Knockdown of lncRNA DLX6-AS1 inhibits cell proliferation, migration and invasion while promotes apoptosis by downregulating PRR11 expression and upregulating miR-144 in non-small cell lung cancer

Biomedicine & Pharmacotherapy 109 (2019) 1851–1859

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Knockdown of lncRNA DLX6-AS1 inhibits cell proliferation, migration and invasion while promotes apoptosis by downregulating PRR11 expression and upregulating miR-144 in non-small cell lung cancer

T

Yongjie Huanga, Ran Nib, Jing Wangb, , Ying Liuc ⁎

a

Department of Senile Respiratory and Sleep, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China Department Two of Respiratory Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China c Department Five of Respiratory Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China b

ARTICLE INFO

ABSTRACT

Key words: DLX6-AS1 miR-144 PRR11 Proliferation NSCLC

Background: Long non-coding RNA (lncRNA) distal-less homeobox 6 antisense 1 (DLX6-AS1) was reported to be dysregulated in lung cancer. However, detailed roles of DLX6-AS1 in the pathogenesis of non-small cell lung cancer (NSCLC) were largely unknown. Methods: The expression of DLX6-AS1 was measured in NSCLC tissues and cells by quantitative real-time PCR (qRT-PCR). The abundance of proline rich 11 (PRR11) were detected by qRT-PCR and western blot, respectively. The effects of DLX6-AS1 and PRR11 on cell proliferation, migration, invasion and apoptosis were explored by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), transwell and flow cytometry analysis, respectively. Luciferase reporter assay, qRT-PCR and western blot were performed to confirm the interaction between miR-144 and DLX6-AS1 or PRR11. Tumor xenograft assay was performed to verify the role of DLX6-AS1 in NSCLC in vivo. Results: DLX6-AS1 and PRR11 were elevated in NSCLC tissues and cells. DLX6-AS1 was positively correlated with PRR11 mRNA expression in NSCLC tissues. Knockdown of DLX6-AS1 and PRR11 significantly suppressed cell proliferation, migration and invasion and induced apoptosis in NSCLC cells, which was reversed by PRR11 overexpression. In addition, DLX6-AS1 and PRR11 were demonstrated to interact with microRNA-144 (miR-144) and DLX6-AS1 upregulated PRR11 expression by acting as a competing endogenous RNA (ceRNA) of miR-144 in NSCLC cells. Furthermore, DLX6-AS1 knockdown suppressed tumor growth in NSCLC in vivo by upregulating miR-144 and downregulating PRR11. Conclusion: Knockdown of DLX6-AS1 inhibited cell proliferation, migration, invasion and promoted apoptosis by downregulating PRR11 expression and upregulating miR-144 in NSCLC.

1. Introduction Lung cancer is one of the most common devastating malignancies with the leading cause of cancer-associated mortality worldwide [1]. Non-small cell lung carcinoma (NSCLC) is the most common histopathological type accounting for approximately 85% of all lung cancer cases and generally diagnosed at an advanced stage [2]. It has been proposed that the low 5-year survival rate (about 15%) is largely attributable to advanced local invasion and/or distant metastasis, and recurrence of NSCLC [3,4]. Although the enormous improvement has been made in early diagnosis and clinical treatment strategies of NSCLC, the prognosis of the patients with NSCLC is still dismal [3].

Compelling evidence reveals that the tumorigenesis and progression for NSCLC are complicated processes through accumulation of multiple epigenetic and genetic alterations [5,6]. Therefore, understanding the molecular mechanism underlying NSCLC is crucial to identify better therapeutic targets and strategies for the treatment of NSCLC. Long non-coding RNAs (lncRNAs) are most commonly defined as non-coding RNAs (ncRNAs) that are more than 200 nucleotides in length and lack of protein-coding capacity, being able to regulate gene expression at transcriptional, posttranscriptional and epigenetic levels [7]. It has been well acknowledged that lncRNAs play functional roles in diverse biological processes, such as cell differentiation, proliferation, invasion and migration [8]. Accumulating evidence has suggested

⁎ Corresponding author at: Department Two of Respiratory Medicine, Floor 8, Building 3, the First Affiliated Hospital of Zhengzhou University, No.1, East Jianshe Rd, Zhengzhou, 450052, China. E-mail address: [email protected] (J. Wang).

