Long noncoding RNA DLX6-AS1 promotes neuroblastoma progression by regulating miR-107/BDNF pathway

Our collected samples and clinical data were obtained with the informed consent of all legal guardians of the patients. Approval to conduct this study was obtained from the Ethics Committee of the Affiliated Hospital of Qingdao University. Human normal dorsal root ganglia were collected from interrupted pregnancies. Fresh tumor specimens of a total of 36 NB patients were collected at surgery and stored at − 80 °C until use. Total RNAs of normal human dorsal ganglia pooling from 18 male/female Caucasians were obtained from Clontech (Mountain View, CA, USA).

The Gene Expression Omnibus (GEO) database (https://​www.​ncbi.​nlm.​nih.​gov/​geo/​) was used to retrieve the relevant datasets. The differentially expressed lncRNAs between different groups were screened by unpaired Student’s t test and correction multiple testing method. Log-rank test and correction multiple testing analyses were applied to evaluate the survival significance of each lncRNA in NB patients.

Human NB cell lines NB-1643, NB-1691, SK-N-AS, IMR-32, SH-SY5Y and SK-N-SH were obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in RPMI1640 medium (Life Technologies, USA) containing 10% fetal bovine serum (Gibco Termo Fisher Scientifc, USA), penicillin (100 U/mL), and streptomycin (100 mg/mL) in a humidified incubator at 37 °C with 5% CO₂.

Lentivirus expressing shDLX6-AS1 or shRNA control were designed and packaged by Genechem (Shanghai, China). Stable cell lines were established by infecting lentivirus into SK-N-SH cells and selected by puromycin (Sigma, St. Louis, USA). The small interfering RNAs (siRNAs) for DLX6-AS1 (siDLX6-AS) and the negative control siRNA, miR-107 mimics, miR-107 inhibitors and the negative control miRNAs were purchased from RiboBio (Guangzhou, China). Human BDNF overexpression plasmid (pcDNA-BDNF) and blank plasmid (pcDNA3.1) were obtained from GenePharma (Shanghai, China). The transiently transfection for Small interfering RNA or pcDNA plasmid was conducted using Lipofectamine 2000 Transfection Reagent (15596‐026; Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and the transfection efficiency was confirmed using quantitative real-time PCR (qRT-PCR). Small interfering RNAs contain si-DLX6-AS1, 5′-AAUAAAGAACACUUACACUACUG-3′; miR-107 mimics, 5′-AGCAGCAUUGUACAGGGCUAUCAAUAGCCCUGUACAAUGCUGCUUU-3′; miR-107 inhibitors,5′-UGAUAGCCCUGUACAAUGCUGCU-3′. Each experiment was performed in triplicate.

Total RNA isolation from NB cells and frozen tissue was performed using TRIzol reagent (Invitrogen). Then, cDNA was constructed from total RNA using the PrimeScript first Strand cDNA Synthesis Kit (6110A; TaKaRa, Dalian, China). Real-time PCR was carried out using One Step SYBR RT‐PCR Kit (RR086A; TaKaRa) and measured by ABI 7900 system. The following primers were used to detect the expression of DLX6-AS, miR-107, BDNF, and internal control U6 and GAPDH: DLX6-AS1, forward: 5′-CCAAATGCTACCATCCAGCC-3′, reverse: 5′-TCTGGCTTCCCTTAACCAAAA-3′; miR-107, RT, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGATAG-3′, forward: 5′- AGCAGCATTGTACAGGG-3′, reverse: 5′- GTGCAGGGTCCGAGGT-3′; BDNF, forward: 5′-TCCCTGGCTGACACTTTT-3′, reverse: 5′-ATTGGGTAGTTCGGCATT-3′; U6, RT, 5′-AACGCTTCACGAATTTGCGT-3′, forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH, forward, 5′-CGACACTTTATCATGGCTA-3′, reverse, 5′-TTGTTGCCGATCACTGAAT-3′. The transcript levels were analyzed by 2^−∆∆Ct method. Each experiment was performed in triplicate.

Cell proliferation status was determined with the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) assay and colony formation assay. For MTT assay, the transfected cells were seeded in 96-well plates at the density of 1 × 10³ per well and then cultured for 1, 2, 3, 4 and 5 days. Then, 20 μL of MTT (5 mg/mL) (Roche, Basel, Switzerland) solution was added and incubated at 37 °C for 2 h. The optical density (OD) values were read at 490 nm by a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). Percent viability was defined as the relative absorbance of intervened cells versus control cells. For colony formation assay, the transfected cells were seeded in 6-well plates at the density of 500 per well for 2 weeks. After 14 days, colonies were fixed with 4% paraformaldehyde for 5 min and stained with 0.1% crystal violet for 10 min. Each experiment was performed in triplicate.

