Long Noncoding RNA LAMTOR5-AS1 Interference Affects MicroRNA-506-3p/E2F6-Mediated Behavior of Non-Small Cell Lung Cancer Cells.

Long noncoding RNA LAMTOR5 antisense RNA 1 (LAMTOR5-AS1) has been certified as a risk predictor and diagnostic biomarker of prostate cancer. However, the expression and exact roles of LAMTOR5-AS1 in non-small cell lung cancer (NSCLC) remain unclear. Thus, we measured LAMTOR5-AS1 expression in NSCLC and gauged its clinical value. The detailed roles and downstream working mechanism of LAMTOR5-AS1 in NSCLC were comprehensively unraveled. qRT-PCR was applied to measure gene expression. Functionally, utilizing small interfering RNA, LAMTOR5-AS1 was ablated, and the functional alterations were addressed by means of different experiments. The targeting activities between LAMTOR5-AS1 and microRNA-506-3p (miR-506-3p) and between miR-506-3p and E2F transcription factor 6 (E2F6) were confirmed by RNA immunoprecipitation and luciferase reporter assays. LAMTOR5-AS1 overexpression in NSCLC was verified in TCGA datasets and our own cohort and manifested an evident relationship with poor prognosis. Interference with LAMTOR5-AS1 led to repression of the proliferation, cloning, and metastasis abilities of NSCLC cells in vitro. We further confirmed an obvious increase in LAMTOR5-AS1-silenced NSCLC cell apoptosis. Furthermore, the absence of LAMTOR5-AS1 restricted tumor growth in vivo. Mechanistically, LAMTOR5-AS1 sponged miR-506-3p in NSCLC cells. Furthermore, E2F6, a downstream target of miR-506-3p, was under the control of LAMTOR5-AS1, which was realized by decoying miR-506-3p. Rescue experiments showed that miR-506-3p suppression or E2F6 reintroduction was capable of remitting LAMTOR5-AS1 deficiency-triggered anticarcinogenic actions in NSCLC. Our study confirmed the exact roles of LAMTOR5-AS1 for the first time and revealed that LAMTOR5-AS1 knockdown disrupts the malignancy of NSCLC by targeting the miR-506-3p/E2F6 axis. Targeting the LAMTOR5-AS1/miR-506-3p/E2F6 pathway may be instrumental for managing patients with NSCLC.

INTRODUCTION

Lung cancer is the most frequently occurring cancer and the leading cause of cancer-related deaths around the world1. According to global cancer statistics, over 2 million novel lung cancer cases occur globally, resulting in nearly 1.7 million deaths1. Lung cancer has two completely different histological types: non-small cell lung cancer (NSCLC) and small cell lung cancer, with the former accounting for over 85% of all lung cancer cases2. Although great advancements have been realized in modern diagnostic techniques over the past few decades, only approximately 20%–25% of NSCLC cases can be diagnosed at an early stage, at which point implementation of surgical excision is suitable3. However, when thoracalgia, chest discomfort, cough, or weight loss occurs, most patients are likely to present with late stage disease4. The 5-year overall survival rate for patients with early stage disease is approximately 14% to 49% but decreases to 5% for patients with stage IIIB/IV disease5. Even after conventional therapy, approximately 15% of patients with NSCLC experience a second primary cancer6. Furthermore, local or distant metastasis is another major obstacle in improving clinical efficiency and prognosis7. In this regard, determining the detailed molecular events that occur during NSCLC pathogenesis may assist in finding novel therapeutic targets.

Long noncoding RNAs (lncRNAs) are a cluster of noncoding RNA transcripts that are at least 200 nucleotides in length8. Previously, owing to the absence of coding regions, lncRNAs were judged to be nonfunctional or “noise” during transcription events; nevertheless, recent studies have revealed their significant functions in a wide variety of aggressive processes9–11. lncRNAs perform critical regulatory actions in the process of NSCLC oncogenesis and progression12,13. lncRNAs have tumor-inhibiting or tumor-promoting capabilities and are implicated in the control of many aggressive tumor properties14.

MicroRNAs (miRNAs) refer to a group of evolutionarily conserved and noncoding short RNA transcripts consisting of 17–22 nucleotides15. They have been certified as essential epigenetic factors that regulate gene expression via interacting with the 3’-untranslated region of their downstream target genes, ultimately triggering translation suppression or mRNA degradation16. Aberrant miRNA expression is widely observed in NSCLC occurrence and development and contributes to an extensive range of malignant behaviors17. Recently, an increasing number of studies have highlighted that the competing endogenous RNA (ceRNA) regulatory network has a role in NSCLC18–20. According to this theory, lncRNAs act as “miRNA sponges” or ceRNAs and thus control downstream target genes.