https://doi.org/10.1016/j.biopha.2018.09.151 Received 6 July 2018; Received in revised form 17 September 2018; Accepted 26 September 2018 0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Fig. 1. The expression of DLX6-AS1 and PRR11 in NSCLC tissues. qRT-PCR analysis of DLX6AS1(A) and PRR11 mRNA (B) in NSCLC tissue samples (n = 48) and adjacent normal tissues (n = 48). (C) Western blot analysis of PRR11 protein level in NSCLC tissue samples and adjacent normal tissues. (D) The correlation between DLX6-AS1 and PRR11 mRNA expression in NSCLC tissues. *P < 0.05.

the important roles of dysregulated lncRNAs in tumorigenesis and progression of various cancers, including NSCLC [9,10]. Interestingly, lncRNA distal-less homeobox 6 antisense 1 (DLX6-AS1) has been reported to be ectopic and high expression of DLX6-AS1 was significantly associated with histological differentiation and TNM stage in lung adenocarcinoma tissues [11]. However, the detailed roles of DLX6-AS1 and molecular mechanism in the pathogenesis of NSCLC were elusive. MicroRNAs (miRNAs) are a class of endogenous, small ncRNAs with 18 to 25 nucleotides in length, which result in mRNAs degradation or translational inhibition by base pairing to the 3′ untranslated region (3′UTR) of the target mRNAs [12]. MiRNAs are well-known to participate in the regulation of various cellular biological processes, such as tumorigenesis and cancer metastasis [13,14]. MiR-144 was previously documented to be downregulated in NSCLC tissues, overexpression of which inhibited NSCLC tumor cell growth and induced apoptosis [15,16]. However, the molecular mechanism of miR-144 in NSCLC is far from being addressed. Proline rich 11 (PRR11), a newly discovered gene encoding a 360amino acid protein, is located on human chromosome 17q22 [17]. PRR11 was first identified during a screen for novel cancer-associated genes [18]. It has been suggested that PRR11 was abnormally upregulated in various tumors and its high expression was associated with tumor development and progression, including lung cancer [19,20]. Therefore, we hypothesized miR-144 and PRR11 might be required for DLX6-AS1-mediated progression of NSCLC. In our study, we measured the expressions of DLX6-AS1 and PRR11, and then investigated the functional roles of DLX6-AS1 and PRR11 as well as the underlying regulatory mechanism in NSCLC tissues and cells.

2. Materials and methods 2.1. Patients and tissue samples A total of 48 paired NSCLC tissues and adjacent normal tissues were obtained from patients with NSCLC undergoing surgery at the First Affiliated Hospital of Zhengzhou University between January 2015 and March 2016. Patients with NSCLC were diagnosed based on histopathological evaluation and none of these patients received any chemotherapy or radiotherapy prior to surgery. All the collected tissue samples were immediately snap frozen in liquid nitrogen and then stored at −80 °C until use. This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University and written informed consent was obtained from all patients recruited. 2.2. Cell culture and transfection NSCLC cell lines (H1975 and A549) and human bronchial epithelial cell line (16HBE) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin and 100 U/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Small interfering RNA (siRNA) against DLX6-AS1 (si-DLX6-AS1), siRNA against PRR11 (si-PRR11), siRNA control (si-Control), shorthairpin RNA plasmid targeting DLX6-AS1 (sh-DLX6-AS1), pcDNA control, pcDNA-PRR11, miR-144 mimics (miR-144), and miRNA control (miR-control) were designed and synthesized by GenePharma (Shanghai, China). Transient transfection into H1975 and A549 cells 1852

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Fig. 2. The effects of DLX6-AS1 knockdown on cell proliferation, migration, invasion and apoptosis in NSCLC cells. (A) The expression of DLX-AS1 in NSCLC cell lines (H1975 and A549) and human bronchial epithelial cell line (16HBE) by qRT-PCR analysis. MTT assay was conducted to detect cell proliferation at 24 h, 48 h and 72 h in H1975 (B) and A549 (C) cells transfected with si-DLX6-AS1 or si-Control. Cell migration was investigated in si-DLX6-AS1-transfected H1975 (D) and A549 (E) cells. Cell invasion assay was carried out to evaluate the invasion abilities of H1975 (F) and A549 (G) cells introduced with si-DLX6-AS1 or si-Control. Flow cytometry analysis was employed to analyze the apoptosis of H1975 (H) and A549 (I) cells transfected with si-DLX6-AS1 or si-Control. *P < 0.05.

was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested at 48 h posttransfection.

then electroblotted onto nitrocellulose membranes (Millipore, Madison, WI, USA). After being saturated for 2 h with 5% skim milk in Trisbuffered saline containing 0.1% Tween-20 (TBST), the membranes were incubated with the primary antibody against PRR11 (Abcam, Cambridge, UK) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Finally, the protein signals were detected using enhanced chemiluminescence detection system (Pierce Biotechnology, Rockford, IL, USA).

