LncRNA DLEU2 accelerates the tumorigenesis and invasion of non–small cell lung cancer by sponging miR‐30a‐5p

Abstract Long non‐coding RNAs (lncRNAs) have been reported to participate in the pathogenesis of non–small cell lung cancer (NSCLC). However, how lncRNA deleted in lymphocytic leukaemia 2 (DLEU2) contributes to NSCLC remains undocumented. The clinical significance of lncRNA DLEU2 and miR‐30a‐5p expression in NSCLC was analysed by using fluorescence in situ hybridization and TCGA cohorts. Gain‐ and loss‐of‐function experiments as well as a NSCLC tumour model were executed to determine the role of lncRNA DLEU2 in NSCLC. DLEU2‐sponged miR‐30a‐5p was verified by luciferase reporter, and RIP assays. Herein, the expression of lncRNA DLEU2 was elevated in NSCLC tissues, and its high expression or low expression of miR‐30a‐5p acted as an independent prognostic factor of poor survival and tumour recurrence in NSCLC. Silencing of lncRNA DLEU2 repressed the tumorigenesis and invasive potential of NSCLC, whereas re‐expression of lncRNA DLEU2 showed the opposite effects. Furthermore, lncRNA DLEU2 harboured a negative correlation with miR‐30a‐5p expression in NSCLC tissues and acted as a sponge of miR‐30a‐5p, which reversed the tumour‐promoting effects of lncRNA DLEU2 by targeting putative homeodomain transcription factor 2 in NSCLC. Altogether, lncRNA DLEU2 promoted the tumorigenesis and invasion of NSCLC by sponging miR‐30a‐5p.

In addition, lncRNAs act as the sponges of miRNAs in NSCLC.

| Materials
NSCLC cell lines (A549 and NCI-H460) used in our study were purchased from the Institute of Chemistry and Cell Biology. Lentivirusmediated sh-DLEU2 and negative control (sh-NC) vectors were F I G U R E 1 Up-regulation of lncRNA DLEU2 expression was associated with poor survival in patients with NSCLC. A, FISH analysis of the localization and expression levels of lncRNA DLEU2 in LAC tissues. B, qRT-PCR analysis of the expression levels of lncRNA DLEU2 in LAC tissue samples. C, TCGA analysis of the expression levels of lncRNA DLEU2 in paired and unpaired LAC tissues. D, ROC curve analysis of the cut-off value, AUC, sensitivity and specificity of lncRNA DLEU2 in LAC patients. E, The cut-off value divided the LAC patients into high DLEU2 expression and low DLEU2 expression groups. F, Kaplan-Meier analysis of the association of high or low lncRNA DLEU2 expression with the poor survival and tumour recurrence in LAC patients purchased from Genechem. LncRNA DLEU2 plasmid, pcDNA3.1 and miR-30a-5p mimic or inhibitor were from GenePharma. The antibodies against PCNA, MMP-2 and PHTF2 were from Abcam.

| Clinical samples
The RNA-seq data including the clinicopathological and prognostic information of NSCLC and the expression levels of lncRNA DLEU2, miR-30a-5p/-30b-5p/-30c-5p/-30d-5p/-30e-5p and PHTF2 were downloaded from the TCGA dataset. A tissue microarray (TMA) containing 20 paired lung adenocarcinoma (LAC) tissues (Cat No. ZL-LUC1601) was from Superbiotek, and another 15 paired frozen LAC tissue samples were from our hospital. These protocols were approved by the ethics committee of our hospital.

| Bioinformatic analysis
LncRNA DLEU2-specific binding with miRNAs and the targets of miR-30a-5p were identified using the StarBase v2.0 on the basis of the high stringency (>5) and the number of cancers types (≥5).

| Cell culture, transfection and quantitative realtime PCR
The expression levels of lncRNA DLEU2 in NSCLC cell lines and tissue samples were detected by quantitative real-time PCR (qRT-PCR) analysis. Cell culture, transfection and qRT-PCR analysis were carried out as previously described. 18 The primers used are listed in Table S1.

| Cell viability and transwell invasion assays
Cell viability and Transwell invasion assays were conducted as previously described. 18

| Dual-luciferase report and RNA immunoprecipitation assays
Luciferase gene report and RNA immunoprecipitation (RIP) assays were executed according to the previous report. 19

| Animal experiments
Six-week-old female BALB/c-nu mice were injected subcutaneously with 5 × 10 6 A549 cells stably transfected with sh-DLEU2 or sh-NC and were monitored daily so as to develop a subcutaneous xenograft tumour. The detailed detection measures were performed as previously described. 18 This animal study was approved by the ethics committee of our hospital.

