Long noncoding RNA VPS9D1-AS1 augments the malignant phenotype of non-small cell lung cancer by sponging microRNA-532-3p and thereby enhancing HMGA2 expression

High level of VPS9D1-AS1 expression in NSCLC

Expression profiles of VPS9D1-AS1 in 51 pairs of NSCLC samples and corresponding normal lung tissues were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). VPS9D1-AS1 expression was higher in NSCLC tissue samples than in normal lung tissues (Figure 1A, P < 0.05). By using the median level of VPS9D1-AS1 expression in the NSCLC tissue samples as a cutoff, all samples from the 51 NSCLC patients were classified into either VPS9D1-AS1 high-expression or VPS9D1-AS1 low-expression groups. The analysis of the correlation between VPS9D1-AS1 expression level and clinicopathological characteristics revealed that increased VPS9D1-AS1 expression correlated significantly with tumor size (P = 0.025), TNM stage (P = 0.002), and lymph node metastasis (P = 0.012; Table 1). In particular, patients with NSCLC in the VPS9D1-AS1 high-expression group showed shorter overall survival than patients in the VPS9D1-AS1 low-expression group (Figure 1B, P = 0.030). Furthermore, the expression of VPS9D1-AS1 was measured using RT-qPCR in five NSCLC cell lines (H522, H460, H1299, A549, and SK-MES-1). The normal, non-tumorigenic, bronchial epithelium cell line BEAS-2B was chosen as the control. VPS9D1-AS1 expression levels were higher in all tested NSCLC cell lines than in BEAS-2B cells (Figure 1C, P < 0.05). These data indicated that VPS9D1-AS1 is upregulated in NSCLC and that its expression level may correlate with tumor progression.

Figure 1. High expression of VPS9D1-AS1 in NSCLC indicating poor prognosis in NSCLC patients. (A) RT-qPCR analysis of VPS9D1-AS1 expression in 51 pairs of NSCLC samples and corresponding normal lung tissues. *P < 0.05 vs. normal lung tissues. (B) Relationship between VPS9D1-AS1 expression and overall survival of patients with NSCLC analyzed by the Kaplan–Meier method and log-rank test. P = 0.030. (C) Determination of VPS9D1-AS1 expression by RT-qPCR in total RNA from five NSCLC cell lines (H522, H460, H1299, A549, and SK-MES-1) and one normal nontumorigenic bronchial epithelium cell line (BEAS-2B). *P < 0.05 vs. BEAS-2B cells.

VPS9D1-AS1 knockdown inhibits the proliferation, migration, and invasiveness of NSCLC cells and promotes their apoptosis

The observed relationship between VPS9D1-AS1 expression level and malignancy prompted us to investigate the biological effects of VPS9D1-AS1 on the malignant phenotype of NSCLC H460 and A549 cells, which showed the highest expression of VPS9D1-AS1 among the five NSCLC cell lines. H460 and A549 cells were transfected with the small interfering RNA (siRNA) si-VPS9D1-AS1 targeting VPS9D1-AS1 or negative control siRNA (si-NC). Successful knock-down of VPS9D1-AS1 after transfecting H460 and A549 cells with si-VPS9D1-AS1 was confirmed by RT-qPCR (Figure 2A, P < 0.05).

Figure 2. VPS9D1-AS1 knockdown inhibits proliferation, colony-forming capacity, migration, and invasiveness of H460 and A549 cells but promotes their apoptosis. (A) Evaluation of the transfection efficiency of H460 and A549 cells with si-VPS9D1-AS1 or si-NC at ~48 h post-transfection using RT-qPCR. *P < 0.05 vs. the si-NC group. (B–D) Differences in the proliferation, colony-forming capacity, and apoptosis rate of H460 and A549 cells transfected with si-VPS9D1-AS1 or si-NC determined by the CCK-8 assay, the colony formation assay, and flow cytometry, respectively. *P < 0.05 vs. the si-NC group. (E, F) Effects of treatment with si-VPS9D1-AS1 or si-NC on the migration and invasiveness of H460 and A549 cells assessed by the Transwell migration and invasiveness assays, respectively. *P < 0.05 vs. the si-NC group.

