EGFR-AS1 Promotes Nonsmall Cell Lung Cancer (NSCLC) Progression via Downregulating the miR-524-5p/DRAM1 Axis and Inhibiting Autophagic Lysosomal Degradation

Nonsmall cell lung cancer (NSCLC) accounts for the majority of lung cancers. Studies have revealed the regulatory role of lncRNAs in cancer pathogenesis and their potential use as diagnostic and prognostic biomarkers. The epidermal growth factor receptor antisense RNA 1 (EGFR-AS1) has been reported to be upregulated in NSCLC tissues, while its detailed mechanism in lung cancer needs to be explored. DNA damage-regulated autophagy modulator 1 (DRAM1) has been known to act as a tumor suppressor in NSCLC, and miR-524-5p has been reported to be a biomarker in idiopathic pulmonary fibrosis and different lung disorders. Our investigation revealed that EGFR-AS1 is highly expressed in lung cancer tissues, and its knockdown inhibited lung cancer cell invasion and viability and reduced tumor growth in vivo. We also found that EGFR-AS1 targets miR-524-5p, and there was a negative correlation between their expressions in lung cancer tissues. Simultaneously, miR-524-5p has been found to promote DRAM1 expression. In addition, the inhibition of miR-524-5p diminished DRAM1 protein expression and promoted lung cancer cell invasion. Our study has revealed that EGFR-AS1 contributes to the pathogenesis of NSCLC by inhibiting autophagic-lysosomal degradation via targeting the miR-524-5p/DRAM1 axis. This finding elucidated for the first time the role of EGFR-AS1 in lung cancer progression and the positive regulatory function of miR-524-5p in regulating DRAM1 protein and suppressing lung cancer progression. This novel mechanism provided a better insight into the pathogenesis of lung cancer and presented a better strategy for the treatment of lung cancer.


Introduction
Lung cancer is explicated as small cell lung cancer or nonsmall cell lung cancer (NSCLC), where NSCLC accounts for the majority of lung cancers and is a leading global cause of cancer-related deaths [1,2]. NSCLC is a heterogeneous disorder, with various subtypes representing different clinical indexes requiring different treatment strategies. Distinct clinical outcomes accompany these different histological subtypes, disclosing heterogeneity in disease aggressiveness and underlying prognostic alterations [3][4][5].
Indeed, complex cellular signaling and tumor microenvironment factors are associated with poor prognosis, imparting a distinctive biological basis to an individual's disorder [1]. e identification of oncogenic driver modulations has helped ameliorate the outcomes in lung cancer patients. However, most lung cancer patients do not have an actionable molecular abnormality [6,7]. erefore, identifying new biomarkers and alternative treatments is of great necessity.
MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that positively or negatively regulate gene expression [25]. Riveting evidence has elucidated the distinctly dysregulated miRNA expression in human cancers, including deletion or amplification of miRNA genes, dysregulated epigenetic changes, transcriptional control of miRNAs, and the flawed miRNA biogenesis mechanism [26]. miR-524-5p was reported to be essential in the pathogenesis of gliomas [27]. It has been recently found to be highly expressed in idiopathic pulmonary fibrosis (IPF) and can be utilized as a biomarker for the diagnosis and prognosis of IPF [28]. is finding indicated that miR-524-5p could potentially have a role in the pathogenesis of other lung-related disorders.
DNA damage-regulated autophagy modulator 1 (DRAM1) is known to induce autophagy and is downregulated in multiple human cancers [29]. In the presence of growth factors, DRAM1 regulates the activation of the IGF-1 receptor and inhibits the downstream PI3K-AKT-mTOR pathway, promoting autophagy activation and suppressing cell proliferation in various human cancers [30]. DRAM1 was reported to be decreased in NSCLC and was negatively correlated with EGFR levels. In addition, DRAM1 overexpression inhibited the proliferation, invasion, migration, and EMT of NSCLC cell lines harboring mutant EGFR in vitro and in vivo [31]. Nevertheless, the underlying mechanism of DRAM1 in the pathogenesis of NSCLC remains unclear.
e mechanism of EGFR-AS1 and miR-524-5p in lung cancer requires detailed analysis. On the other hand, the role of DRAM1 in lung cancer remains unclear. e present study identified EGFR-AS1/miR-524-5p/DRAM1 as a novel signaling pathway associated with lung cancer progression and comprehended the mechanism of miR-524-5p and DRAM1 in lung cancer pathogenesis.

