Long noncoding RNA PXN-AS1-L promotes non-small cell lung cancer progression via regulating PXN

The human normal bronchial epithelial cell line 16HBE, human NSCLC cell lines NCI-H1975, A549, NCI-H1299, and SK-MES-1 were obtained from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 16HBE cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA). NCI-H1975 and NCI-H1299 cells were cultured in RPMI-1640 Medium (Invitrogen). A549 cells were cultured in F-12K Medium (Invitrogen). SK-MES-1 cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen). All the cells were maintained in the above described medium supplemented with 10% fetal bovine serum (Invitrogen) in a humidified incubator at 37 °C with 5% CO₂. Where indicated, NSCLC cells were treated with 50 µM α-amanitin (Sigma-Aldrich, Saint Louis, MO, USA) for 0–24 h as shown in the article.

Sixty-six pairs of NSCLC tissues and adjacent noncancerous lung tissues, and ten NSCLC bone metastases tissues were acquired from NSCLC patients who underwent surgery at the General Hospital of Chinese People’s Liberation Army (Beijing, China). All the tissues were diagnosed and histologically confirmed by two pathologists. The resected specimens were immediately frozen in liquid nitrogen and stored at − 80 °C until use. The study was approved by the ethics committee of the General Hospital of Chinese People’s Liberation Army (Beijing, China) and written informed consent was obtained from all patients.

PXN-AS1-L overexpression plasmid pcDNA3.1-PXN-AS1-L was constructed as previously described [38]. Briefly, PXN-AS1-L full-length transcript was PCR amplified by Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo-Fisher Scientific, Waltham, MA, USA) and subcloned into the BamH I and EcoR V sites of pcDNA3.1 plasmid (Invitrogen). The primers sequences were: 5′-GGTACCGAGCTCGGATCCTCGCGTTGGAGGAGCTTG-3′ (forward) and 5′-GCCACTGTGCTGGATATCCTACAAAAAAAATTTATTTAATAAAA-3′ (reverse). The cDNA oligonucleotides suppressing PXN-AS1-L or PXN expression were designed and synthesized by GenePharma (Shanghai, China). After annealing, double strand oligonucleotides were inserted to the SuperSilencing shRNA expression plasmid pGPU6/Neo (GenePharma). A scrambled shRNA was used as a negative control and designated as shControl. The shRNA sequences were: for shPXN, 5′-CACCCCTGACGAAAGAGAAGCCTAATTCAAGAGATTAGGCTTCTCTTTCGTCAGGTTTTTTG-3′ (sense), 5′-GATCCAAAAAACCTGACGAAAGAGAAGCCTAATCTCTTGAATTAGGCTTCTCTTTCGTCAGG-3′ (anti-sense); for shRXN-AS1-L, 5′-CACCGCCCAGAGGAAATCAACAAGATTCAAGAGATCTTGTTGATTTCCTCTGGGCTTTTTTG-3′ (sense), 5′-GATCCAAAAAAGCCCAGAGGAAATCAACAAGATCTCTTGAATCTTGTTGATTTCCTCTGGGC-3′ (anti-sense); for shControl, 5′-CACCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG-3′ (sense), 5′-GATCCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAC-3′ (anti-sense). Transient transfection was performed using Lipofectamine 3000 (Invitrogen) in accordance with the manufacturer’s instruction.

To constructing PXN-AS1-L stably overexpressed A549 cells, pcDNA3.1-PXN-AS1-L or pcDNA3.1 was transfected into A549 cells and selected with neomycin (800 µg/ml) for 4 weeks. To constructing PXN-AS1-L stably depleted NCI-H1299 cells, shPXN-AS1-L or shControl was transfected into NCI-H1299 cells and selected with neomycin (1000 µg/ml) for 4 weeks.