LAMTOR5-AS1 has been identified as a risk prediction and diagnostic biomarker of prostate cancer21. Nonetheless, the exact functions of LAMTOR5-AS1 in NSCLC remain poorly understood and demand further investigation. In view of this, in our current study, we aimed to probe the LAMTOR5-AS1 level in NSCLC and assess its clinical significance. Furthermore, we comprehensively unraveled the detailed roles and downstream working mechanism of LAMTOR5-AS1 in NSCLC.

MATERIALS AND METHODS

Clinical Specimens

The present study received approval from the Ethics Committee of The First People’s Hospital of Yancheng and was implemented in accordance with the Declaration of Helsinki. We collected NSCLC tissues and paired adjacent normal tissues from 49 patients in our hospital. All patients offered written informed consent before tissue collection. The exclusion criteria included patients who had received radiochemotherapy or other anticancer treatments, patients with other cancer types, and patients who refused to take part in the research.

Cell Culture

The NSCLC cell lines NCI-H460 (H460) and NCI-H522 [H522; American Type Culture Collection (ATCC), Manassas, VA, USA] were grown in F-12K medium (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA). The NSCLC cell lines A549 (ATCC) and NCI-H1299 (H1299; National Collection of Authenticated Cell cultures, Shanghai, China) were cultured in RPMI-1640 medium. Minimum essential medium was applied for culture of the NSCLC cell line SK-MES-1 (ATCC). BEAS-2B (ATCC), an immortalized human bronchial epithelial mesothelial cell line, was maintained in bronchial epithelial cell growth medium (Lonza, Walkersville, MD, USA). Meanwhile, 10% fetal bovine serum (FBS; Gibco) was used in the cultures of all of the above cell lines, which were cultivated at 37°C in a humidified atmosphere with 5% CO2.

Transfection

Shanghai GenePharma Co. Ltd. (Shanghai, China) chemically synthesized three specific small interfering (si)RNAs targeting LAMTOR5-AS1 (si-LAMTOR5-AS1s) and a scramble control siRNA (si-NC). The si-LAMTOR5-AS1#1 sequence was 5′-TTGTAAAATTGAAATATTTGAAT-3′, the si-LAMTOR5-AS1#2 sequence was 5′-TACCAAAAAATAAAAATATGAAC-3′, the si-LAMTOR5-AS1#3 sequence was 5′-GGCATACTTAGTTTTACATTTTT-3′, and the si-NC sequence was 5′-CACGATAAGACAATGTATTT-3′. Increases and decreases in miR-506-3p were achieved through an miR-506-3p mimic and an miR-506-3p inhibitor, respectively (Shanghai GenePharma Co. Ltd.). An miRNA mimic control (miR-NC) and a negative control (NC) inhibitor served as the controls. GenScript Biotech Corp. (Nanjing, China) prepared full-length E2F6 and then ligated it into a pcDNA™ 3.1 vector, consequently yielding pcDNA3.1-E2F6. When the cell density reached approximately 60%–70% confluence, cell transfection was conducted applying Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

TRIzol (Beyotime, Shanghai, China) was adopted for total RNA abstraction. The concentration and quality of total RNA were tested with a NanoDrop® 2000 spectrophotometer (Thermo Fisher Scientific Inc.). For detection of LAMTOR5-AS1 and E2F6, we reverse transcribed total RNA into first-strand complementary DNA with a PrimeScript™ RT reagent kit (Takara, Dalian, China). Next, PCR amplification was carried out by means of TB Green® Premix Ex Taq™ II (Takara). To determine the miR-506-3p level, we conducted reverse transcription and quantitative PCR utilizing an miRcute miRNA First-Strand cDNA Synthesis Kit and an miRcute miRNA qPCR Detection Kit SYBR Green (Tiangen Biotech, Beijing, China), respectively. The 2−ΔΔCq method22 was employed for quantification of the expression of all genes.

Cell Counting Kit-8 (CCK-8) and Colony Formation Assays

We collected the transfected cells and resuspended them in complete culture medium at a concentration of 3 × 104 cells/ml. Each well from 96-well plates was covered with 100 μl of cell suspension. At the set time points after cell inoculation, the cells were probed with 10 μl of CCK-8 solution (Beyotime) at 37°C for 2 h. The absorbance was read at 450-nm wavelength using a microplate reader (Bio-Rad, Hercules, CA, USA).