Cell Counting Kit-8 (CCK-8) and colony formation assays showed that the proliferative (Figure 2B, P < 0.05) and colony-forming (Figure 2C, P < 0.05) capabilities of both H460 and A549 cells were significantly attenuated by VPS9D1-AS1 knockdown. Flow cytometry revealed that H460 and A549 cells transfected with si-VPS9D1-AS1 manifested a higher rate of apoptosis than those transfected with si-NC (Figure 2D, P < 0.05). These data suggested that the decrease in NSCLC cell proliferation induced by VPS9D1-AS1 knockdown was due to apoptosis induction. Transwell migration and invasiveness assays showed that transfection with si-VPS9D1-AS1 significantly decreased the migratory (Figure 2E, P < 0.05) and invasive (Figure 2F, P < 0.05) capabilities of H460 and A549 cells. Overall, these results suggested that VPS9D1-AS1 expression may exert oncogenic effects in NSCLC cells in vitro.

VPS9D1-AS1 acts as a molecular sponge of miR-532-3p in NSCLC cells

Numerous cytoplasmic lncRNAs can function as competing endogenous RNAs (ceRNAs) by competitively binding to specific miRNAs [27]. To elucidate the molecular mechanisms underlying the oncogenic effects of VPS9D1-AS1 in NSCLC cells, we first investigated the subcellular localization of VPS9D1-AS1 in NSCLC cells. VPS9D1-AS1 was mainly localized in the cytoplasm of H460 and A549 cells (Figure 3A, P < 0.05), suggesting that this lncRNA may function as a ceRNA of some miRNAs in NSCLC. To test this hypothesis, potential interactions between VPS9D1-AS1 and miRNAs were explored using bioinformatics analysis. We focused on the changes in the expression of miR-532-3p as our analysis predicted that this miRNA (Figure 3B) has a high probability of binding to VPS9D1-AS1; in addition, miR-532-3p has been reported to play a crucial role in NSCLC progression [28].

Figure 3. VPS9D1-AS1 functions as a sponge for miR-532-3p in NSCLC cells. (A) Relative expression of VPS9D1-AS1 in nuclear and cytoplasmic fractions of H460 and A549 cells determined by subcellular fractionation and RT-qPCR analysis. (B) Predicted wild-type and mutant complementary sites of miR-532-3p in a VPS9D1-AS1 fragment. (C) RT-qPCR analysis of miR-532-3p expression in 51 pairs of NSCLC samples and corresponding normal lung tissues. *P < 0.05 vs. normal lung tissues. (D) Negative correlation between VPS9D1-AS1 and miR-532-3p levels in the 51 NSCLC tissue samples revealed by the Spearman’s rank correlation analysis. R² = 0.3370, P < 0.0001. (E) RT-qPCR analysis of miR-532-3p expression levels in H460 and A549 cells with VPS9D1-AS1 knockdown. *P < 0.05 vs. the si-NC group. (F) RT-qPCR analysis of miR-532-3p expression levels in H460 and A549 cells transfected with agomir-532-3p or agomir-NC. *P < 0.05 vs. the agomir-NC group. (G) Interaction between miR-532-3p and VPS9D1-AS1 in NSCLC cells revealed by the luciferase reporter assay. H460 and A549 cells were co-transfected with a luciferase reporter plasmid carrying wild-type (wt) or mutant (mt) VPS9D1-AS1 and agomir-532-3p or agomir-NC. *P < 0.05 vs. the agomir-NC group. (H) miR-532-3p and VPS9D1-AS1 enrichment in AGO2 immunoprecipitates from H460 and A549 cell lysates determined by the RIP assay. IgG served as negative control. *P < 0.05 vs. the IgG group.