2.3. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated from tissues or cultured cells using TRIzol reagent (Thermo Fisher Scientific). Total RNA was reverse transcribed into the first-strand cDNA using the Reverse Transcription System Kit (Takara, Dalian, China). For DLX6-AS1 and PRR11 mRNA quantification, qRT-PCR was performed using SYBR Premix Ex Taq (Takara) on the ABI PRISM® 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA), with GAPDH as an endogenous control. For miR-144 detection, qRT-PCR was conducted using TaqMan miRNA assays (Applied Biosystems) on the ABI PRISM® 7300 real-time PCR system (Applied Biosystems), with U6 small nuclear RNA (snRNA) as an internal control. PCR reaction conditions were performed as follows: denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, annealing at 60 °C for 1 min, and a final extension at 75 °C for 30 s. The relative gene expression was calculated by the 2−ΔΔCt method.

2.5. Cell proliferation assay Cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, H1975 and A549 cells were inoculated into 96-well plates at a density of 2 × 104 cells/well and then transfected with si-DLS6-AS1, si-PRR11, si-Control, si-DLS6-AS1 + pcDNA-PRR11, or si-DLS3-AS1 + pcDNA-Control, followed by incubation for 48 h. Subsequently, 20 μL MTT (5 mg/mL, Sigma-Aldrich) was added into each well and incubated for 4 h in a humidified atmosphere containing 5% CO2 at 37 °C. The reaction was then terminated by removal of the supernatant and 150 μL of dimethylsulfoxide (DMSO) solution was added to dissolve the purple formazan crystals. The optical density was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.4. Western blot Protein samples were extracted from tissues or cultured cells using RIPA lysis buffer (Beyotime, Jiangsu, China) containing 1% proteinase inhibitor (Sigma-Aldrich, St Louis, MO, USA) and quantified using a bicinchoninic acid (BCA) protein quantification kit (Beyotime). Equal doses of protein lysates (25 μg each lane) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 1853

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Fig. 3. The effects of PRR11 knockdown on NSCLC cell proliferation, migration, invasion and apoptosis. (A and B) The mRNA and protein levels of PRR11 in H1975, A549, and 16HBE cells were detected by qRT-PCR and western blot. PRR11protein level in H1975 (C) and A549 (D) cells transfected with si-Control or si-PRR11 was estimated by western blot. MTT assay was conducted to detect cell proliferation at 24 h, 48 h, and 72 h in H1975 (E) and A549 (F) cells transfected with si-Control or si-PRR11. Cell migration was measured in H1975 (G) and A549 (H) cells after treatment with si-Control or si-PRR11. Cell invasion assay was carried out to determine invasion ability of H1975 (I) and A549 (J) cells after treatment with si-Control or si-PRR11. Flow cytometry analysis was conducted to evaluate apoptosis of H1975 (K) and A549 (L) cells with si-Control or si-PRR11 transfection. *P < 0.05.

2.6. Transwell assay

buffer. Then the cells were stained with 10 μl Annexin V/FITC for 10 min and 5 μl PI for 5 min in the dark at room temperature. The apoptotic cells were analyzed with a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with CellQuest software (BD Biosciences).

Cell migration and invasion were determined by transwell assay. For invasion assay, transwell chambers (8 μm, Corning, Inc., Corning, NY, USA) were pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, the transfected H1975 and A549 cells resuspended in serum-free medium were seeded in the upper transwell chamber, while 500 μl DMEM containing 10% FBS was added into the lower chamber as the chemoattractant. Following incubation at 37 °C for 24 h, cells remaining on the upper chamber were wiped out using a cotton swab and cells that had invaded to the bottom chamber were fixed in 100% methanol and stained with 0.05% crystal violet (SigmaAldrich). The number of invasive cells was counted from 5 randomly selected fields and imaged using an inverted microscope (Olympus Corp., Tokyo, Japan). Cell migration was investigated in transwell chambers without matrigel following the similar instructions.

