Additional file 1: of Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression

TableS1. The list of primers and the sequence of siRNAs. (XLS 8 kb

Additional file 2: of Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression

TableS2. mRNAs increased abundance (≥2-fold) in AFAP1-AS1 knockdown A549 cells. (XLS 125 kb

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

Identification of Suz-12 as a functional target of miR-200b in CSCs.

<p>(<b>A</b>) qRT-PCR detection of relative Suz-12 mRNA expression in the indicated cells. Data were normalized to GAPDH RNA and determined relative to the corresponding parental-cell groups. (<b>B</b>) The protein levels of Suz-12 as measured by Western blotting in the indicated cells. GAPDH was used as an internal control. (<b>C</b>) qRT-PCR detection of Suz-12 mRNA expression in CSCs after transfection of the indicated vectors. (<b>D</b>) Western blotting detection of Suz-12 protein expression in CSCs after transfection with the indicated vectors. GAPDH was used as an internal control. (<b>E</b>) Luciferase activity of the CSCs (SPC-A1/DTX) co-transfected with pcDNA/miR-200b (or pcDNA/miR-NC) or pLUC/Suz-12/3′-UTR-wt (or pLUC/Suz-12/3′-UTR-mut). Data were mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Properties of CSCs obtained from docetaxel-resistant human lung adenocarcinoma cells (SPC-A1/DTX and H1299/DTX).

<p>(A₁, A₂, A₃) Percentage of CD133⁺/CD326⁺ CSCs as analyzed by Flow cytometry at the indicated time after cultivating CD133⁺/CD326⁺ CSCs (acquired by sorting SPC-A1/DTX and H1299/DTX cells, respectively) under differentiation conditions. (B) Mammosphere forming ability of the indicated cells (1000 cells) after 7 days under CSC-cultivating conditions. (C) qRT-PCR detection of relative mRNA levels of the CSC-related markers in the indicated cells. Data was calculated with the 2^−△△Ct method using GAPDH RNA as the reference gene. The expression level was measured relative to the fold change of the parental cells groups that were defined as 1.0. (D₁, D₂) The protein levels of the CSC-related markers in the indicated cells. GAPDH was used as an internal control. (E) Tumor incidence in nude mice that were subcutaneously injected with the indicated cells. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

MiR-200b has effects on CSCs by regulating Suz-12/E-cadherin.

<p>(<b>A</b>) Western blotting detection of E-cadherin protein expression in the indicated cells transfected with the indicated vectors. (<b>B</b>) qRT-PCR detection of E-cadherin mRNA expression as measured by in the CSCs transfected with the indicated vectors. (<b>C</b>) Western blotting detection of E-cadherin protein expression in the CSCs transfected with the indicated vectors. (<b>D</b>) and (<b>E</b>) Suz-12 overexpression rescued the increased level of both mRNA and protein expression of E-cadherin in CSCs (SPC-A1/DTX) mediated by miR-200b upregulation, while silencing of Suz-12 rescued the decreased expression of mRNA and protein of E-cadherin in CSCs (SPC-A1/DTX) induced by miR-200b repression. (<b>F</b>) qRT-PCR detection of miR-200b and Suz-12 mRNA expression at the indicated time after plating CD133⁺/CD326⁺ CSCs under differentiation conditions. (<b>G₁, G₂</b>) The protein levels of Suz-12 and E-cadherin at the indicated time after cultivating CD133⁺/CD326⁺ CSCs under differentiation conditions. (<b>H</b>) MiR-200b upregulation decreased the amount of Suz-12 binding to the promoter of E-cadherin in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. (<b>I</b>) MiR-200b overexpression reduced trimethylation of histone H3-lysine-27 (H3-K27) at E-cadherin promoter in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01; ^#<i>p</i><0.05, ^##<i>p</i><0.01, compared with corresponding 0 day groups.</p

HDAC1 repression or miR-200b overexpression reduces tumorigenicity and reverses chemoresistance of CSCs <i>in vivo</i>.

<p>(<b>A</b>) Tumorigenicity in nude mice subcutaneously injected with CSCs from SPC-A1/DTX cells (5000 cells/mouse, n = 5) that were stably transfected with the indicated vectors previously. (<b>B</b>) Tumor volume in nude mice injected with H1299/DTX cells that were stably transfected with the indicated vectors and were combined with DTX treatment. Data were presented as mean ± SD. (<b>C</b>) The protein level of E-cadherin and Suz-12 in tumors of the indicated groups that were provided at 5 weeks after the inoculation. GAPDH was used as an internal control. (<b>D</b>) Flow cytometry detection of percentage of CD133⁺/CD326⁺ CSCs in tumors of the indicated groups. (<b>E</b>) The mRNA level of miR-200b and Suz-12 in tumors of the indicated groups. Data were normalized to U6 RNA and GAPDH, respectively. Data were presented as mean ± SD of at least three independent experiments. **<i>p</i><0.01.</p

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

siRNA/p21 or pcDNA/21 reverses the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the chemosensitivity of LAD cells to cisplatin.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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). <i>N.S</i> indicates <i>P</i>>0.05 and * or ** indicates <i>P</i><0.05 or <0.01, respectively.</p

Effects of HOTAIR expression on the in vivo sensitivity of A549/DDP cells to cisplatin.

<p>(<b>A</b>) Tumor volume was calculated twice weekly following injection of A549/DDP cells transfected with siRNA/control or siRNA/HOTAIR after cisplatin treatment. Points, mean (<i>n</i>=3); bars indicate SD. (<b>B</b>) After 28 days, tumor weights are represented as means of tumor weights ± SD. (<b>C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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×. (<b>F</b>) 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 <i>P</i><0.05 or <0.01, respectively.</p