Transfected cells were collected, and a cell suspension was prepared in the same manner as described above. Cells were spread evenly in six-well plates at a density of 1,000 cells/well. We allowed the cells to form colonies at 37°C for 14 days. Finally, the colonies were rinsed with phosphate-buffered saline (PBS), fixed using 100% methanol, and stained with 0.05% crystal violet. The colonies were counted under a microscope (Nikon, Tokyo, Japan).

Transwell Experiments

To detect migration ability, 1 × 105 cells were added to the upper compartments of the Transwell chambers (8-μm pores; Corning Costar, Cambridge, MA, USA) containing 100 μl of FBS-free medium. Twenty percent FBS-supplemented culture medium was placed into the lower chambers. We incubated cells at 37°C for 1 day and then used a cotton bud to manually wipe away the nonmigrated cells. The migrated cells were treated with 100% methanol for fixation and 0.05% crystal violet for coloration. Next, we employed an inverted light microscope to take photos and count the migrated cells. To assess invasion capacity, the top layer surface of the membrane was coated with Matrigel (BD Bioscience, San Jose, CA, USA) in advance, and the subsequent experimental steps were the same as those described above.

Flow Cytometry Analysis

Transfected cells were harvested via treatment with 0.25% EDTA-free trypsin, washed with PBS, and centrifuged to remove the supernatant fluid. Evaluation of cell apoptosis was achieved with the help of an Annexin-V–FITC/PI Apoptosis Kit (KeyGEN, Nanjing, China). In total, 500 μl of binding buffer was utilized to resuspend the transfected cells, which were then mixed with 5 μl of annexin V–fluorescein isothiocyanate (FITC) and 5 μl of propidium iodide. After cultivation at room temperature without light, the proportion of apoptotic cells was measured with a FACScan flow cytometer (BD Bioscience).

Tumor Xenograft Experiment

The experiments involving animals were implemented with approval from the Animal Ethics Committee of The First People’s Hospital of Yancheng and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Shanghai GenePharma Co. Ltd. synthesized short-hairpin RNA (shRNA) targeting LAMTOR5-AS1 (sh-LAMTOR5-AS1) and scramble control shRNA (sh-NC) and inserted these constructs into a lentiviral vector, producing recombinant lentiviral vectors. The sh-LAMTOR5-AS1 sequence was 5′-CCGGTACCAAAAAATAAAAATATGAACCTCGAGGTTCATATTTTTATTTTTTGGTATTTTTG-3′, and the sh-NC sequence was 5′-CCGGCACGATAAGACAATGTATTTCTCGAGAAATACATTGTCTTATCGTGTTTTTG-3′. After transfection of 293T cells with the appropriate lentiviral vector along with psPAX2 and pMD2.G, the lentiviral particles were harvested after 48 h of culture at 37°C. For acquisition of a stable LAMTOR5-AS1-knockdown cell line, A549 cells were cultivated with culture medium containing lentiviral particles and selected with puromycin.

Male BALB/c nude mice, aged 4–6 weeks, were acquired from Vital River Laboratory (Beijing, China) and maintained under specific pathogen-free conditions at 25°C and 50% humidity with a 10:14 light/dark cycle and ad libitum access to food and water. A total of 1 × 106 stable LAMTOR5-AS1-knockdown A549 cells were resuspended in 100 μl of PBS and subcutaneously injected into the flank of mice. One week after cell injection, tumor size was recorded every 4 days and used to determine tumor volume based on the following equation: volume = 0.5 × length × width2. At 31 days after cell injection, all mice were euthanized by cervical dislocation, and we excised the tumor xenografts for tumor weight assessment and further immunohistochemistry analyses.

Fluorescence In Situ Hybridization

We analyzed the in situ localization of LAMTOR5-AS1 in NSCLC cells using a Fluorescent In Situ Hybridization (FISH) Kit (Guangzhou RiboBio Co. Ltd., Science City, Guangzhou, China). Briefly, we rinsed NSCLC cells twice with PBS, fixed them with 4% paraformaldehyde, and treated them with probes specifically targeting LAMTOR5-AS1 (Guangzhou RiboBio Co. Ltd.) at 37°C overnight without light. Next, DNA staining was done using Hoechst solution. Finally, imaging was implemented under a confocal laser scanning microscope (Leica, Solms, Germany).

Nuclear/Cytoplasmic Fractionation Assay

We collected cells in the logarithmic growth phase and subjected these cells to a PARIS™ Kit (Thermo Fisher Scientific Inc.) to obtain nuclear/cytoplasmic fractions. After RNA extraction, qRT-PCR was carried out to determine the subcellular localization of LAMTOR5-AS1.