To examine the correlation between VPS9D1-AS1 and miR-532-3p expression levels in NSCLC, miR-532-3p expression levels were quantified by RT-qPCR in 51 pairs of NSCLC samples and corresponding normal lung tissues. We found that miR-532-3p expression was lower in NSCLC tissue samples than in normal lung tissue samples (Figure 3C, P < 0.05). Of note, an inverse correlation between VPS9D1-AS1 and miR-532-3p expression levels was observed in NSCLC tissue samples, as evidenced by Spearman’s rank correlation analysis (Figure 3D; R² = 0.3370, P < 0.0001). We also examined whether miR-532-3p expression was affected by VPS9D1-AS1 levels in NSCLC cells and established that VPS9D1-AS1 knockdown significantly increased miR-532-3p expression in H460 and A549 cells (Figure 3E, P < 0.05).

Direct binding of miR-532-3p to VPS9D1-AS1 in NSCLC cells was verified by the luciferase reporter assay. Luciferase reporter plasmids carrying the wild-type (wt) or mutant (mut) binding site for miR-532-3p were constructed (VPS9D1-AS1-wt and VPS9D1-AS1-mut, respectively) and separately co-transfected with agomir-532-3p, an miR-532-3p mimic increasing its expression, or agomir-NC into H460 and A549 cells. Agomir-532-3p transfection-mediated upregulation of miR-532-3p (Figure 3F, P < 0.05) dramatically decreased VPS9D1-AS1-wt luciferase activity (P < 0.05) in H460 and A549 cells, whereas no such change was detected in cells transfected with VPS9D1-AS1-mut (Figure 3G). An RNA immunoprecipitation (RIP) assay confirmed that VPS9D1-AS1 and miR-532-3p were substantially enriched in the immunoprecipitated AGO2 protein complex, suggesting that AGO2 bound to VPS9D1-AS1 and miR-532-3p directly in NSCLC cells (Figure 3H, P < 0.05). Collectively, these data provided sufficient evidence that VPS9D1-AS1 directly interacts with miR-532-3p in NSCLC cells.

HMGA2 is a direct target gene of miR-532-3p in NSCLC cells

To explore the biological effects of miR-532-3p on NSCLC tumorigenesis, agomir-532-3p or agomir-NC was introduced into H460 and A549 cells. Next, the influence of miR-532-3p upregulation on cell proliferation was examined using the CCK-8 and colony formation assays. Ectopic miR-532-3p expression strongly decreased the proliferative (Figure 4A, P < 0.05) and colony-forming (Figure 4B, P < 0.05) abilities of H460 and A549 cells. Furthermore, transfection with agomir-532-3p markedly increased the apoptotic rate of H460 and A549 cells (Figure 4C, P < 0.05). In addition, the results of the Transwell migration and invasiveness assays indicated that overexpression of miR-532-3p significantly attenuated the migration (Figure 4D, P < 0.05) and invasiveness (Figure 4E, P < 0.05) of H460 and A549 cells.

Figure 4. miR-532-3p directly targets HMGA2 mRNA to inhibit the malignant phenotype of NSCLC cells. (A–C) Cell proliferation, colony formation, and apoptosis parameters determined by the CCK-8 assay, the colony formation assay, and flow cytometry in H460 and A549 cells transfected with agomir-532-3p or agomir-NC. *P < 0.05 vs. the agomir-NC group. (D, E) Migratory and invasive capabilities of H460 and A549 cells after transfection with agomir-532-3p or agomir-NC analyzed by the Transwell migration and invasiveness assays. *P < 0.05 vs. the agomir-NC group. (F) Wild-type and mutant miR-532-3p-binding site in the 3′-UTR of HMGA2 mRNA. (G) Relative firefly luciferase activity determined in H460 and A549 cells at 48 h after co-transfection with agomir-532-3p or agomir-NC and HMGA2-wt or HMGA2-mut. *P < 0.05 vs. the agomir-NC group. (H) RT-qPCR analysis of HMGA2 mRNA expression level in 51 pairs of NSCLC samples and corresponding normal lung tissues. *P < 0.05 vs. normal lung tissue. (I) Negative correlation between HMGA2 mRNA and miR-532-3p levels in NSCLC tissue samples revealed by the Spearman’s correlation analysis (R² = 0.3659, P < 0.0001). (J, K) HMGA2 gene and protein expression levels in H460 and A549 cells determined by RT-qPCR and western blot, respectively, following transfection with agomir-532-3p or agomir-NC. *P < 0.05 vs. the agomir-NC group.