| Haematoxylin and eosin and Immunohistochemistry analysis
Xenograft tumour tissues were harvested and fixed in 4% paraformaldehyde and preserved in optimal cutting temperature compound.
The tumour tissues were stained with haematoxylin and eosin (HE) for the histological analysis, and immune-stained for Ki-67 (Abcam) as previously described. 18

| Statistical analysis
Statistical analysis was carried out as previously described. 19

| Elevated expression of lncRNA DLEU2 is associated with poor survival in patients with NSCLC
As shown by FISH analysis, we found that lncRNA DLEU2 expression, predominantly in the cytoplasm, was dramatically raised in lung adenocarcinoma (LAC) tissue samples compared with the normal tissues (n = 20, P < .0001; Figure 1A). qRT-PCR analysis also showed the up-regulation of lncRNA DLEU2 in LAC tissue samples as compared with the adjacent normal tissues (n = 15, P = .0003; Figure 1B). These results were further validated in paired (n = 7, P = .013) and unpaired LAC tissues (n = 331, P = .0004) in TCGA cohort ( Figure 1C).
To analyse the association between lncRNA DLEU2 expression and clinicopathological characteristics and prognosis in patients with LAC based on the lncRNA DLEU2 expression levels, survival time and survival status, we obtained the cut-off value (4.918), AUC (0.51), sensitivity (81.0%) and specificity (20.2%) of DLEU2 in LAC patients ( Figure 1D), suggesting that lncRNA DLEU2 might be a potential marker for LAC patients.
According to the cut-off value of lncRNA DLEU2, the patients were divided into high DLEU2 expression group (n = 243) and low DLEU2 expression group (n = 60) ( Figure 1E). High expression of lncRNA DLEU2 had no association with the clinicopathological  Table S2). Subsequently, as indicated by Kaplan-Meier analysis, we found that the patients with high DLEU2 expression had a poorer survival (P = .0329), but showed no difference in tumour recurrence (P = .1308), as compared with those with low DLEU2 expression ( Figure 1F).
According to the univariate and multivariate analysis, we found that high DLEU2 expression as well as lymph node metastasis was an independent prognostic factor of poor survival in LAC patients (Tables S3).

| lncRNA DLEU2 enhances the proliferation and invasion of NSCLC cells
The knockdown effect of sh-DLEU2 vector in A549 and NCI-H460 cell lines was determined by qRT-PCR analysis (Figure 2A). We found that knockdown of DLEU2 reduced the proliferative viability ( Figure 2B) and invasive potential ( Figure 2C) in A549 and NCI-H460 cells. In addition, the expression levels of PCNA and MMP-2, as evaluated by Western blot analysis, were decreased by knockdown of lncRNA DLEU2 in these two cell lines ( Figure 2D).
Then, the overexpression effect of lncRNA DLEU2 plasmid in these two lines was assessed by qRT-PCR analysis ( Figure 2E).
Overexpression of lncRNA DLEU2 promoted the proliferative viability ( Figure 2F) and invasive potential ( Figure 2G) in A549 and NCI-H460 cells. The expression levels of PCNA and MMP-2, as estimated by Western blot analysis, were increased by overexpression of ln-cRNA DLEU2 in these two cells ( Figure 2H).