Clinical Samples.
Eighty pairs of cancerous and adjacent normal lung tissues were collected from patients diagnosed with lung cancer at Jinan Central Hospital Affiliated to Shandong University. e tissue specimens were collected postsurgical resection and promptly transferred and stored in liquid nitrogen. All patients had written informed consent.
is study was approved by Jinan Central Hospital Affiliated to Shandong University.

Cell Lines and Cell Culture.
e human nontumorigenic lung epithelial cell line BEAS-2B was purchased from the American Type Culture Collection (ATCC) and was cultured in BEBM complete medium supplemented with 10% FBS and incubated at 37°C and 5% CO 2 atmosphere along with penicillin (100 U/ml) and streptomycin (100 mg/ml, HyClone, USA). e adenocarcinoma lung cancer cell lines HCC827 and NCI-H1650 were purchased from ATCC and were cultured in RPMI-1640 medium supplemented with 10% FBS and incubated at 37°C and 5% CO 2 atmosphere along with penicillin (100 U/ml) and streptomycin (100 mg/ ml, HyClone, USA) at 37°C with 5% CO 2 .

Cell
Transfection. HCC827 and NCI-H1650 cells were transfected with constructed lentivirus vectors by RiboBio (Guangzhou, China) to knockdown the expression of miR-524-5p. According to the manufacturer's protocols, the cells were transfected accordingly using Lipofectamine 2000 transfection reagents (Invitrogen, Shanghai, China). e cell culture medium was replaced with a fresh medium after 24 h. In addition, cells were transfected with pcDNA3.1-si-EGFR-AS1 or miR-524-5p inhibitor (GenePharma, Shanghai, China). e empty vectors in this experiment were considered as the negative control. Lentiviruses (lv-shEGFR-AS1 and lv-oeEGFR-AS1) were purchased from Shanghai Heyuan Biotechnology and were transduced into HCC827 cells as previously described [32].

Cell Viability and Invasion Assays.
e cell viability of HCC827 and NCI-H1650 cells was measured using the CCK-8 reagent (Sigma-Aldrich, St. Louis, MO, USA). ree thousand cells/well were seeded into the 96-well plate and then cultured for 24, 48, and 96 h. Cell viability was measured by detecting the absorbance of cultured cells using a microplate reader at 450 nm after the addition of CCK-8 for two hours. e invasion ability of HCC827 and NCI-H1650 cells was assayed using the Matrigel-coated Transwell (Corning, MA, USA). Cells (2 × 10 4 ) in 200 μL of serum-free medium were added into the upper Transwell chambers. e bottom chamber was added with the complete medium. After incubation for 48 h in a humidified atmosphere (37°C and 5% CO 2 ), cells on the upper surface of the membrane were completely removed using a cotton swab. e remaining cells were fixed with methanol and stained for 20 min with 0.1% crystal violet. e stained cells were counted and assessed from five randomly selected fields under a microscope, and the data were presented from triplicate experiments.

Luciferase Report Assay.
e binding of miR-524-5p to DRAM1 as well as their binding sequences were predicted from the miRDB database [33]. e wild-type EGFR-AS1 or DRAM1 3′untranslated region (3′UTR) sequences, which have binding sites with miR-524-5p, were inserted into the pmirGLO vector (Promega, Madison, WI, USA) to construct the WT-EGFR-AS1 or WT-DRAM1 vector. e mut-EGFR-AS1 or mut-DRAM1 vector was also constructed by inserting the mutated binding sequences into the pmirGLO vector. HCC827 and NCI-H1650 cells were cotransfected with luciferase reporter vectors and miR-524-5p mimic or negative vectors. Lastly, the relative luciferase activities were measured with a Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) after 48 h of transfection.