Bioinformatics Prediction and Luciferase Reporter Assay

The putative targets of LAMTOR5-AS1 were predicted with starBase 3.0 (http://starbase.sysu.edu.cn/). The prediction of the miR-506-3p downstream targets was conducted using two algorithms, including miRDB (http://mirdb.org/) and TargetScan (http://www.targetscan.org/).

Partial fragments of LAMTOR5-AS1 carrying wild-type (WT) or mutant (MT) miR-506-3p-binding sites were synthesized by Shanghai GenePharma Co. Ltd. and cloned into a psiCHECK2 vector (Promega, Madison, WI, USA). The generated luciferase reporter plasmids were labeled WT-LAMTOR5-AS1 and MUT-LAMTOR5-AS1. In a similar manner, WT-E2F6 and MUT-E2F6 reporter plasmids were produced. For the reporter assay, NSCLC cells were transiently transfected with the indicated plasmids alongside miR-506-3p mimic or miR-NC. Two days after transfection, the luciferase activity was examined utilizing a Dual-Luciferase Reporter Assay System (Promega).

RNA Immunoprecipitation (RIP)

By utilizing a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Merck Millipore, Darmstadt, Germany), we examined the interaction among LAMTOR5-AS1, miR-506-3p, and E2F6. In brief, whole NSCLC cell lysates were obtained by treatment of cells with complete RIP lysis buffer and probed overnight at 4°C with RIP buffer supplemented with magnetic beads containing anti-Argonaute2 (Ago2) antibody or IgG control (Millipore). Then the magnetic beads were harvested and rinsed using RIP washing buffer, followed by treatment with Proteinase K buffer. The target immunoprecipitated RNA was extracted and detected via qRT-PCR.

Western Blot Analysis

We isolated total protein using RIP assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride (Beyotime). After quantification with a BCA Protein Assay Kit (Beyotime), the same amounts of protein were loaded and subjected to separation in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membranes were immersed in 5% nonfat dry milk at room temperature for 2 h and incubated overnight at 4°C with primary antibodies targeting E2F6 (Cat. No. ab11952) or GAPDH (Cat. No. ab8245; both from Abcam, Cambridge, UK). Next, a further 2-h incubation at room temperature was conducted with a horseradish peroxidase (HRP)-conjugated secondary antibody (Cat. No. ab205719; Abcam). Finally, Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific Inc.) was adopted for target signal visualization.

Statistical Analysis

All data were obtained from three independent experiments and are expressed as the mean ± standard deviation. Survival analysis was implemented utilizing the Kaplan–Meier method and a log rank test. The evaluation of differences between two groups was achieved using paired or unpaired Student’s t-tests. One-way analysis of variance (ANOVA) followed by Tukey’s test was employed to assess differences between multiple groups. Gene expression correlation was tested using Pearson’s correlation coefficient. A value of p < 0.05 was considered to indicate a statistically significant difference.

RESULTS

LAMTOR5-AS1 Depletion Induces Inhibition of Malignant Characteristics in NSCLC

The expression status of LAMTOR5-AS1 in NSCLC was discovered using The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Compared with normal tissues, LAMTOR5-AS1 was overexpressed in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (Fig. 1A). Next, we measured LAMTOR5-AS1 expression in our own cohort. LAMTOR5-AS1 was observably overexpressed in NSCLC tissues compared to adjacent normal tissues (Fig. 1B). In contrast to BEAS-2B cells, the tested NSCLC cell lines manifested higher LAMTOR5-AS1 levels (Fig. 1C). Based on the median value of LAMTOR5-AS1 in NSCLC tumor tissues, all patients with NSCLC were divided into either the low LAMTOR5-AS1 group (n = 24) or the high LAMTOR5-AS1 group (n = 25). NSCLC cases with high LAMTOR5-AS1 levels presented evidently shorter overall survival than cases with low LAMTOR5-AS1 levels (Fig. 1D).

Figure 1

LAMTOR5-AS1 is overexpressed in non-small cell lung cancer (NSCLC). (A) The fragments per kilobase per million (FPKM) expression values of LAMTOR5-AS in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were determined using The Cancer Genome Atlas (TCGA). (B) Assessment of LAMTOR5-AS1 levels in NSCLC tissues was performed via quantitative real-time polymerase chain reaction (qRT-PCR). (C) qRT-PCR was adopted to measure LAMTOR5-AS1 levels in NSCLC cell lines. (D) Kaplan–Meier curves revealed the prognosis of NSCLC cases with high or low LAMTOR5-AS1 expression. **p < 0.01.