Total RNA was extracted from indicted tissues or cells using TRIzol Reagent (Invitrogen) in accordance with the manufacturer’s protocol. Reverse transcription was performed using the M-MLV Reverse Transcriptase (Invitrogen) in accordance with the manufacturer’s protocol. Quantitative real-time PCR (qPCR) was performed in the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the SYBR^® Premix Ex Taq™ II kit (Takara, Dalian, China) in accordance with the manufacturer’s protocols. β-actin was used as an endogenous control. The primers sequences were: for PXN-AS1-L, 5′-ACCCATCCTCAACTACCCC-3′ (forward) and 5′-ACTTCGTCTGTGCCTTCTGC-3′ (reverse); for PXN, 5′-TATCTCAGCCCTCAACACGC-3′ (forward) and 5′-GGCAGAAGGCACAGACGAA-3′ (reverse); for β-actin, 5′-GGGAAATCGTGCGTGACATTAAG-3′ (forward) and 5′-TGTGTTGGCGTACAGGTCTTTG-3′ (reverse). The relative expression of RNAs was calculated using the comparative Ct method.

Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China). Identical quantities of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes. After being blocked with 5% not-fat milk, the membranes were incubated with primary antibodies specific for PXN (Abcam, Hong Kong, China) or β-actin (Proteintech, Rosemont, IL, USA). After being washed, the membranes were incubated with IRdye 700-conjugated goat anti-mouse IgG or IRdye 800-conjugated goat anti-rabbit IgG and were detected using an Odyssey infrared scanner (Li-Cor, Lincoln, NE, USA).

Cell proliferation was detected using Glo cell viability assay and Ethynyl deoxyuridine (EdU) incorporation assay. For Glo cell viability assay, 3000 indicated NSCLC cells were seeded each well in 96-well plate. At indicated time, cell viability was detected using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) in accordance with the manufacturer’s protocol. EdU incorporation assay was carried out using the EdU kit (Roche, Mannheim, Germany) in accordance with the manufacturer’s protocol. The results were acquired and quantified with the Zeiss AxioPhot Photomicroscope (Carl Zeiss, Oberkochen, Germany) based on at least ten random fields. Cell apoptosis was detected using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. Indicated NSCLC cells were treated with 25 ng/ml doxorubicin (Selleck, Houston, TX, USA) for 24 h. Then, cell apoptosis was detected using the TUNEL Cell Apoptosis Detection Kit (Beyotime) in accordance with the manufacturer’s protocol.

Cell migration was detected using transwell assay. 5 × 10⁴ indicated NSCLC cells in serum-free medium were plated in the top chamber a 24-well transwell chamber (BD Biosciences, San Jose, CA, USA). Complete medium containing 10% fetal bovine serum was placed into the lower chamber. After incubation for 24 h, cells remaining on the top chamber were wiped off using a cotton swab, and the cells that had traversed the membranes were stained by crystal violet and counted.

3 × 10⁶ indicted NSCLC cells were subcutaneously injected into 6-week-old athymic BALB/c nude mice purchased from the Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). Subcutaneous xenograft growth was measured weekly with a caliper, and the tumor volume was calculated as a × b² × 0.5 (a, longest diameter; b, shortest diameter). The animal studies were approved by the ethics committee of the General Hospital of Chinese People’s Liberation Army (Beijing, China).

For IHC, the subcutaneous xenografts were formalin-fixed, paraffin-embedded, and cut into 4 µm sections. The sections were incubated with primary antibodies specific for Ki67 (Abcam) or cleaved caspase-3 (Cell Signaling Technology, Boston, MA, USA). After being washed, the sections were incubated with horseradish peroxidase-conjugated second antibody (Abcam) and visualized using DAB Horseradish Peroxidase Color Development Kit (Beyotime).

All statistical analyses were performed using the GraphPad Prism Software. For comparisons, Student’s t test (two-sided), Wilcoxon signed-rank test, Mann–Whitney test, Pearson Chi square test, Log-rank test, and Pearson correlation analysis were performed as indicated. P values < 0.05 were considered as statistically significant.