Transfected cells were seeded in six‐well plates, and grown to 100% confluency. The confluent monolayer of cells was scratched with a plastic apparatus to create a clear cell‐free zone. Subsequently, the cells were incubated in serum-free medium and cultured for 24 h. Wound closure was measured using Nikon NIS‐Element Basic Research v3.2 software. The migration assays were performed independently in triplicate.

The invasion assay was performed using Transwell chambers with Matrigel (8 µm pore size, BD Biosciences, Franklin Lakes, NJ, CA, USA). At 48 h after transfection, 1 × 10⁵ NB cells were resuspended in 200 μL serum-free medium and placed on upper chambers and 600 μL medium containing 10% FBS was added into lower chambers. After 24 h of incubation, cells were fixed with polyoxymethylene. Finally, the invaded cells were counted from five randomly fields under a microscope. Each experiment was performed in triplicate.

Apoptosis ratios of cells were determined by Annexin V/FITC and PI staining flow cytometry. Briefly, the transfected NB cells were harvested, digested and suspended in 100 μL binding buffer. Then the cells were stained with 10 μL Annexin V/FITC (BD Pharmingen, San Diego, CA) for 10 min and 5 μL PI (20 mg/mL) for 5 min on ice in the dark at room temperature. The apoptotic cells were analyzed by a flow cytometer (BD Biosciences, San Jose, CA, USA) at a wavelength of 488 nm.

Proteins were extracted from tissues using RIPA lysis buffer (Roche, Diagnostics, Mannheim, Germany). Then, the lysate was loaded into SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). After blocking with 5% milk at room temperature, the membranes were incubated with primary antibodies against BDNF (ab220679, 1:1000; Abcam Inc., Cambridge, UK), GAP43 (ab11136, 1:1000; Abcam Inc.), NF-200 (ab8135, 1:1000; Abcam Inc.) or GAPDH (ab9485, 1:3000; Abcam Inc.) at 4 °C overnight. Subsequently, the membranes were incubated in HRP‐conjugated secondary antibody, goat antirabbit to IgG (ab205718, 1: 20,000; Abcam Inc.) for 1 h at room temperature. The expression was tested with enhanced chemiluminescence (ECL, Millipore, Bredford, USA) and ImageJ (NIH, USA) was used for image densitometric analysis and GAPDH was used as the internal control. Each experiment was performed in triplicate.

Fragments of DLX6-AS1 and BDNF 3′-UTR containing miR-107 binding sites (WT) were amplified by RT-PCR and cloned into the luciferase report vector pGL3-basic (Promega, Madison, WI., USA). Point mutations were generated in the miR-107 binding site of the DLX6-AS1 fragment and BDNF 3′-UTR using the Site-Directed Mutagenesis kit (Invitrogen, Carlsbad, CA, USA). HEK293T cells were co-transfected with luciferase plasmids and miR-107 mimics, or mimics NC by using Lipofectamine 2000 reagent according to the manufacturer’s instruction (Invitrogen). The luciferase activity of Firefly luciferase compared to Renilla luciferase was measured 24 h after transfection according to the manufacturer’s protocol (Promega). Each experiment was performed in triplicate.

All animal experiments were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and approved by the Animal Care Committee of the Affiliated Hospital of Qingdao University. Male BALB/c athymic nude mice at 4–6 weeks were purchased from Shanghai SLAC Laboratory Animal Co. All mice were maintained in a barrier facility at Animal Center of Qingdao University. For tumor formation assay, 1 × 10⁶ SK-N-SH cells stably expressing shDLX6-AS1 or shNC were subcutaneously injected into the right flank of mice (n = 5 per group). Tumor size was measured every 4 days using (length × width²) × 0.5. Tumor weight was measured after sacrifice of mice. For survival analysis, mice were subcutaneously inoculated with 5 × 10⁶ stable transfected SK-N-SH cells (n = 12 per group), and survival time of the mice were monitored at the end of observation.