The high level of LAMTOR5-AS1 expression in NSCLC and its correlation with overall survival compelled us to uncover its role in NSCLC progression. A549 and SK-MES-1 cells manifested the highest LAMTOR5-AS1 level among the five NSCLC cell lines; therefore, they were applied in subsequent functional experiments. Through siRNAs, LAMTOR5-AS1 was depleted in A549 and SK-MES-1 cells (Fig. 2A). si-LAMTOR5-AS1#1 and si-LAMTOR5-AS1#2 were applied for further investigation because their knockdown efficiency was higher than that of si-LAMTOR5-AS1#3. Interference with LAMTOR5-AS1 expression impeded the proliferation (Fig. 2B) and cloning ability (Fig. 2C) of NSCLC cells. In addition, flow cytometry analysis was implemented, verifying that cell apoptosis was increased after knockdown of LAMTOR5-AS1 (Fig. 2D). Furthermore, depletion of LAMTOR5-AS1 led to a clear decrease in NSCLC cell migration and invasion (Fig. 3A and B). Overall, LAMTOR5-AS1 knockdown disrupted the malignancy of NSCLC.

Figure 2

LAMTOR5-AS1 downregulation impairs the malignant progression of NSCLC. (A) qRT-PCR confirmed the silencing efficiency of siRNAs targeting LAMTOR5-AS1. (B, C) Cell Counting Kit-8 (CCK-8) and colony formation assays illustrated the growth of NSCLC cells after LAMTOR5-AS1 depletion. (D) The proportion of apoptotic LAMTOR5-AS1-deficient NSCLC cells was tested via flow cytometry analysis. **p < 0.01.

Figure 3

LAMTOR5-AS1 knockdown decreases the migration and invasion of NSCLC. (A, B) Transwell experiments revealed the motility of NSCLC cells after transfection with siRNA to induce LAMTOR5-AS1 downregulation. **p < 0.01.

LAMTOR5-AS1 Competitively Binds to miR-506-3p in NSCLC

Based on the results of the nuclear/cytoplasmic fractionation experiment and fluorescence in situ hybridization, LAMTOR5-AS1 was confirmed to be mainly expressed in the cell cytoplasm (Fig. 4A and B), indicating that LAMTOR5-AS1 may perform its cancer-promoting actions by acting as a molecular sponge or ceRNA. As predicted by the starBase 3.0 tool, 46 miRNAs were identified as possible downstream targets of LAMTOR5-AS1. We then analyzed the expression profile of the 46 candidates in NSCLC by analyzing TCGA datasets. miR-506-3p was downregulated in both LUAD and LUSC (Fig. 4C) and therefore was selected for subsequent studies. For further verification, qRT-PCR was implemented to measure the expression of miR-506-3p in LAMTOR5-AS1-silenced NSCLC cells. Transfection with si-LAMTOR5-AS1 increased the level of miR-506-3p in NSCLC cells (Fig. 4D).

Figure 4

LAMTOR5-AS1 acts as an miR-506-3p sponge in NSCLC. (A, B) Localization of LAMTOR5-AS1 in A549 and SK-MES-1 cells. (C) miR-506-3p expression in LUAD and LUSC was examined using TCGA databases. (D) After LAMTOR5-AS1 ablation, the expression of miR-506-3p was examined via qRT-PCR. (E) Wild-type (WT) and mutant (MUT) binding sequences of miR-506-3p within LAMTOR5-AS1 sequences. (F) Luciferase activity was evaluated in miR-506-3p-overexpressing NSCLC cells after transfection with WT-LAMTOR5-AS1 or MUT-LAMTOR5-AS1. (G) The enrichment of LAMTOR5-AS1 and miR-506-3p by Ago2 antibody was confirmed with a RNA immunoprecipitation (RIP) assay. (H) qRT-PCR was applied to measure miR-506-3p expression in NSCLC tissues. (I) Pearson’s correlation analysis illustrated the relation between LAMTOR5-AS1 and miR-506-3p in NSCLC tissues. **p < 0.01.

Later, the direct binding between LAMTOR5-AS1 and miR-506-3p (Fig. 4E) was explored using a luciferase reporter assay. The luciferase activity of WT-LAMTOR5-AS1 was diminished upon miR-506-3p overexpression, but when the binding sequences were mutated, the miR-506-3p mimic was unable to alter the luciferase activity (Fig. 4F). Ago2 is a key element of the RNA-induced silencing complex and can directly trigger the degradation of targets through its catalytic activity in gene silencing processes guided by miRNAs. In a RIPA, miR-506-3p and LAMTOR5-AS1 were evidently enriched in Ago2-containing beads compared with the IgG group (Fig. 4G). In addition, the miR-506-3p level was reduced in NSCLC tissues (Fig. 4H) and manifested an inverse relationship with LAMTOR5-AS1 expression (Fig. 4I). Undoubtedly, LAMTOR5-AS1 operated as a miR-506-3p sponge in NSCLC.