2.8. Luciferase reporter assay The wild-type or mutated fragment of DLX6-AS1 and 3′UTR of PRR11 containing the miR-144 binding sites were synthesized by Genescript (Nanjing, Jiangsu, China) and cloned into the downstream of the luciferase gene of pmirGLO luciferase reporter vector (Promega, Madison, WI, USA). H1975 and A549 cells (3 × 104 cells per well) were seeded into 24-well plates and then transfected with the constructed luciferase reporter vectors and miR-144 or miR-Control using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) at 48 h posttransfection. Firefly luciferase activity was normalized to Renilla luciferase activity.

2.7. Flow cytometry analysis Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BestBio, Shanghai China). Briefly, the transfected H1975 and A549 cells were harvested, digested and resuspended in 100 μL binding

2.9. Tumor xenograft formation assay All animal procedures in this study were manipulated with the 1854

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Fig. 4. PRR11 overexpression reversed the effects of DLX6-AS1 knockdown on cell proliferation, migration, invasion, and apoptosis in NSCLC cells. H1975 and A549 cells were transfected with si-DLX6-AS1, si-Control, si-DLX6-AS1 + pcDNA-PRR11, or si-DLX6-AS1 + pcDNA-Control, followed by incubation for 48 h. The abundance of PRR11 was measured in transfected H1975 (A) and A549 (B) cells. MTT assay was performed to detect cell proliferation at 24 h, 48 h, 72 h in the transfected H1975 (C) and A549 (D) cells. Cell migration was investigated in H1975 (E) and A549 (F) cells after si-DLX6-AS1 or (and) pcDNA-PRR11 ransfection. Cell invasion assay was applied to evaluate the invasion ability of the treated H1975 (G) and A549 (H) cells. Flow cytometry analysis was conducted to analyze the apoptosis rates of the introduced H1975 (I) and A549 (J) cells. *P < 0.05.

approval of the Animal Research Committee of the First Affiliated Hospital of Zhengzhou University. Four-week-old male BALB/c nude mice were purchased from the Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China) and maintained under specific pathogen-free conditions. A549 cells (5 × 106) stably transfected with pcDNA-DLX6-AS1 or sh-DLX6-AS1 were subcutaneously injected into the posterior flank of the nude mice. After 7 days of injection, tumor volumes were measured every 3 day using calipers and calculated using the following formula: volume (mm3) = width2 × length/2. After 22 days following the injection, the mice were killed and tumor tissues were excised and weighted. The excised tumor tissues were harvested for qRT-PCR and western blot analysis.

3. Results 3.1. DLX6-AS1 and PRR11 were significantly upregulated in NSCLC tissues We initially detected the expressions of DLX6-AS1 and PRR11 in 48 NSCLC tissues. As demonstrated by qRT-PCR, DLX6-AS1 (Fig. 1A) and PRR11 mRNA (Fig. 1B) expressions were robustly enhanced in NSCLC tissues compared with that in the pair-matched normal tissues. Meanwhile, western blot analysis also demonstrated that the protein level of PRR11 was remarkably higher in NSCLC tissues than that in the corresponding adjacent normal tissues (Fig. 1C). Interestingly, we found that there was a positive correlation between DLX6-AS1 and PRR11 mRNA expression in NSCLC tissues (Fig. 1D). These above results suggested that dysregulated DLX6-AS1 and PRR11 might be involved in the development of NSCLC.

2.10. Statistical analysis All data were shown as mean ± standard deviation (SD) from three independent experiments. Statistical analysis was conducted using SPSS 13.0 statistical software (SPSS, Inc. Chicago, IL, USA). The significance of differences was estimated by Student’s t test or one-way analysis of variance (ANOVA). Differences were considered statistically significant when P value < 0.05.