To uncover the molecular events underpinning miR-532-3p-mediated inhibition of NSCLC progression, the putative targets of this miRNA were searched in two bioinformatics databases. The bioinformatics prediction indicated that a site in the 3′-UTR of HMGA2 mRNA matched the “seed sequence” of miR-532-3p (Figure 4F). HMGA2 was selected for further verification because that this gene was reported to be overexpressed in NSCLC and exert crucial roles during NSCLC initation and progression [29–32]. A luciferase reporter assay was then performed to demonstrate the possibility of miR-532-3p binding to the 3′-UTR of HMGA2 mRNA in NSCLC cells. The luciferase activity of the HMGA2-wt reporter plasmid, which contained a wild-type miR-532-3p-binding site in the HMGA2 3′-UTR driving the expression of the luciferase gene, was notably weakened upon co-transfection of H460 and A549 cells with agomir-532-3p (P < 0.05). In contrast, the luciferase activity of HMGA2-mut was not altered by miR-532-3p upregulation (Figure 4G). In addition, RT-qPCR analysis confirmed that the mRNA expression of HMGA2 was higher in the NSCLC tissue samples (Figure 4H, P < 0.05), revealing its negative correlation with the miR-532-3p expression level (Figure 4I; R² = 0.3659, P < 0.0001). Moreover, transfection with agomir-532-3p significantly downregulated the mRNA (Figure 4J, P < 0.05) and protein (Figure 4K, P < 0.05) expression of HMGA2 in H460 and A549 cells. Collectively, these data implied that miR-532-3p suppresses NSCLC progression and that HMGA2 is a direct target gene of miR-532-3p.

VPS9D1-AS1 positively regulates HMGA2 expression in NSCLC via sponging of miR-532-3p

As we established that HMGA2 was a direct target gene of miR-532-3p in NSCLC cells, we next explored whether VPS9D1-AS1 could modulate HMGA2 expression in NSCLC cells. mRNA (Figure 5A, P < 0.05) and protein (Figure 5B, P < 0.05) levels of HMGA2 were low in H460 and A549 cells deficient in VPS9D1-AS1, as revealed by RT-qPCR and western blot, respectively. To investigate whether miR-532-3p sponging mediated the positive effect of VPS9D1-AS1 on HMGA2 expression, rescue experiments were conducted in H460 and A549 cells by co-transfecting them with si-VPS9D1-AS1 and antagomir-532-3p (inhibitor of miR-532-3p) or antagomir-NC. Transfection with antagomir-532-3p potently silenced miR-532-3p expression in H460 and A549 cells, as evidenced by RT-qPCR data (Figure 5C, P < 0.05). VPS9D1-AS1 knockdown significantly increased miR-532-3p expression and decreased HMGA2 mRNA and protein levels in H460 and A549 cells. In contrast, co-transfection with antagomir-532-3p abrogated the influence of VPS9D1-AS1 knockdown on miR-532-3p (Figure 5D, P < 0.05) as well as on HMGA2 mRNA (Figure 5E, P < 0.05) and protein (Figure 5F, P < 0.05) expression levels. These findings indicated that VPS9D1-AS1 sponges miR-532-3p in NSCLC cells, thereby enhancing HMGA2 expression.