All Data were shown as mean ± standard deviation (SD). The cutoff values were determined by average gene expression levels. The data analysis was performed by using GraphPad Prism (Version 7.0, GraphPad, San Diego, USA). Correlations between expression and clinicopathological characteristics were analyzed by Chi-square test. Student’s t test or one-way ANOVA were used for intra-group comparison, as appropriate. Kaplan–Meier’s method and the log-rank test were performed for survival rate p value < 0.05 was considered statistically significant.

Initially, microarray dataset (GSE16476) [12] of 88 NB patients was acquired from GEO. We identified 93, 82, 271, and 167 differentially expressed (fold change > 1.3, p < 0.05) lncRNAs in NB tissues with varied status of death, clinical progression, and international neuroblastoma staging system (INSS) stages (stage 1 versus 4 and stage 4S versus 4), respectively (Fig. 1a). To further search for the differentially expressed genes in NB, 9 lncRNAs were found to be consistently associated with the above clinical parameters via over-lapping analysis of these lncRNAs (p < 0.001) (Fig. 1a). Log-rank test analysis revealed top four lncRNA predicting prognosis of NB patients (Fig. 1a). Among them, the functions of lncRNAs pancEts-1 [5] and MYCNOS [13] in NB have been reported, while the roles of DLX6-AS1 in the tumorigenesis and aggressiveness of NB still remain elusive. The expression of DLX6-AS1 in NB tissues was further divided into low expression group and high expression group based on the median values. Kaplan–Meier curves of these 88 NB cases (GSE16476) showed significant difference in overall survival (p = 5 × 10⁻³) between the high and low DLX6-AS1 expression groups (Fig. 1b), which was accordant with our collected samples with 36 NB patients (Fig. 1c). We validated that the expression of DLX6-AS1 was significantly increased in an independent cohort of 36 primary NB tumors compared to those in normal dorsal ganglia (Fig. 1d). High expression of DLX6-AS1 was positively correlated with poor differentiation or advanced TNM stage (Table 1). We then performed qRT-PCR to analyze DLX6-AS1 expression in NB cell lines. As compared with normal dorsal ganglia, higher levels of DLX6-AS1 expression was seen in all NB cell lines (Fig. 1e). To explore the roles of DLX6-AS1 in NB progression, NB cell lines representing higher expression levels were applied. These observations suggested that DLX6-AS1 might be involved in the regulation of NB development.

As we revealed that DLX6-AS1 was significantly upregulated in NB, we then sought the mechanistic regulation of DLX6-AS1 in human NB development. To do so, we transfected SK-N-SH and SH-SY5Y cells with DLX6-AS1 siRNA (siDLX6-AS1) or scrambled siRNA (siNC). qPCR confirmed that, endogenous DLX6-AS1 genes were markedly downregulated in SK-N-SH and SH-SY5Y cells transfected with siDLX6-AS1 than in those transfected with siNC (Fig. 2a). For the proliferation capacity, MTT assay and colony formation assay together demonstrated that DLX6-AS1 knockdown inhibited the proliferation vitality of NB cells (Fig. 2b, c). Wound‐healing assays demonstrated that knockdown of DLX6-AS1 suppressed the migration ability of SK-N-SH and SH-SY5Y cells (Fig. 2d). Depletion of DLX6-AS1 led to decrease in invasion ability of SK-N-SH and SH-SY5Y cells in vitro (Fig. 2e). Furthermore, Annexin V/FITC and PI staining flow cytometry assay indicated that DLX6-AS1 knockdown induced the apoptosis of NB cells (Fig. 2f). Moreover, increased expression of neuronal differentiation markers growth associated protein 43(GAP43)and neurofilament heavy polypeptide (NF-200) were observed in SK-N-SH and SH-SY5Y cells following DLX6-AS1 suppression (Fig. 2g). Together, these findings demonstrated that knockdown of DLX6-AS1 suppressed the progression of NB.

We identified that miR-107 as a potential target of DLX6-AS1 with Starbase (http://​starbase.​sysu.​edu.​cn). There is a putative binding sites of DLX6-AS1 and miR-107 (Fig. 3a). Luciferase reporter assays showed that miR-107 mimics significantly repressed the luciferase activity of which transfected with the reporter plasmid, which containing wild type DLX6-AS1 in the downstream of luciferase gene, but had no effect on the mutant one in HEK293T cells (Fig. 3b). Meanwhile, the miR-107 expression level in NB tissues and NB cells was lower than those in normal dorsal ganglia (Fig. 3c, d). Spearman’s correlation analysis revealed that miR‐107 expression was negatively correlated with DLX6-AS1 expression (Fig. 3e). qRT-PCR further validated that the silencing of DLX6-AS1 by siDLX6-AS1 increased the expression level of miR-107 in SK-N-SH and SH-SY5Y cell lines (Fig. 3f). The efficacy of miR-107 inhibitor or NC was also confirmed by qRT-PCR in these 2 NB cell lines. We observed miR-107 inhibitor significantly suppressed the expression of miR-107 when compared with cells transfected with NC (Fig. 3f).