In Vivo Xenograft Model.
e present in vivo experiments were approved by the Animal Research Ethics Committee of the Jinan Central Hospital Affiliated to Shandong University. e 5-week-old female BALB/c nude mice were purchased from Charles River (Beijing, China) and subsequently randomly divided into the designated groups consisting of five mice in each group. HCC827 cells (2 × 10 7 ) were injected subcutaneously into the right flank of each mouse. e tumor size was calculated and recorded once a week. Six weeks later, the tumors were extracted, photographed, and weighed.
2.9. Statistical Analysis. All data presented were analyzed for statistical significance using the Student's t-test and analysis of variance (ANOVA). Spearman's correlation analysis was used for expression correlation analysis in NSCLC specimens. Data in this study were obtained from three independent experiments and were presented as mean ± standard deviation (S.D). e software GraphPad Prism 8.0 (San Diego, CA) was used for all statistical analyses, and a p < 0.05 was considered significant.

Silencing EGFR-AS1 Inhibited Lung Cancer Cell Viability and Invasion and Reduced Tumor Size In Vivo.
We compared the expression level of EGFR-AS1 in normal lung cells and tumor cells. EGFR-AS1 is upregulated in HCC827 and NCI-H1650 lung cancer cells compared to normal BEAS-2B cells (Figure 1(a)). EGFR-AS1 was highly expressed in tumor tissues compared to healthy tissues (Figure 1(b)). EGFR-AS1 was more prominent in the cytoplasm in HCC827 and NCI-H1650 cells than in the nucleus (Figure 1(c)). We knocked down EGFR-AS1 expression in NSCLC cells, and it significantly inhibited its expression compared to the negative control group (Figure 1(d)). Next, we determined the effect of silencing EGFR-AS1 on cell viability and invasion. We found that silencing EGFR-AS1 significantly inhibited the viability and invasion of HCC827 and NCI-H1650 cells (Figures 1(e) and 1(f )). Simultaneously, silencing EGFR-AS1 reduced tumor size in nude mice bearing xenografts (Figure 1(g)).

EGFR-AS1 Expression Is Negatively Correlated with the
Expression of miR-524-5p. We utilized in silico analysis to predict if there could be a potential relation/interaction between EGFR-AS1 and miR-524-5p. As shown in Figure 2(a), EGFR-AS1 and miR-524-5p have been shown to interact potentially. Comparing the enrichment levels of EGFR-AS1 and miR-524-5p, EGFR-AS1 is more enriched than miR-524-5p in HCC827 and NCI-H1650 cells (Figure 2(b)). To confirm the binding between EGFR-AS1 and miR-524-5p, we conducted a luciferase activity assay. Luciferase activity of wt-EGFR-AS1 was suppressed by miR-524-5p mimics, while that of mut-EGFR-AS1 showed no significant response (Figure 2(c)). On the other hand, the expression of miR-524-5p was found to be higher in healthy lung cells and tissues than in control cells and tissues (Figures 2(d) and 2(e)). Moreover, we found that EGFR-AS1  Comparison of the different relative expressions of EGFR-AS1 and miR-524-5p enriched in Ago2 in NSCLC cells. e unpaired-student t test was applied. (c) e binding of miR-524-5p to EGFR-AS1 in NSCLC cells was assessed by luciferase reporter assay. e unpaired-student t test was applied. (d) e expression level of miR-524-5p in normal lung cells and NSCLC cells. One-way ANOVA was applied. (e) e expression level of miR-524-5p in 50 samples of normal lung tissues and NSCLC tissues. e paired-student t test was applied. (f) A negative correlation was found between miR-524-5p and EGFR-AS1 in NSCLC samples. Spearman's correlation analysis was conducted. e data are represented as mean ± S.D.