Figure 5. VPS9D1-AS1 acts as a ceRNA of miR-532-3p and thereby upregulates HMGA2 expression. (A, B) HMGA2 mRNA and protein levels in H460 and A549 cells determined by RT-qPCR and western blot, respectively, after transfection with si-VPS9D1-AS1 or si-NC. *P < 0.05 vs. the si-NC group. (C) Efficiency of transfection of H460 and A549 cells with antagomir-532-3p determined by RT-qPCR. *P < 0.05 vs. the antagomir-NC group. (D–F) Expression levels of miR-532-3p, HMGA2 mRNA, and HMGA2 protein in H460 and A549 cells after co-transfection with si-VPS9D1-AS1 and antagomir-532-3p or antagomir-NC determined using RT-qPCR and western blot, respectively. *P < 0.05 vs. the si-NC group. ^#P < 0.05 vs. the si-VPS9D1-AS1 + antagomir-NC group.

VPS9D1-AS1 knockdown inhibits the malignant characteristics of NSCLC cells by reducing the miR-532-3p-HMGA2 axis output

We carried out rescue experiments to further confirm that VPS9D1-AS1 exerts its oncogenic effects in NSCLC cells by regulating the miR-532-3p-HMGA2 axis. For this purpose, si-VPS9D1-AS1 was co-transfected into H460 and A549 cells with antagomir-532-3p or antagomir-NC. As revealed by the CCK-8 assay, the colony formation assay, and flow cytometry, co-transfection with antagomir-532-3p reversed the modulatory influence of VPS9D1-AS1 knockdown on the proliferation (Figure 6A, P < 0.05), colony-forming capacity (Figure 6B, P < 0.05), and apoptosis (Figure 6C, P < 0.05) of H460 and A549 cells, respectively. In addition, the suppression of H460 and A549 cell migration (Figure 6D, P < 0.05) and invasiveness (Figure 6E, P < 0.05) induced by VPS9D1-AS1 knockdown was strongly attenuated by miR-532-3p inhibition.

Figure 6. Inhibition of miR-532-3p strongly attenuates the influence of VPS9D1-AS1 knockdown on the malignant characteristics of H460 and A549 cells. (A–E) Proliferation, colony-forming capacity, apoptosis, migration, and invasiveness parameters of H460 and A549 cells measured by the CCK-8 assay, the colony formation assay, flow cytometry, Transwell migration and invasiveness assays, respectively. *P < 0.05 vs. the si-NC group. ^#P < 0.05 vs. the si-VPS9D1-AS1 + antagomir-NC group.

Rescue experiments were also performed in H460 and A549 cells after co-transfecting them with si-VPS9D1-AS1 and the HMGA2-overexpressing plasmid pc-HMGA2 or the corresponding empty pcDNA3.1 vector. Western blotting indicated that the downregulation of HMGA2 protein expression in VPS9D1-AS1-depleted H460 and A549 cells was almost completely reversed by the reintroduction of HMGA2 (Figure 7A, P < 0.05). The CCK-8 and colony formation assays indicated that the restoration of HMGA2 expression abrogated the inhibitory effects of VPS9D1-AS1 knockdown on H460 and A549 cell proliferation (Figure 7B, P < 0.05) and colony formation (Figure 7C, P < 0.05). Similarly, the potentiation of H460 and A549 cell apoptosis by VPS9D1-AS1 knockdown was prevented by co-transfection with pc-HMGA2 (Figure 7D, P < 0.05). Transwell migration and invasiveness assays indicated that the restoration of HMGA2 expression reversed the si-VPS9D1-AS1-induced inhibition of migration (Figure 7E, P < 0.05) and invasiveness (Figure 7F, P < 0.05) of H460 and A549 cells. In summary, VPS9D1-AS1 knockdown attenuated HMGA2 expression by reducing the sponging of miR-532-3p and thereby decreased NSCLC malignancy.