3.2. DLX6-AS1 silencing suppressed proliferation, migration and invasion while induced apoptosis of NSCLC cells Due to the abnormal expression of DLX6-AS1 in NSCLC tissues, we further examined the expression profile of DLX6-AS1 in NSCLC cells. The results showed that DLX6-AS1 exhibited a marked upregulation in NSCLC cell lines (H1975 and A549) compared with human bronchial epithelial cell line (16HBE) (Fig. 2A). To further decipher the regulatory 1855

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Fig. 5. DLX6-AS1 upregulated PRR11 expression by acting as a ceRNA of miR-144 in NSCLC cells. (A) The wild-type or mutated miR-144 binding sites in DLX6-AS1. Luciferase reporter assay was conducted to detect the luciferase activity in H1975 (B) and A549 (C) cells after cotransfection with miR-Control or miR-144 and DLX6-AS1-WT or DLX6-AS1-MUT. (D) The wild-type or mutated miR-144 binding sites in the 3′UTR of PRR11. Luciferase reporter assay was performed to detect the luciferase activity in H1975 (E) and A549 (F) cells cotransfected with PRR11 3′UTR-WT or PRR11 3′UTR-MUT and miR-144 or miR-Control. The expression of miR144 in H1975 (G) and A549 (H) cells transfected with si-Control, si-DLX6AS1, pcDNA-Control, or pcDNA-DLX6-AS1 was measured by qRT-PCR. The protein level of PRR11 in H1975 (I) and A549 (J) cells transfected with miR144, miR-Control, miR-144 + pcDNA-Control, miR-144 + pcDNA-DLX6AS1 was examined by western blot. *P < 0.05.

role of DLX-AS1 in the development of NSCLC, we decreased DLX6-AS1 expression in H1975 and A549 cells by transfecting with si-DLX6-AS1. MTT assay showed that DLX6-AS1 depletion led to a remarkable

reduction of cell proliferation in H1975 (Fig. 2B) and A549 (Fig. 2C) cells compared with that in si-Control group. Moreover, knockdown of DLX6-AS1 inhibited cell migration compared with si-control treatment 1856

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Fig. 6. DLX6-AS1 knockdown suppressed tumor growth in NSCLC in vivo by upregulating miR-144 and downregulating PRR11. A549 cells stably transfected with pcDNA-DLX6-AS1 or sh-DLX6-AS1 were subcutaneously injected into the posterior flank of the nude mice. (A) After 7 days of injection, tumor volumes were measured every 3 day using calipers. (B) After 22 days following the injection, the mice were killed and tumor tissues were excised and weighted. (C) The expressions of DLX6-AS1 (C), miR-144 (D), and PRR11 at mRNA (E) and protein (F) levels in the resected tumor tissues were determined by qRT-PCR and western blot. *P < 0.05.

in H1975 (Fig. 2D) and A549 (Fig. 2E) cells. Cell invasion assay displayed that H1975 (Fig. 2F) and A549 (Fig. 2G) cells transfected with si-DLX6-AS1 exhibited an evident suppression of invasion abilities with respect to that in si-Control-introduced cells. Furthermore, flow cytometry analysis demonstrated that DLX6-AS1 knockdown drastically promoted apoptosis of H1975 (Fig. 2H) and A549 (Fig. 2I) cells relative to control group. Collectively, these results demonstrated that DLX6AS1 knockdown impeded the progression of NSCLC, revealed by the reduced proliferation, migration, invasion and increased apoptosis.

Together, these findings indicated that PRR11 knockdown restrained proliferation, migration, invasion and promoted apoptosis of NSCLC cells. 3.4. PRR11 overexpression reversed the effects of DLX6-AS1 knockdown on cell proliferation, migration, invasion and apoptosis in NSCLC cells To explore the relationship between DLX6-AS1 and PRR11 in NSCLC cells, rescue experiments were performed in H1975 and A549 cells by transfecting with si-DLX6-AS1, si-Control, si-DLX6-AS1 + pcDNA-PRR11, or si-DLX6-AS1 + pcDNA-Control. As a result, inhibition of DLX6-AS1 led to a loss of PRR11 level, whereas PRR11 overexpression supported the abundance at protein level in H1975 (Fig. 4A) and A549 cells (Fig. 4B). DLX6-AS1 knockdown led to an obvious suppression of cell proliferation in H1975 (Fig. 4C) and A549 (Fig. 4D) cells compared with si-Control, while increased PRR11 expression distinctly abrogated the inhibitory effect of DLX6-AS1 silencing on cell proliferation. In addition, addition of PRR11 abated the inhibitory effect of DLX6-AS1 on cell migration in H1975 (Fig. 4E) and A549 (Fig. 4F) cells. Moreover, a significant decrease of the invasion ability was shown in si-DLX6-AS1-transfected H1975 (Fig. 4G) and A549 (Fig. 4H) cells relative to si-Control-treated cells, which was remarkably restored by ectopic expression of PRR11. Besides, overexpression of PRR11 prominently abated DLX6-AS1 depletion-induced apoptosis in H1975 (Fig. 4I) and A549 (Fig. 4J) cells. Therefore, these results demonstrated that DLX6-AS1 knockdown suppressed cell proliferation, migration and invasion while induced apoptosis by downregulating PRR11.