LAMTOR5-AS1 Positively Regulates E2F6 Expression in NSCLC by Sequestering miR-506-3p

To illuminate the role of miR-506-3p in NSCLC, we overexpressed miR-506-3p in NSCLC cells using a miR-506-3p mimic (Fig. 5A). Through functional experiments, exogenous miR-506-3p was found to restrict NSCLC cell proliferation (Fig. 5B and C), trigger cell apoptosis accumulation (Fig. 5D), and hinder cell motility (Fig. 5E and F). To identify potential targets of miR-506-3p, bioinformatics prediction was conducted and revealed that E2F6, a well-known oncogene in various human cancer types, may be a possible candidate target of miR-506-3p (Fig. 6A). miR-506-3p upregulation observably downregulated E2F6 in NSCLC cells (Fig. 6B and C). Moreover, E2F6 overexpression in NSCLC (Fig. 6D) presented a clear negative correlation with miR-506-3p expression (Fig. 6E). Moreover, the miR-506-3p mimic weakened the luciferase activity of WT-E2F6, whereas the activity caused by MUT-E2F6 remained largely unchanged despite the same cotransfection status (Fig. 6F).

Figure 5

miR-506-3p reintroduction attenuates the malignant behaviors of NSCLC cells. (A) The transfection efficiency of miR-506-3p mimic in NSCLC. (B, C) miR-506-3p-overexpressing NSCLC cell proliferation and colony formation. (D) Apoptosis of NSCLC cells transfected with the miR-506-3p mimic was examined. (E, F) The migratory and invasive properties of miR-506-3p-overexpressing NSCLC cells were determined via Transwell experiments. **p < 0.01.

Figure 6

E2F6 is controlled by the LAMTOR5-AS1/miR-506-3p axis in NSCLC. (A) The WT and MUT binding sequences of miR-506-3p within the E2F6 3′-untranslated region (3′-UTR). (B, C) E2F6 expression was measured in miR-506-3p mimic-transfected NSCLC cells. (D) E2F6 mRNA levels in NSCLC tissues were determined via qRT-PCR. (E) The expression relationship between miR-506-3p and E2F6 in NSCLC tissues. (F) Luciferase activity was evaluated in miR-506-3p-overexpressing NSCLC cells after transfection with WT-E2F6 or MUT-E2F6. (G, H) Detection of E2F6 levels was conducted in NSCLC cells when LAMTOR5-AS1 was ablated. (I, J) LAMTOR5-AS1-silenced NSCLC cells were further probed with NC inhibitor or miR-506-3p inhibitor and subjected to assessment of E2F6 levels. (K) Expression correlation between LAMTOR5-AS1 and E2F6 in NSCLC tissues. (L) The enrichment of LAMTOR5-AS1, miR-506-3p, and E2F6 by Ago2 antibody was confirmed with a RIP assay. **p < 0.01.

We next attempted to examine whether LAMTOR5-AS1 is implicated in modulating E2F6 expression by acting as an miR-506-3p sponge. Loss of LAMTOR5-AS1 resulted in a decrease in E2F6 expression (Fig. 6G and H), which was abrogated by cotransfection with the miR-506-3p inhibitor (Fig. 6I and J). Additionally, a positive expression relationship was affirmed between LAMTOR5-AS1 and E2F6 in NSCLC tissues (Fig. 6K). Finally, as evidenced by the RIP results, the enrichment of LAMTOR5-AS1, miR-506-3p, and E2F6 was confirmed in Ago2-precipitated products (Fig. 6L), indicating the coexistence of the three RNAs in the RNA-induced silencing complex. Thus, LAMTOR5-AS1 positively controlled E2F6 expression in NSCLC by decoying miR-506-3p.