| MiR-30a-5p displays a negative correlation with lncRNA DLEU2 expression in NSCLC tissues
To elucidate the mechanisms by which lncRNA DLEU2 contributes to NSCLC, we used a starBasev2.0 tool to screen five miRNAs that may have the strongest binding potential with lncRNA DLEU2 in cancers (Table S4). The expression levels of these miRNAs were detected in LAC tissues using the TCGA data set. We found that F I G U R E 4 LncRNA DLEU2 acted as a sponge of miR-30a-5p in NSCLC cells. A, Schematic representation of the binding sites between miR-30a-5p and WT or Mut lncRNA DLEU2. B, Luciferase activity of WT or Mut DLEU2 after the cotransfection with the miR-30a-5p mimic or inhibitor and WT or Mut DLEU2 vector in A549 and NCI-H460 cells. C, qRT-PCR analysis of the effects of lncRNA DLEU2 on the expression levels of miR-30a-5p in A549 and NCI-H460 cells. D, RIP analysis of the amount of lncRNA DLEU2 and miR-30a-5p pulled down from the Ago2 protein in A549 and NCI-H460 cells (E and F). MTT analysis of the proliferation viability after cotransfection with DLEU2 plasmid and miR-30a-5p mimic or sh-DLEU2 and miR-30a-5p inhibitor in A549 and NCI-H460 cells. Data shown are the mean ± SEM of three experiments. *P < .05 miR-30a-5p/-30d-5p rather than other miRNAs had a decreased expression in paired and unpaired LAC tissues ( Figure 3A). Pearson correlation analysis showed that miR-30a-5p harboured a most significantly negative correlation with lncRNA DLEU2 expression (r = −0.2444, P < .0001; Figure 3B), as compared with other miRNAs in LAC tissues ( Figure S1). As shown by FISH analysis, we also validated that miR-30a-5p, mainly localized in the cytoplasm, was down-regulated (P = .026) and had a negative correlation with lncRNA DLEU2 expression in LAC tissues (r = −0.573, P = .008; Figure 3C).
We further obtained a cut-off value (14.49), AUC (0.58), sensitivity (23.2%) and specificity (92%) of miR-30a-5p in LAC patients ( Figure 3D) and divided the patients into high and low miR-30a-5p expression groups ( Figure S2). As indicated in Table S5, low expression of miR-30a-5p had an association with lymph node metastasis (P = .005), rather than other parameters in LAC (each P > .05). Subsequently, Kaplan-Meier analysis demonstrated that the patients with low miR-30a-5p expression possessed a lower survival (P = .0182) and a higher tumour recurrence (P = .0007) in comparison with those with high miR-30a-5p expression ( Figure 3E). Univariate and multivariate analysis unveiled that low miR-30a-5p expression as well as lymph node metastasis was an independent prognostic factor of poor survival and tumour recurrence in LAC patients (Tables S6,7).

| lncRNA DLEU2 acts as a sponge of miR-30a-5p in NSCLC cells
The binding sites between miR-30a-5p and WT or Mut DLEU2 are indicated in Figure 4A. We cotransfected A549 and NCI-H460 F I G U R E 5 miR-30a-5p reversed lncRNA DLEU2-induced PHTF2 expression in NSCLC cells. A, Identification of the targets of miR-30a-5p using a starBasev2.0 prediction tool. B, TCGA analysis of the expression levels of these target genes in unpaired LAC tissues. C, Pearson analysis of the correlation of miR-30a-5p with lncRNA PHTF2 expression in LAC tissues. D, Schematic representation of the binding sites between miR-30a-5p and WT or Mut PHTF2 3'UTR. E, Luciferase activity of WT or Mut PHTF2 3'UTR after the cotransfection with miR-30a-5p mimic or inhibitor and WT or Mut PHTF2 3'UTR in A549 and NCI-H460 cells (F and G). qRT-PCR and western blot analysis of the expression levels of PHTF2 after cotransfection with lncRNA DLEU2 plasmid and miR-30a-5p mimic or sh-DLEU2 and miR-30a-5p inhibitor in A549 and NCI-H460 cells. Data shown are the mean ± SEM of three experiments. *P < .05 and ***P < .0001 cells with WT or Mut DLEU2 reporter and miR-30a-5p mimic or inhibitor, and found that miR-30a-5p mimic reduced the luciferase activity of WT DLEU2, while miR-30a-5p inhibitor reversed this effect ( Figure 4B). Re-expression of lncRNA DLEU2 reduced the expression levels of miR-30a-5p, indicated by qRT-PCR analysis, but knockdown of lncRNA DLEU2 had an opposite effect ( Figure 4C). Furthermore, RIP assay was confucted for Ago2 protein in A549 and NCI-H460 cells, and endogenous lncRNA DLEU2 and miR-30a-5p pulled down from Ago2-expressed cell lines were enriched in Ago2 pellet as compared with the input control ( Figure 4D). The miR-30a-5p mimic and DLEU2 plasmid or miR-30a-5p inhibitor and sh-DLEU2 vector were cotransfected into A549 and NCI-H460 cells, indicating that miR-30a-5p mimic repressed the proliferative viability and attenuated the proliferation-promoting effects of lncRNA DLEU2 ( Figure 4E), but miR-30a-5p inhibitor reversed these effects ( Figure 4F).
We found that CPSF6, TMCC1 and PHTF2 had an increased expression in paired ( Figure S3A) and unpaired LAC tissues ( Figure 5B).
Pearson correlation analysis showed that PHTF2 rather than CPSF6 and TMCC1 had a most obviously negative correlation with miR-30a-5p expression in LAC tissues ( Figure 5C and Figure S3B).
We further obtained a cut-off value (10.31), AUC (0.52), sensitivity (10.5%) and specificity (94.22%) of PHTF2 in LAC patients and divided the patients into high and low expression groups ( Figure   S4A). As shown in Table S8, high expression of PHTF2 had no association with the clinicopathological factors in LAC patients (each P > .05). Kaplan-Meier analysis demonstrated that the patients with high PHTF2 expression had a lower survival (P = .042) but showed no difference in tumour recurrence (P = .615; Figure S4B).
Univariate and multivariate analysis uncovered that high PHTF2 expression was not an independent prognostic factor of poor survival in LAC patients (Tables S9). The binding sites between miR-30a-5p and the WT or Mut PHTF2 3'UTR are demonstrated in Figure 5D. We cotransfected A549 and NCI-H460 cell lines with WT or Mut PHTF2 3'UTR and miR-30a-5p mimic or inhibitor, and found that miR-30a-5p mimic decreased the luciferase activity of WT PHTF2 3′UTR, while miR-30a-5p inhibitor showed the opposite effects. Both of miR-30a-5p mimic and inhibitor had no impact on the luciferase activity of Mut PHTF2 3'UTR as compared with the control group ( Figure 5E). qRT-PCR and Western blot analysis showed that overexpression of DLEU2 increased the expression levels of PHTF2, and miR-30a-5p reversed DLEU2-induced PHTF2 expression ( Figure 5F). Knockdown of DLEU2 inhibited the expression levels of PHTF2, and miR-30a-5p inhibitor reversed this effect ( Figure 5G). qRT-PCR analysis also showed that the expression levels of lncRNA DLEU2 were reduced in sh-DLEU2 group as compared with the sh-NC group ( Figure 6E).