Furthermore, to explore whether DLX6-AS1 promoted cell progression through miR-107, SK-N-SH and SH-SY5Y cells were treated with siDLX6-AS1, together with miR-107 inhibitor or NC. MTT assay showed that miR-107 inhibitor reversed cell viability as well as colony formation ability caused by DLX6-AS1 knockdown (Fig. 3g, h). In addition, cell migration and invasion assay showed that miR-107 inhibitor abrogated the inhibitory effect of DLX6-AS1 knockdown on cell migration and invasion in SK-N-SH and SH-SY5Y cells (Fig. 3i, j). Besides, knockdown of miR-107 abolished DLX6-AS1 depletion-induced apoptosis in NB cells (Fig. 3k). Taken together, these results supported that DLX6-AS1 promotes NB progression by targeting miR‐107 in NB.

Bioinformatic analysis indicated that BDNF contains a miR-107 binding site in its 3′UTR (Fig. 4a). MiR-107 significantly reduced the luciferase activity in HEK293T cells transfected with the wild-type BDNF plasmid, but it failed to suppress luciferase activity in cells transfected with the mutant plasmid (Fig. 4b). The mRNA expression level of BDNF was upregulated in NB cases (GSE16476) with the status of death and in our collected 36 NB patient tissues compared with normal dorsal ganglia (Additional file 1: Figure S1a, b). As compared with normal dorsal ganglia, higher protein level of BDNF was also seen in all NB cell lines (Additional file 1: Figure S1c). The Spearman’s correlation analysis suggested that BDNF expression was negatively correlated with miR‐107 level but positively correlated with DLX6-AS1 expression in NB tissues (Fig. 4c, d). The mRNA and protein levels of BDNF was markedly reduced in SK-N-SH and SH-SY5Y cells transfected with the miR-107 mimics or siDLX6-AS1, while increased by transfected with miR-107 inhibitor (Fig. 4e and Additional file 1: Figure S1d). Then the effect of BDNF over-expression plasmid on SK-N-SH and SH-SY5Y cells was confirmed by western blot (Fig. 4f and Additional file 1: Figure S1e). Interestingly, restoration of BDNF rescued the inhibitory effect on cell proliferation, migration, and invasion in the SK-N-SH and SH-SY5Y cells induced by knockdown of DLX6-AS1 (Fig. 4g–j). Besides, overexpression of BDNF prominently abated DLX6-AS1 depletion-induced apoptosis in NB cells (Additional file 1: Figure S1f). These data illustrated that DLX6-AS1 functions in NB progression through modulating the miR‐107/BDNF pathway.

To investigate whether knockdown of DLX6-AS1 could inhibit tumor growth in vivo, SK-N-SH cells stably infected with sh‐DLX6-AS1 or sh‐NC were injected subcutaneously into nude mice. qPCR confirmed that, endogenous DLX6-AS1 genes were markedly downregulated in SK-N-SH cells transfected with shDLX6-AS1 than in those transfected with shNC (Fig. 5a). Tumor volumes were measured every 4 days until the mice were killed at 28 days. Compared with sh‐NC cell‐derived xenograft tumors, sh‐DLX6-AS1 cell‐derived xenograft tumors grew more slowly (Fig. 5b). Moreover, the mean weight of sh‐DLX6-AS1 cell‐derived xenograft tumors were also significantly decreased compared with sh‐NC cell‐derived xenograft tumors (Fig. 5c). The qRT‐PCR assay revealed that DLX6-AS1 expression was downregulated, while miR‐107 expression was upregulated in sh‐DLX6-AS1 cell‐derived xenograft tumors compared with sh‐NC cell‐derived xenograft tumors (Fig. 5d, e). In addition, the protein level of BDNF was decreased while GAP43 and NF-200 was upregulated in shDLX6-AS1 group (Fig. 5f). Moreover, mice injected with sh‐DLX6-AS1 cells had a longer survival time than their controls (Fig. 5f).