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

Abstract

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.

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.

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.

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.

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.

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.

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.

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.