Figure 7. Recovery of HMGA2 expression reverses inhibitory actions of si-VPS9D1-AS1 on NSCLC cells. (A) Quantification of HMGA2 protein expression in H460 and A549 cells transfected with plasmid pc-HMGA2 or the empty pcDNA3.1 vector along with si-VPS9D1-AS1 by western blot. *P < 0.05 vs. the si-NC group. ^#P < 0.05 vs. the si-VPS9D1-AS1 + pcDNA3.1 group. (B–F) Cell proliferation, colony formation, apoptosis, migration, and invasiveness parameters of H460 and A549 cells, treated as described above, determined by the CCK-8 assay, the colony formation assay, flow cytometry, and Transwell migration and invasiveness assays, respectively. *P < 0.05 vs. the si-NC group. ^#P < 0.05 vs. the si-VPS9D1-AS1 + pcDNA3.1 group.

VPS9D1-AS1 knockdown slows down tumor growth of NSCLC cells in vivo by increasing miR-532-3p expression and decreasing HMGA2 expression

We finally investigated the impact of VPS9D1-AS1 knockdown on tumor growth in vivo in the xenograft experiment. H460 cells transfected with si-VPS9D1-AS1 or si-NC were subcutaneously injected into nude mice. Tumor xenograft volume was much smaller in the si-VPS9D1-AS1 group than in the si-NC group (Figure 8A, 8B; P < 0.05). In addition, tumor weight was significantly lower in the si-VPS9D1-AS1 group than in the si-NC group (Figure 8C, P < 0.05) at 4 weeks after the injection of cells. VPS9D1-AS1 expression was significantly downregulated in tumor xenografts from the si-VPS9D1-AS1 group, compared with that in xenografts from the si-NC group (Figure 8D, P < 0.05). Meanwhile, RT-qPCR revealed that miR-532-3p expression was higher in tumor xenografts derived from H460 cells transfected with si-VPS9D1-AS1 cells (Figure 8E, P < 0.05). Furthermore, HMGA2 protein expression was significantly lower in the si-VPS9D1-AS1 group relative to that in the si-NC group (Figure 8F, P < 0.05). These results indicated that VPS9D1-AS1 knockdown attenuated tumor growth of NSCLC cells through the downregulation of the miR-532-3p-HMGA2 axis output.

Figure 8. VPS9D1-AS1 knockdown suppresses tumor growth of NSCLC cells in vivo. (A) Nude mice were inoculated with H460 cells transfected with si-VPS9D1-AS1 or si-NC. Tumor volumes were measured 4 weeks after the injection, and tumor growth curves were plotted. *P < 0.05 vs. the si-NC group. (B) Representative images of tumor xenografts in the two groups. (C) Weights of tumor xenografts collected at the end of the experiment. *P < 0.05 vs. the si-NC group. (D, E) RT-qPCR analysis of VPS9D1-AS1 and miR-532-3p expression levels in tumor xenografts from si-VPS9D1-AS1 and si-NC groups. *P < 0.05 vs. the si-NC group. (F) Western blot data illustrating the HMGA2 protein expression level in the tumor xenografts derived from H460 cells transfected with si-VPS9D1-AS1 or si-NC. *P < 0.05 vs. the si-NC group.

Ethics statement

This study was conducted in accordance with the ethical standards recommended by the Declaration of Helsinki and according to national and international guidelines. The study was approved by the institutional review boards of the authors' institutions. In particular, the experimental procedures of this study were approved by the Ethics Committee of the Cancer Hospital of the Guangxi Medical University. Written informed consent was also obtained from all patients before their enrolment in this study. The experimental procedures involving animals were approved by the Animal Ethics Committee of the Cancer Hospital of the Guangxi Medical University, and the assays were carried out in accordance with the Animal Protection Law of the People’s Republic of China-2009 for experimental animals.

Clinical tissue specimens

Paired samples of NSCLC and corresponding normal lung tissues were obtained from 51 patients with NSCLC who underwent surgical resection at the Cancer Hospital of the Guangxi Medical University. None of the patients were treated with chemotherapy, radiotherapy, or other anticancer modalities prior to the surgical intervention. All tissue specimens were immediately frozen in liquid nitrogen and preserved at −80°C until further use.