To investigate the expression pattern of PXN-AS1-L in NSCLC, we first measured the expression of PXN-AS1-L in normal bronchial epithelial cell line 16HBE and NSCLC cell lines NCI-H1975, A549, NCI-H1299, SK-MES-1. The results displayed that PXN-AS1-L was significantly up-regulated in NSCLC cell lines compared with that in normal bronchial epithelial cell line, and further up-regulated in NSCLC cell lines derived from metastatic sites (NCI-H1299 and SK-MES-1) (Fig. 1a). Then, we collected 66 pairs of NSCLC tissues and adjacent noncancerous lung tissues and measured the expression of PXN-AS1-L in these tissues. The results displayed that the expression of PXN-AS1-L was significantly higher in NSCLC tissues than that in adjacent noncancerous lung tissues (Fig. 1b). Furthermore, we collected 10 NSCLC bone metastases tissues and also measured the expression of PXN-AS1-L. The results displayed that the expression of PXN-AS1-L was further higher in bone metastases tissues than that in primary NSCLC tissues (Fig. 1c).

The correlations between the expression of PXN-AS1-L in NSCLC tissues and clinicopathological characteristics of these 66 NSCLC patients were analyzed. As shown in Table 1, increased expression of PXN-AS1-L was positively associated with larger tumor size, positive lymph nodes metastasis, and advanced TNM stages, but not associated with age, gender, and histologic subtype. Kaplan–Meier survival analysis displayed that NSCLC patients with higher PXN-AS1-L expression had worse survival than those with lower PXN-AS1-L expression (Fig. 1d). All these data together demonstrated that PXN-AS1-L was up-regulated in NSCLC, further up-regulated in metastatic NSCLC cells and tissues. Increased expression of PXN-AS1-L predicted poor outcome of NSCLC patients.

To reveal the biological effects of PXN-AS1-L on NSCLC, we stably overexpressed PXN-AS1-L in A549 cells which has a relative low expression of PXN-AS1-L among NSCLC cell lines by transfecting PXN-AS1-L overexpression plasmid (Fig. 2a). Glo cell viability assays displayed that PXN-AS1-L overexpression increased cell viability of A549 cells (Fig. 2b). EdU incorporation assays also displayed that PXN-AS1-L overexpression promoted cell proliferation of A549 cells (Fig. 2c). TUNEL assays displayed that PXN-AS1-L overexpression inhibited cell apoptosis of A549 cells (Fig. 2d). Transwell assays displayed that PXN-AS1-L overexpression promoted cell migration of A549 cells (Fig. 2e). All these data together demonstrated that PXN-AS1-L overexpression promoted cell proliferation, inhibited cell apoptosis, and promoted cell migration of NSCLC cells, suggesting that PXN-AS1-L has oncogenic roles in NSCLC.

To further confirm the oncogenic roles of PXN-AS1-L on NSCLC, we stably knocked down PXN-AS1-L expression in NCI-H1299 cells which has a relative high expression of PXN-AS1-L among NSCLC cell lines by transfecting PXN-AS1-L specific shRNA (Fig. 3a). Glo cell viability assays displayed that PXN-AS1-L knockdown reduced cell viability of NCI-H1299 cells (Fig. 3b). EdU incorporation assays also displayed that PXN-AS1-L knockdown inhibited cell proliferation of NCI-H1299 cells (Fig. 3c). TUNEL assays displayed that PXN-AS1-L knockdown promoted cell apoptosis of NCI-H1299 cells (Fig. 3d). Transwell assays displayed that PXN-AS1-L knockdown inhibited cell migration of NCI-H1299 cells (Fig. 3e). All these data together demonstrated that PXN-AS1-L knockdown inhibited cell proliferation, promoted cell apoptosis, and inhibited cell migration of NSCLC cells, supporting the oncogenic roles of PXN-AS1-L in NSCLC.

To explore whether PXN-AS1-L also have oncogenic roles in vivo, PXN-AS1-L stably overexpressed and control A549 cells were subcutaneously injected into nude mice. Xenografts growth rates were measured every 7 days. At the 28th day, the xenografts were excised and weighed. The results displayed that PXN-AS1-L overexpression markedly promoted xenograft growth in vivo, and PXN-AS1-L overexpressed A549 cells formed much larger tumor than those formed by control A549 cells (Fig. 4a, b). Ki67 staining of xenograft displayed that PXN-AS1-L overexpression promoted A549 cell proliferation in vivo (Fig. 4c). Cleaved caspase-3 staining of xenograft displayed that PXN-AS1-L overexpression inhibited A549 cell apoptosis in vivo (Fig. 4d). All these data together demonstrated that PXN-AS1-L also had oncogenic roles in vivo.