LAMTOR5-AS1 Ablation Hinders NSCLC Cell Behaviors by Increasing the Output of the miR-506-3p/E2F6 Axis

Rescue experiments were carried out to determine whether the miR-506-3p/E2F6 axis is required for the anticarcinogenic actions of si-LAMTOR5-AS1 in NSCLC cells. First, the knockdown efficiency of the miR-506-3p inhibitor was verified via qRT-PCR (Fig. 7A). Treatment with the miR-506-3p inhibitor reversed the decrease in NSCLC cell proliferation and colony formation mediated by LAMTOR5-AS1 downregulation (Fig. 7B and C). Furthermore, downregulation of LAMTOR5-AS1 promoted cell apoptosis, which was neutralized after miR-506-3p depletion (Fig. 7D). Additionally, the impaired migratory and invasive properties of LAMTOR5-AS1-deficient NSCLC cells were restored by miR-506-3p inhibition (Fig. 7E).

Figure 7

Inhibition of miR-506-3p abrogates the suppressive actions of si-LAMTOR5-AS1 in NSCLC. (A) The efficiency of the miR-506-3p inhibitor was determined by qRT-PCR. (B–D) NSCLC cells were transfected with si-NC, si-LAMTOR5-AS1+ NC inhibitor, or si-LAMTOR5-AS1+ miR-506-3p inhibitor. Cellular proliferation, colony formation, and apoptosis were assessed using CCK-8, colony formation, and flow cytometry assays. (E) Transwell experiments were performed to evaluate the motility of NSCLC cells treated as described above. *p < 0.05 and **p < 0.01.

In addition, Western blotting verified the overexpression efficiency of pcDNA3.1-E2F6 in NSCLC cells (Fig. 8A). Reintroduction of E2F6 reversed the si-LAMTOR5-AS1-mediated effects on NSCLC cell proliferation and cloning abilities (Fig. 8B and C) as well as on cell apoptosis (Fig. 8D). Additionally, the decreased migration and invasion induced by si-LAMTOR5-AS1 were recovered by E2F6 restoration (Fig. 8E). Collectively, the results indicate that the absence of LAMTOR5-AS1 repressed the malignant progression of NSCLC cells via control of the miR-506-3p/E2F6 axis.

Figure 8

The tumor-inhibiting actions of si-LAMTOR5-AS1 in NSCLC are neutralized by E2F6 overexpression. (A) Successful transfection of pcDNA3.1-E2F6 was demonstrated by Western blotting. (B–D) pcDNA3.1-E2F6 or pcDNA3.1 alongside si-LAMTOR5-AS1 was transfected into NSCLC cells. CCK-8 assay, colony formation assay, and flow cytometry were implemented to assess cell proliferation, colony formation, and apoptosis, respectively. (E) NSCLC cells treated as described above were subjected to Transwell experiments for cell migration and invasion determination. **p < 0.01.

LAMTOR5-AS1 Downregulation Impedes Tumor Growth In Vivo

Finally, a tumor xenograft experiment was carried out to characterize the effect of LAMTOR5-AS1 on tumor growth in vivo. At 31 days after cell injection, tumor xenografts were excised (Fig. 9A). Tumor xenografts from the sh-LAMTOR5-AS1 group featured slower growth (Fig. 9B) and a lower weight (Fig. 9C) than those from the sh-NC group. Furthermore, compared with the sh-NC group, LAMTOR5-AS1 was downregulated (Fig. 9D), whereas miR-506-3p was overexpressed (Fig. 9E) in sh-LAMTOR5-AS1-injected tumors. Additionally, E2F6 protein was evidently downregulated in xenografts from the sh-LAMTOR5-AS1 group (Fig. 9F). Immunohistochemistry analysis showed that the levels of E2F6 and the proliferation marker Ki-67 were reduced, while those of the apoptotic marker cleaved caspase-3 were increased in LAMTOR5-AS1-depleted tumors (Fig. 9G). In short, LAMTOR5-AS1 downregulation led to in vivo tumor growth suppression.

Figure 9

Knocking down LAMTOR5-AS1 represses tumor growth in vivo. (A) Images of the representative tumor xenografts. A total of six mice were used. Each group had three nude mice. (B) Tumor volume was recorded and applied to plot a growth curve. (C) The weight of tumor xenografts. (D–F) Levels of LAMTOR5-AS1, miR-506-3p, and E2F6 in tumors were determined. (G) Immunohistochemistry analysis of E2F6, Ki-67, and cleaved caspase-3 levels in tumor xenografts. **p < 0.01.

DISCUSSION

Numerous studies have authenticated the multifaceted functions of lncRNAs in the onset and progression of NSCLC23–25. With the great advancements made in next-generation sequencing technology, a considerable number of lncRNAs have been found to induce dysregulation in NSCLC26; however, the exact functions of the vast majority of lncRNAs in NSCLC await further exploration. Thus, identifying lncRNAs that contribute to NSCLC pathogenesis has become an area of interest for many researchers and would be of instrumental significance for managing NSCLC patients. In this study, we measured LAMTOR5-AS1 expression in NSCLC and gauged its clinical value. Furthermore, LAMTOR5-AS1 was knocked down to assess the functional alterations in NSCLC cells in response to LAMTOR5-AS1 depletion.