Cell culture

Normal non-tumorigenic bronchial epithelium cell line BEAS-2B and five human NSCLC cell lines (H522, H460, H1299, A549, and SK-MES-1) were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% (v/v) of a penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was utilized for cell culture. All cells were maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.

Transfection procedures

The siRNA that specifically reduced the expression of VPS9D1-AS1 (si-VPS9D1-AS1) and si-NC were acquired from RiboBio Biotechnology (Guangzhou, China). miR-532-3p agomir (agomir-532-3p), negative control agomir (agomir-NC), antagomir-532-3p, and antagomir-NC were designed and chemically produced by GenePharma (Shanghai, China). The constructed HMGA2 overexpression plasmid pcDNA3.1-HMGA2 (pc-HMGA2) and the empty pcDNA3.1 vector were purchased from IBSbio (Shanghai, China). The cells were seeded into 6-well plates, and all transfection procedures were conducted using the transfection reagent Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Subcellular fractionation and RT-qPCR

The cytoplasmic and nuclear fractions of NSCLC cells were separated using a PARIS kit (Ambion; Thermo Fisher Scientific, Inc.). RNA was isolated from both fractions and analyzed by RT-qPCR.

Total RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., USA) and quantified on a NanoDrop 2000c spectrophotometer (Invitrogen; Thermo Fisher Scientific, Inc.). An All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, Md, USA) was used to determine the miR-532-3p expression, with U6 small nuclear RNA as control. HMGA2 mRNA and VPS9D1-AS1 expression levels were measured using a PrimeScript RT Reagent Kit and SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd., Japan). The GAPDH mRNA level served as the internal reference for HMGA2 mRNA and VPS9D1-AS1 expression levels. The relative gene expression level was calculated by the 2^−ΔΔCq method [50].

CCK-8 assay

Transfected cells were detached using 0.25% trypsin, counted, and seeded into 96-well plates at a density of 2 × 10³ cells/well. The cells were maintained at 37°C and 95% O₂/5% CO₂. The medium in each well was supplemented with 10 μL of the CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) at 0, 24, 48, and 72 h post seeding. Cell proliferation rate was inferred from the optical density values at 450 nm measured on a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Colony formation assay

In total, 2 mL of a cell suspension containing 1,000 transfected cells was seeded into each well of 6-well plates. After 2 weeks of incubation in a humidified atmosphere of 95% air and 5% CO₂ at 37°C, the cells were fixed in 100% methanol, stained with methyl violet (Beyotime Institute of Biotechnology, Inc., Shanghai, China), and extensively washed with phosphate buffered saline (Gibco; Thermo Fisher Scientific, Inc.). The number of colonies (≥50 cells) was determined under an inverted microscope (Olympus, Tokyo, Japan).

Flow cytometry

After 48 h of culture, transfected cells were harvested with 0.25% trypsin without EDTA, washed thrice with phosphate buffered saline, and centrifuged. An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Biolegend, San Diego, CA, USA) was employed to determine cell apoptosis rates. Cells were resuspended in 100 μL of binding buffer and stained with 5 μL of Annexin V-FITC and 5 μL of propidium iodide solution. Following a 15 min incubation in the dark at room temperature, the apoptotic cells were scored using a FACScan flow cytometer (BD Biosciences, CA, USA).

Transwell migration and invasiveness assays

Matrigel (BD Biosciences) diluted with DMEM was utilized to coat the Transwell chambers (BD Biosciences) for the Transwell invasiveness assay but not for the migration assay. Transfected cells were harvested at 48 h post-transfection, washed twice with phosphate-buffered saline, and resuspended in FBS-free DMEM. In total, 50,000 cells were plated into the upper compartments and 500 μL of DMEM supplemented with 20% FBS was added into the lower compartments. Later, the chambers were placed into a 37°C incubator supplied with 95% air/5% CO₂ for 24 h incubation; then, the cells remaining on the inner side of the membranes were wiped off with cotton balls. Migratory and invasive cells were fixed with 100% methanol and stained with 0.1% crystal violet. After extensive washing, migratory and invasive cells were photographed under an inverted microscope. Five fields of view were randomly selected, and the average numbers of migratory and invasive cells were determined.

