Overexpression of lncRNA HOXA11-AS promotes cell epithelial–mesenchymal transition by repressing miR-200b in non-small cell lung cancer

We collected paired NSCLC tissue and adjacent normal lung tissues from 78 cases of patients who underwent radical surgical resection between January 2010 and June 2014 at Huai’an Second People’s Hospital. No patient had received local or systemic treatment before any operation. All collected tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA analysis. Written consent was obtained from each patient before tissue collection. The protocol was approved by the Institutional Research Ethics Committee of Huai’an Second People’s Hospital.

The human NSCLC cells A549, H1299, 95D and normal human bronchial epithelial cells 16HBE cell lines were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the RPMI1640 medium (Hyclone, USA) and added with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C, high humidity, and 5% CO₂.

2 × 10⁵ A549 and H1299 cells were seeded in 6-well plates and were incubated overnight, and then transfected using 100 nmol/L of small-interfering si-HOXA11-AS-1 or si-HOXA11-AS-2, miR-200b inhibitor and a negative control (NC) that were purchased from RiboBio (Guangzhou, China). Cells were transfected using Lipofectamine^® 3000 transfection reagent (Invitrogen, USA).

The invasive activity was detected by transwell invasion assays using 24-well transwell insert coated with Matrigel (8-μm pore size; Millipore). The 1 × 10⁵ cells that were transfected with si-HOXA11-AS or si-NC were seeded in serum-free medium and added on the upper chamber. In the lower chamber, cells were cultured with Medium supplemented with 10% fetal bovine serum. After 48 h of incubation, the cells on the upper surface of the filter were removed using a cotton swab, Cells in lower surface of the filter were fixed and stained with 0.5% crystal violet and counted under a light microscope.

The RNA was isolated from cell lines and lysed with Trizol (Life Technologies, Carlsbad, CA) and RNA isolation was performed according to the manufacturer’s protocol. RNA was reversed into cDNA using the PrimeScript™ RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. QRT-PCR assays were performed using SYBR^® Premix Ex Taq™ II (Takara) in the ABI PRISM^® 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). GADPH were used as endogenous controls. The primer sequences for relative mRNA were HOXA11-AS-F:5′-GATTTCTCCAGCCTCCCTTC-3′ and HOXA11-AS-R:5′-AGAAATTGGACGAGACTGCG-3′. ZEB1-F:5′-TCCATGCTTAAGAGCGCTAGCT-3′, ZEB2-R:5′-ACCGTAGTTGAGTAGGTGTATGCCA-3′. ZEB2-F:5′-GGCGCAAACAAGCCAATCCCA-3′, ZEB2-R:5′-TTCACTGGACCATCTACAGAGGCTT-3′. Snail1-F:5′-CAAGGAATACCTCAGCCTGG-3′, Snail1-R:5′-ATTCACATCCAGCACATCCA-3′. Snail2-F:5′-CTACAGCGAACTGGACACACA-3′, Snail2-R:5′-GGAATGGAGCAGCGGTAGT-3′GAPDH-F:5′-TGGTATCGTGGAAGGACTCAT-3′, GAPDH-R:5′-GTGGGTGTCGCTGTTGAAGTC-3′. Data were collected and were analyzed using 2^−ΔΔCt method for quantification of the relative mRNA expression levels.

Cells were transfected with si-HOXA11-AS-1 or si-HOXA11-AS-2 and si-negative control (NC) at 48 h and then cells were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Beijing, China). About 40 μg of protein extracts were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA), and incubated with specific antibodies with ZEB1 (Santa Cruz Biotechnology Inc. Dallas, TX, USA), ZEB2 (Santa Cruz Biotechnology Inc. Dallas, TX, USA), Snail1 (Santa Cruz Biotechnology Inc. Dallas, TX, USA), Snail2 (Santa Cruz Biotechnology Inc. Dallas, TX, USA), E-cadherin (Cell Signaling, San Jose, CA, USA), N-cadherin (Cell Signaling, San Jose, CA, USA) and GAPDH (Cell Signaling, San Jose, CA, USA). The membrane was blocked with 5% skimmed milk in Tris-buffered saline and then incubated with second antibody for 1 h. An enhanced chemiluminescent (ECL) chromogenic substrate was performed to visualize the bands. The relative protein levels were normalized to GAPDH.

RIP assays were carried out using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) following the manufacturer’s instructions in A549 and H1299 cells. Antibody for RIP assays of EZH2 (CST, USA) and DNMT1 (CST,USA) or control IgG (Millipore) were used in the study.

CHIP assays was performed using a EZ-Magna ChIP kit (Millipore) according to manufacturer’s protocol, the A549 and H1299 cells were fixed with 4% paraformaldehyde and treated with glycine for 20 min to generate DNA–protein cross-links. Then, cells were lysed by lysis Buffer and Nuclear Lysis Buffer and sonicated to product chromatin fragments of 200–300 bp and cell lysates were immunoprecipitated with Magnetic Protein A Beads conjugated with EZH2 (CST, USA) and DNMT1(CST, USA) antibody.

Statistical analysis was determined using the SPSS 18.0 software. All data were expressed as means  ±  standard deviation (SD). For comparisons between two samples, an unpaired two-tailed t test was performed. P values of less than 0.05 were considered as statistically significance.