| D ISCUSS I ON
Accumulating data show that down-regulation of lncRNA NKILA or up-regulation of XLOC_008466 and FAL1 is linked to lymph node metastasis and TNM stage, 12,13,15 and lncRNA AFAP1-AS1, linc00673 and PVT1 predict a poor prognosis in NSCLC. 10,16,17 lncRNA DLEU2 is identified to have a differential expression in LAC, 20 acute myeloid leukaemia 21 and laryngeal carcinoma. 22 Herein, we found that lncRNA DLEU2 expression levels were remarkably elevated in NSCLC tissue samples, but had no association with the clinicopathological parameters. High expression of lncRNA DLEU2 was an independent prognostic factor of poor survival in patients with NSCLC.
Functionally, lncRNA DLEU2 inhibits the proliferation, migration and invasion of laryngeal carcinoma 22 and limits lymphocytic leukaemia. 23 However, we found that lncRNA DLEU2 promoted the proliferation and invasion of NSCLC cells, but inhibition of lncRNA DLEU2 reversed these effects. PCNA and MMP-2, tumour proliferation and invasion related markers, act as the poor prognostic factors in NSCLC. 24 We found that lncRNA DLEU2 induced their expression levels and acted as an oncogene in NSCLC.
Mounting evidence shows that miR-30a-5p represses tumour progression by targeting E2F7 26 or IGF1R. 27 Reduced expression of miR-30a-5p is a poor indicator in breast cancer. 28 In accordance, we found that miR-30a-5p was down-regulated in NSCLC tissues, and decreased expression of miR-30a-5p was associated with poor survival and tumour recurrence in patients with NSCLC. PHTF2 was further identified as a direct target of miR-30a-5p in NSCLC cells and harboured a negative correlation with miR-30a-5p expression in NSCLC tissues. MiR-145-5p also had a negative correlation with lncRNA DLEU2 expression and attenuated lncRNA DLEU2-induced cell proliferation and PHTF2 expression in NSCLC cells. Our results suggested that lncRNA DLEU2 acted as a sponge of miR-30a-5p to promote PHTF2 expression, leading to the tumorigenesis of NSCLC ( Figure 7).
Taken together, lncRNA DLEU2 facilitates the tumorigenesis and invasion of NSCLC by sponging miR-30a-5p and provides a potential biomarker for predicting the survival of NSCLC.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.
F I G U R E 7 Schematic representation of the underlying mechanism of lncRNA DLEU2/miR-30a-5p/PHTF2 axis in NSCLC. DLEU2 sponged miR-30a-5p, and thereby up-regulated PHTF2 expression, contributing to the tumorigenesis of NSCLC Xiang Guo and Tiancheng Zhao carried out the statistical analysis.
Weiming Wu wrote the paper, and Weiwei He revised the paper. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used to support the findings of this study are available from the corresponding authors upon request.