Tumor xenograft experiment

Four-week-old nude mice were bought from the Guangdong Medical Laboratory Animal Center (Foshan, Guangdong, China) and randomly divided into two groups. One group was subcutaneously injected with si-VPS9D1-AS1-transfected H460 cells, whereas the other group was similarly injected with si-NC-transfected H460 cells. The volume of tumor xenografts was calculated according to the following formula: volume (mm³) = (L × W²)/2; where L and W represent the length (longest dimension) and width (shortest dimension), respectively. At the end of the experiment, all mice were euthanized, and tumor xenografts were collected for further analysis.

RIP assay

The RIP assay was conducted using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Inc., Billerica, MA, USA). In brief, cells were lysed with RIPA lysis buffer and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was incubated with magnetic beads conjugated with anti-Argonaute 2 (AGO2) or anti-IgG antibodies (Millipore Inc.). After overnight incubation at 4°C, the immunoprecipitated RNA was purified using proteinase K digestion and analyzed by RT-qPCR.

Bioinformatics prediction

We searched for miRNA(s) that could be sponged by VPS9D1-AS1 using starBase 3.0 (http://starbase.sysu.edu.cn/). The putative targets of miR-532-3p were predicted using two publicly available databases: miRDB (http://mirdb.org/) and TargetScan (http://www.targetscan.org/vert_60/).

Luciferase reporter assay

The fragments of VPS9D1-AS1 containing wt or mut miR-532-3p binding site were PCR-amplified by GenePharma and inserted into the pmirGLO luciferase reporter vector (Promega Corporation, Madison, WI, USA) to create recombinant reporter plasmids VPS9D1-AS1-wt and VPS9D1-AS1-mut. The construction of luciferase reporter plasmids HMGA2-wt and HMGA2-mut was performed in a similar manner.

H460 and A549 cells were seeded in 24-well plates one night before the transfection. Wt or mut recombinant plasmids and agomir-532-3p or agomir-NC were co-transfected into NSCLC cells using Lipofectamine 2000. At 48 h post-transfection, the cells were collected and subjected to the Dual-Luciferase Reporter Assay (Promega Corporation) to measure the firefly and Renilla luciferase activities. The activity of the firefly luciferase was normalized to that of the Renilla luciferase.

Western blot analysis

Total protein was extracted using RIPA lysis buffer and quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Unoccupied sites on the membranes were blocked with 5% nonfat milk diluted with Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature. After incubation with primary antibodies against HMGA2 (ab207301; dilution 1:1,000) or GAPDH (ab181602; dilution 1: 1,000; both from Abcam, Cambridge, UK) overnight at 4°C, the membranes were extensively washed with TBST and incubated with a goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (ab205718; dilution 1:5,000; Abcam, Cambridge, UK). After three washes, the signals were detected using an Enhanced Chemiluminescence Western Blotting Detection Kit (Applygen Technologies, Inc., Beijing, China).

Statistical analysis

All data were analyzed using SPSS statistical software (version 11.0; SPSS, Inc.) and are presented as the mean ± standard deviation. Pairwise comparisons were performed using Student’s t test, whereas statistical analysis of multiple groups (≥3) was carried out using one-way analysis of variance, followed by the post hoc Tukey’s test, if appropriate. The correlation between VPS9D1-AS1 and miR-532-3p expression levels in NSCLC tissue samples was established using the Spearman’s rank correlation analysis. The association between VPS9D1-AS1 level and the clinicopathological characteristics of patients with NSCLC was examined using the chi-squared test. The overall survival rates of NSCLC patients were analyzed by the Kaplan–Meier method and compared by the log-rank test. All statistical analyses were conducted at a significance level of α = 0.05 (P < 0.05).