100 or ×200. *<i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

The Long Noncoding RNA HOTAIR Contributes to Cisplatin Resistance of Human Lung Adenocarcinoma Cells via downregualtion of p21^WAF1/CIP1 Expression

<div><p>HOTAIR, a long intervening non-coding RNA (lincRNA), associates with the Polycomb Repressive Complex 2 (PRC2) and is reported to reprogram chromatin organization and promote tumor progression. However, little is known about the roles of this gene in the development of chemoresistance phenotype of lung adenocarcinoma (LAD). Thus, we investigated the involvement of HOTAIR in the resistance of LAD cells to cisplatin. In this study, we show that HOTAIR expression was significantly upregulated in cisplatin-resistant A549/DDP cells compared with in parental A549 cells. Knockdown of HOTAIR by RNA interference could resensitize the responses of A549/DDP cells to cisplatin both <i>in vitro</i> and <i>in vivo</i>. In contrast, overexpression of HOTAIR could decrease the sensitivity of A549 and SPC-A1 cells to cisplatin. We also found that the siRNA/HOTAIR1-mediated chemosensivity enhancement was associated with inhibition of cell proliferation, induction of G₀/G₁ cell-cycle arrest and apoptosis enhancement through regulation of p21^WAF1/CIP1 (p21) expression. Also, pcDNA/p21or siRNA/p21 could mimic the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the sensitivity of LAD cells to cisplatin. Importantly, siRNA/p21 or pcDNA/p21 could partially rescue the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on both p21 expression and cisplatin sensitivity in LAD cells. Further, HOTAIR was observed to be significantly downregulated in cisplatin-responding LAD tissues, and its expression was inversely correlated with p21 mRNA expression. Taken together, our findings suggest that upregulation of HOTAIR contributes to the cisplatin resistance of LAD cells, at least in part, through the regulation of p21 expression. </p> </div

Expression of HOTAIR in cisplatin-resistant A549/DDP cells is significantly upregulated compared with that in parental A549 cells.

<p>(<b>A</b>) Morphologies of A549 and A549/DDP cells. Cells were grown to 70% confluency and then photographe under 40× magnification. (<b>B</b>) The IC₅₀ value of cisplatin to A549/DDP cells was significantly higher than that to A549 cells. (<b>C</b>) Flow cytometric analysis of cell cycle distribution in A549 and A549/DDP cells. (<b>D</b>) 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). (<b>E</b>) qRT-PCR analysis of HOTAIR expression in A549/DDP and A549 cells. (<b>F</b>) 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). <i>N.S</i> indicates <i>P</i>>0.05 and * or ** indicates <i>P</i><0.05 or <0.01, respectively.</p

Additional file 1: of Over-expressed long noncoding RNA HOXA11-AS promotes cell cycle progression and metastasis in gastric cancer

Primers and siRNAs sequences. (XLSX 13 kb

siRNA/HOTAIR significantly increases the in vitro sensitivity of A549/DDP to cisplatin.

<p>(<b>A</b>) 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>B</b>) MTT analysis of the IC₅₀ values of cisplatin to siRNA/HOTAIR1 or siRNA/control-transfected A549/DDP cells. (<b>C</b>) 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). (<b>D</b>) 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). <i>N.S</i> indicates <i>P</i>>0.05 and * or ** indicates <i>P</i><0.05 or <0.01, respectively.</p