miR-524-5p Expression Suppressed Lung Cancer Cell
Viability and Invasion. We analyzed the role of miR-524-5p in the progression of lung cancer cells. We found that silencing EGFR-AS1 promoted the expression of miR-524-5p while miR-524-5p inhibited its expression (Figure 3(a)). Inhibiting miR-524-5p increased lung cancer cell viability, while silencing EGFR-AS1 decreased it compared to the negative control group. ese effects were rescued by the combination of si-EGFR-AS1 and miR-524-5p inhibitor (Figure 3(b)). We determined the effect of inhibiting miR-524-5p on cell invasion. Our results indicated that compared to the negative control group, inhibiting miR-524-5p significantly increased lung cancer cell invasion while silencing EGFR-AS1 significantly inhibited cell invasion. ese effects were rescued by si-EGFR-AS1+miR-524-5p inhibitor (Figure 3(c)). ese results indicated the correlation between EGFR-AS1 and miR-524-5p.

miR-524-5p Negatively Correlated with the Expression of DRAM1 in Lung Cancer Cells.
To identify the potential target of miR-524-5p, we utilized in silico analysis to recognize the potential targets of miR-524-5p (Figure 4(a)). miR-524-5p reduced luciferase activity of DRAM1-wt and had no significant effects on that containing DRAM1-mut (Figure 4(b)). Knowing that DRAM1 is downregulated in lung cancer cells, we confirmed these observations in vitro and in vivo; we found that DRAM1 is upregulated in NSCLC tissues compared with adjacent nontumor tissues. DRAM1 was upregulated in the HCC827 and NCI-H1650 cell lines than the control BEAS-2B cells (Figures 4(c) and 4(d)). Finally, we found that the expression of miR-524-5p was negatively correlated with DRAM1 expression (Figure 4(e)). is result indicated a possibility that miR-524-5p could have a regulatory role on DRAM1.

e miR-524-5p/DRAM1 Axis Is Vital for Lung Cancer
Cell Biological Processes. We further investigated the role of DRAM1 in lung cancer cell biological processes. Western blot analysis showed that both pcDNA-DRAM1 and miR-524-5p inhibitors increased DRAM1 protein expression ( Figure 5(a)). Concerning the role of DRAM1 in autophagy, we examined the autophagic signaling pathway and found that the inhibition of miR-524-5p and overexpression of Journal of Oncology DRAM1 suppressed SQSTM1, Beclin-1, and LC3 protein expression compared to the negative control groups ( Figure 5(b)). CCK-8 and transwell assays showed that overexpression of DRAM1 and inhibition of miR-524-5p significantly promoted lung cancer cell viability and invasion compared to the negative control groups (Figures 5(c) and 5(d)).