LAMTOR5-AS1 has been affirmed to be a risk prediction and diagnostic biomarker of prostate cancer21. However, the expression and biological roles of LAMTOR5-AS1 in NSCLC are unclear. Herein, LAMTOR5-AS1 overexpression in NSCLC was verified in TCGA datasets and our own cohort and manifested an evident relationship with poor prognosis. Functionally, LAMTOR5-AS1 ablation led to repression of the proliferation, cloning, and metastasis abilities of NSCLC cells in vitro. We further observed an obvious increase in LAMTOR5-AS1-silenced NSCLC cell apoptosis. Additionally, the impact of LAMTOR5-AS1 downregulation on in vivo tumor growth was tested, and the inhibitory influence of LAMTOR5-AS1 depletion on NSCLC cell tumor growth was confirmed in vivo. All these observations reveal the importance of LAMTOR5-AS1 in NSCLC oncogenesis and progression.

The operating mode of lncRNAs is defined by their subcellular distribution27. In general, lncRNAs distributed in the cell nucleus affect transcriptional events, whereas cytoplasmic lncRNAs are implicated in posttranscriptional modulation28. The ceRNA theory has been gaining increasing interest in lncRNA research29–31. The theory connects the function of protein-coding mRNAs with that of miRNAs and lncRNAs32. lncRNAs serve as natural sponges for specific miRNAs, which in turn lowers the regulatory action of miRNAs on downstream target mRNAs.

In the current research, LAMTOR5-AS1 was discovered to be located mainly in the cytoplasmic fraction of NSCLC cells, indicating the posttranscriptional activity of this lncRNA. To unravel the molecular events downstream of LAMTOR5-AS1, bioinformatics prediction was implemented and indicated that an miR-506-3p binding site was present in LAMTOR5-AS1. In subsequent experiments, LAMTOR5-AS1 was found to sponge miR-506-3p in NSCLC cells. Furthermore, E2F6, a downstream target of miR-506-3p, was under the control of LAMTOR5-AS1, which was realized by decoying miR-506-3p. In summary, miR-506-3p functions as a bridge between LAMTOR5-AS1 and E2F6, and together, they constitute a new ceRNA pathway in NSCLC, thus contributing to aggressive events.

miR-506-3p presents an aberrant expression pattern in many human cancers. For instance, a high level of miR-506-3p was confirmed in retinoblastoma33 and pancreatic ductal adenocarcinoma34, exerting pro-oncogenic actions. In contrast, osteosarcoma35, papillary thyroid cancer36, ovarian cancer37, and nasopharyngeal carcinoma38 weakly expressed miR-506-3p. In NSCLC, miR-506-3p was downregulated and exhibited a striking association with several aggressive clinicopathological characteristics and poor prognosis39. miR-506-3p was confirmed as a tumor-suppressing miRNA in NSCLC and is involved in malignant properties and gefitinib resistance39,40. In accordance with this, our data revealed a decreased level of miR-506-3p in NSCLC and uncovered its vital roles. More importantly, miR-506-3p directly targeted and lowered E2F6 expression in NSCLC cells. Rescue experiments verified that miR-506-3p suppression or E2F6 reintroduction was capable of remitting LAMTOR5-AS1 deficiency-triggered anticarcinogenic actions in NSCLC. Accordingly, the antineoplastic functions of LAMTOR5-AS1 silencing occurred through its ability to act as a ceRNA for miR-506-3p, consequently weakening the regulatory influences of the latter on E2F6 levels.

The current research contained two limitations. The regulatory effect of LAMTOR5-AS1 on metastasis in vivo was not explored. Furthermore, the related genes contributed to the influences of LAMTOR5-AS1 ablation on NSCLC cell growth, and metastasis should be analyzed. We will resolve the two limitations in the near future. More importantly, we will illuminate the downstream signaling pathway of LAMTOR5-AS1/miR-506-3p/E2F6 in NSCLC.

In summary, our study demonstrated the functions of LAMTOR5-AS1 for the first time and revealed that LAMTOR5-AS1 knockdown disrupted the malignancy of NSCLC by targeting the miR-506-3p/E2F6 axis. Certification of the LAMTOR5-AS1/miR-506-3p/E2F6 pathway may offer novel targets for NSCLC diagnosis and management.