To investigate the clinical significance of lncRNA HOXA11-AS expression in NSCLC, we collected 78 cases of primary NSCLC tissue samples and paired adjacent normal tissues. The lncRNA HOXA11-AS was analyzed by qRT-PCR assays and the results showed that lncRNA HOXA11-AS was significantly up-regulated in the NSCLC tissues, compared with the adjacent normal tissues (Fig. 1a). The median expression was 2.38-fold. According to the median expression of lncRNA HOXA11-AS, we classified the patients into two groups: higher lncRNA HOXA11-AS expression and lower lncRNA HOXA11-AS group. The association between lncRNA HOXA11-AS expression with clinicopathological factors showed that higher lncRNA HOXA11-AS expression levels were positively associated with the lymph node metastasis (P = 0.011) and TNM stage (P = 0.007) (Table 1). Moreover, we showed that the patients who had higher lncRNA HOXA11-AS had a worse survival compared with patients who had lower lncRNA HOXA11-AS (Fig. 1b, P < 0.05 log-rank test). We next examined lncRNA HOXA11-AS expression levels in three human NSCLC cells A549, H1299, 95D and a normal human bronchial epithelial cell 16HBE, the results discovered that lncRNA HOXA11-AS expression levels was higher compared with that in the 16HBE cell (Fig. 1c). Thus, these results demonstrated that lncRNA HOXA11-AS was up-regulated in NSCLC tissues and cells, and higher lncRNA HOXA11-AS predicted a poor prognosis in NSCLC patients.

To assess the biological role of lncRNA HOXA11-AS in NSCLC cells, we down-regulated the expression of lncRNA HOXA11-AS in A549 cells and H1299 cells and determined the si-HOXA11-AS-2 had a better knockdown efficiency for lncRNA HOXA11-AS and were used in the following experiments (Fig. 1d). The transwell cell invasion was used to determine the cell invasion ability in A549 and H1299 cells. The results showed that silencing lncRNA HOXA11-AS significantly inhibited the cell invasion ability, compared with the si-NC group in A549 cells (Fig. 2a, b). Compared with the si-NC group in H1299 cells, the cell invasion ability also been inhibited when lncRNA HOXA11-AS was knocked down (Fig. 2c, d). Thus, our results showed that silencing HOXA11-AS suppressed the cell invasion ability in NSCLC.

Epithelial–mesenchymal transition is an important factor in NSCLC cell invasion and metastasis [16]. Thus, we next determined whether EMT related transcription factors and markers were altered in our study. The mRNA and protein expression of ZEB1, ZEB2, Snail1, Snai2, E-cadherin and N-cadherin were analyzed by qRT-PCR and western blot assays. The results showed that the mRNA and protein expression of ZEB1, ZEB2, Snail1, Snail2 and N-cadherin were significantly decreased while E-cadherin expression was increased when lncRNA HOXA11-AS was knocked down in A549 cells, compared with the si-NC group (Fig. 3a, c). Compared with the si-NC group, the similar results also been found in H1299 cells after lncRNA HOXA11-AS silencing (Fig. 3b, d). Thus, these results demonstrated that lncRNA HOXA11-AS was involved in the EMT process and knockdown of lncRNA HOXA11-AS inhibits cell EMT process in NSCLC cells.

To investigate the underlying mechanisms of lncRNA HOXA11-AS involved in EMT process in NSCLC. We detected the location of lncRNA HOXA11-AS in A549 and H1299 cells and found that lncRNA HOXA11-AS were enrich in nuclear and cytoplasmic fractions by qRT-PCR analysis (Fig. 4a). Many molecular mechanisms have been found to explain lncRNA-mediated epigenetically silencing related gene expression by interacting with EZH2 and DNMT1 [14]. Furthermore, we applied RIP assays to explore whether lncRNA HOXA11-AS interacted with EZH2 and DNMT1 in NSCLC cells. We found that lncRNA HOXA11-AS was enriched in anti-EZH2 and anti-DNMT1 antibodies RIP fraction group, compared with the IgG RIP fraction group (Fig. 4b, c). miR-200b had been found to suppressed cell EMT process and functioned as a target of EZH2, such as, DNMT1 and EZH2 mediated methylation silences the microRNA-200b/a/429 gene and promotes tumor progression [17]. Our results demonstrated that inhibition of lncRNA HOXA11-AS significantly up-regulated the miR-200b expression level in A549 and H1299 cells (Fig. 4d). Moreover, knockdown of EZH2 also increased the miR-200b expression in A549 and H1299 cells (Fig. 4e). Thus, the above results indicated that lncRNA HOXA11-AS could regulate the miR-200b expression interacted with EZH2 and DNMT1in NSCLC cells.

Next, we explored whether lncRNA HOXA11-AS regulated miR-200b expression by recruiting EZH2 and DNMT1 to the promoter of miR-200b. The ChIP assays revealed that EZH2 and DNMT1 could bind to the promoter region of miR-200b in A549 and H1299 cells (Fig. 5a), and knockdown of significantly decreased the binding of EZH2 and DNMT1 to the promoter of miR-200b (Fig. 5b, c). The western blot analysis further showed that knockdown of lncRNA HOXA11-AS down-regulated EMT markers N-cadherin and Vimentin expression and up-regulated the E-cadherin expression, however, co-transfected with si-HOXA11-AS and miR-200b inhibitor dismissed the effects in A549 and H1299 cells (Fig. 5d, e). Moreover, knockdown of lncRNA HOXA11-AS inhibited cell invasion ability, however, co-transfected with si-HOXA11-AS and miR-200b inhibitor dismissed the effects in A549 and H1299 cells (Fig. 6a–d).Thus, these results confirmed that lncRNA HOXA11-AS promoted cell EMT and suppressed miR-200b expression by interacting with EZH2 and DNMT1 in NSCLC cells.