Discussion
Globally, the lung cancer mortality rate is very high. NSCLC represents most of the disease despite improving NSCLC management [34,35]. is burden turned scientists' attention toward lncRNAs as a potential therapeutic target in the treatment of NSCLC, which play essential roles in gene expression and signaling pathways [36][37][38]. Recently, lncRNAs have been dysregulated in different cancer types, resulting in aberrant cell functions. ey could act as tumor suppressors and oncogenes in different cancer types [12,39]. EGFR-AS1 is engaged in the progression of lung cancer [24]. EGFR-AS1 has been reported to be upregulated in lung cancer and has been significantly correlated with the poor survival of lung cancer patients [24]. After knockdown, the EGFR-AS1 gene expression profiling gives more unbiased information concerning pathways at the transcriptional levels. e knockdown of EGFR-AS1 has been shown to suppress lung cancer cell migration, invasion, and proliferation [24]. Our experimental results confirmed the previously reported findings. We have confirmed that the knockdown of EGFR-AS1 inhibited cell viability in HCC827 and NCI-H1650 cell lines and reduced tumor size in vivo in nude mice models bearing the HCC827 cells.
miRNAs can sustain proliferative signaling, evade growth suppressors, avoid immune destruction and tumors, promote inflammation, resist cell death mechanisms, deregulate cell energetics, activate invasion and metastasis, and induce angiogenesis [40]. Increased attention has been drawn toward miR-524 in human cancers. Its downregulation was reported to suppress angiogenesis in colon cancer and promote cell proliferation in osteosarcoma [41,42]. It could be used as a biomarker for the early diagnosis of idiopathic pulmonary fibrosis [28]. Moreover, the overexpression of miR-524-5p is associated with a poor prognosis of patients with idiopathic pulmonary fibrosis e data are represented as mean ± S.D. * p < 0.05, * * p < 0.01, and * * * p < 0.001. [28]. Considering the previously reported findings [24,28,31], we examined the role of miR-524-5p in HCC827 and NCI-H1650 cell lines and found that the enrichment of miR-524-5p was significantly lower than EGFR-AS1, and that they were inversely expressed in HCC827 and NCI-H1650 cell lines. Subsequently, the inhibition of miR-524-5p promoted the viability and invasion of HCC827 and NCI-H1650 cell lines.
ese results indicated that the lncRNA EGFR-AS1 could have a regulatory role over miR-524-5p.
Emerging evidence suggested that DRAM1 is engaged in the biological functions of cancer cells [30,43]. DRAM1 has been found to play a tumor suppressor role in NSCLC [31]. However, DRAM1 expression and clinical significance in lung cancer have not been elucidated. Considering the vital role of miR-524-5p in lung cancer prognosis [28], we explored its potential in regulating DRAM1 expression. We have found that miR-524-5p decreased DRAM1 expression.
ere was a negative expression correlation between miR-524-5p and DRAM1. Inhibiting miR-524-5p or Relative gray density ****** ****** ******  Figure 5: e miR-524-5p/DRAM1 axis is vital for lung cancer cell biological processes. (a) Western blotting analysis was utilized to determine the effect of miR-524-5p inhibition on DRAM1 protein expression in NSCLC cells. One-way ANOVA was applied. (b) Western blot analysis was utilized to determine the effect of miR-524-5p inhibition on autophagy-related protein expression in NSCLC cells. Oneway ANOVA was applied. (c) e CCK-8 assay was used to determine the effect of miR-524-5p inhibition and DRAM1 knockdown on NSCLC cell viability. Two-way ANOVA was applied. (d) e effect of inhibiting miR-524-5p and DRAM1 on NSCLC cell invasion. One-way ANOVA was applied. e data are represented as mean ± S.D. * * p < 0.01.
overexpressing DRAM1 decreased autophagy-related proteins and increased viability and invasion in NSCLC cells.
Our results suggested that EGFR-AS1 could regulate NSCLC progression by the DRAM1/miR-524-5p axis. For the first time, we reported DRAM1 as a potential target of miRNA-524-5p and elucidated its role in lung cancer. e present study has some limitations. We shall investigate the role of EGFR-AS1 concerning the miR-524-5p/DRAM1 axis in vivo and provide a detailed analysis of the underlying mechanism. We also need to validate our results regarding cell death mechanisms.

Conclusion
We spotlighted the vital role of EGFR-AS1 in NSCLC progression and the role of miR-524-5p in regulating DRAM1. For the first time, our results have practically elucidated that EGFR-AS1 contributes to the progression of NSCLC via inhibiting the miR-524-5p/DRAM1 axis. Our findings set forth a novel mechanism of the progression of NSCLC, providing a newly identified target for lung cancer treatment.

Data Availability
Data supporting this research article are available from the corresponding author on reasonable request.
Ethical Approval e experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Jinan Central Hospital Affiliated to Shandong University.

Conflicts of Interest
e authors declare no conflicts of interest in this study.