PXN-AS1-L is previously reported to up-regulate PXN expression in HCC cells [38]. PXN has been revealed to play oncogenic roles in NSCLC in several reports [39‐41]. Therefore, we first investigated whether PXN-AS1-L also regulates PXN expression in NSCLC and whether the oncogenic roles of PXN-AS1-L in NSCLC are dependent on the regulation of PXN. The expressions of PXN in PXN-AS1-L stably overexpressed and control A549 cells, and PXN-AS1-L stably depleted and control NCI-H1299 cells were measured by qRT-PCR and western blot. The results displayed that PXN-AS1-L overexpression up-regulated PXN mRNA level (Fig. 5a). Conversely, PXN-AS1-L knockdown reduced PXN mRNA level (Fig. 5b). Western blot assays also displayed that PXN-AS1-L overexpression up-regulated PXN protein level and while PXN-AS1-L knockdown reduced PXN protein level (Fig. 5c, d). PXN-AS1-L is reported to decrease PXN mRNA degradation and increase PXN mRNA stability in HCC cells [38]. To investigate whether the same mechanism was employed by PXN-AS1-L in NSCLC, we next determined the effects of PXN-AS1-L on PXN mRNA stability in NSCLC cell. After transiently overexpressing PXN-AS1-L in A549 cells or depleting PXN-AS1-L in NCI-H1299 cells, the cells were treated with α-amanitin to block new RNA synthesis. Next, the loss of PXN mRNA was measured. As shown in Fig. 5e, f, PXN-AS1-L overexpression elongated the half-life of PXN mRNA, and conversely, PXN-AS1-L knockdown shortened the half-life of PXN mRNA, which suggested that PXN-AS1-L also increase PXN mRNA stability in NSCLC cells. All these data together demonstrated that PXN-AS1-L up-regulated PXN expression in NSCLC.

To explore whether the regulation of PXN by PXN-AS1-L also exist in vivo, we measured PXN expression in the same 66 pairs of NSCLC tissues and adjacent noncancerous lung tissues used in Fig. 1b. The results displayed that the expression of PXN was also significantly higher in NSCLC tissues than that in adjacent noncancerous lung tissues (Fig. 6a). Furthermore, our results also displayed that the expression of PXN was further higher in bone metastases tissues than that in primary NSCLC tissues (Fig. 6b). Correlation analysis displayed that the expression level of PXN-AS1-L was positively correlated with that of PXN in these 66 NSCLC tissues (Fig. 6c), supporting the positive regulation of PXN by PXN-AS1-L.

To explore whether the oncogenic roles of PXN-AS1-L in NSCLC are dependent on the positive regulation of PXN, we knocked down PXN in PXN-AS1-L stably overexpressed A549 cells by transient transfection of PXN specific shRNA (Fig. 7a). Glo cell viability assays displayed that PXN knockdown attenuated the increasing of cell viability induced by PXN-AS1-L overexpression (Fig. 7b). EdU incorporation assays also displayed that PXN knockdown attenuated the pro-proliferative roles of PXN-AS1-L (Fig. 7c). TUNEL assays displayed that PXN knockdown attenuated the cell apoptosis repressive roles of PXN-AS1-L (Fig. 7d). Transwell assays displayed that PXN knockdown attenuated the increasing of cell migration induced by PXN-AS1-L overexpression (Fig. 7e). All these data together demonstrated that PXN knockdown attenuated the roles of PXN-AS1-L in promoting cell proliferation, inhibiting cell apoptosis, and promoting cell migration of NSCLC cells. These data also suggested that the oncogenic roles of PXN-AS1-L in NSCLC were at least partially dependent on the positive regulation of PXN.