3.3. PRR11 knockdown inhibited proliferation, migration and invasion and promoted apoptosis of NSCLC cells QRT-PCR and western blot results manifested that PRR11 were abnormally increased at mRNA and protein levels in H1975 and A549 cells compared with that in 16HBE cells (Fig. 3A and B). To address the biological function of PRR11 in the progression of NSCLC, knockdown of PRR11 was performed in H1975 and A549 cells by introducing with si-PRR11. As a result, the efficiencies of PRR11 knockdown were confirmed in H1975 (Fig. 3C) and A549 (Fig. 3D) cells by western blot, uncovered by reduced PRR11 protein abundance. Reduced proliferation was observed in H1975 (Fig. 3E) and A549 (Fig. 3F) cells with PRR11 silencing. Moreover, inhibition of PRR11 impaired migrated ability of H1975 (Fig. 3G) and A549 (Fig. 3F). In addition, cell invasion ability was prominently hindered in si-PRR11-transfected H1975 (Fig. 3I) and A549 (Fig. 3J) cells comparison with si-Control-introduced cells. Furthermore, H1975 (Fig. 3K) and A549 (Fig. 3L) cells showed a remarkable enhancement of apoptosis in response to si-PRR11 transfection. 1857

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3.5. DLX6-AS1 upregulated PRR11 expression by acting as a competing endogenous RNAs (ceRNA) of miR-144 in NSCLC cells

between DLX6-AS1 expression and NSCLC. Herein, we demonstrated that DLX6-AS1 expression was aberrantly enhanced in NSCLC tissues and cells, which was in good agreement with the previous study [11]. Functional experiments manifested that decreased expression of DLX6AS1 suppressed cell proliferation, migration and invasion and induced apoptosis in NSCLC cells. Similarly, DLX6-AS1 was highly expressed indicated the poor prognosis of hepatocellular carcinoma (HCC) by regulating proliferation, migration and invasion of HCC cells in vitro [23]. Additionally, DLX6-AS1 was revealed to be upregulated in renal cell carcinoma (RCC) tissues, which promoted RCC cell growth and tumorigenesis [24]. Although DLX6-AS1 has been identified as an oncogene, the potential mechanism by which DLX6-AS1 exerted its oncogenic role in NSCLC remains to be further explored. Increasing studies have proposed a ceRNA hypothesis, by which lncRNAs act as miRNA sponges to suppress the expressions and biological functions of miRNAs, and thereby derepress miRNA target [21]. The lncRNA-miRNA-mRNA interaction network might play an important role in the development of cancer. It was reported that DLX6-AS1 functioned as a ceRNA to absorb miR-203a and thereby regulated matrix metalloproteinase-2 (MMP-2) expression in HCC cells to aggravate HCC carcinogenesis [23]. Additionally, DLX6-AS1, which acted as a miRNA sponge, impeded miR26a to maintain the expression of phosphatase and tensin homologue (PTEN), which contributed to RCC progression [24]. In our study, we found that DLX6-AS1 was positively associated with PRR11 mRNA expression in NSCLC patients. PRR11 has been documented to be implicated in the development of lung cancer by acting as a candidate oncogene [25]. Consistently, we confirmed that PRR11 expression was increased in NSCLC tissues and cells and PRR11 silencing suppressed proliferation, migration, invasion and induced apoptosis in NSCLC cells. We further demonstrated that PRR11 overexpression reversed the effects of DLX6-AS1 knockdown on cell proliferation, migration, invasion and apoptosis in NSCLC cells. To explore whether DLX6-AS1 acted as a ceRNA of miRNA to regulate PRR11 expression, bioinformatics analysis and luciferase reporter assay were performed in NSCLC cell. Results showed that DLX6-AS1 interacted with miR-144 and suppressed miR-144 expression in NSCLC cells. Previous studies have demonstrated that miR-144 is prominently downregulated in malignant tumors including NSCLC, leading to the notion that miR-144 serves as a tumor suppressor [15,26]. Notably, PRR11 was identified as a target of miR-144, which was consistent with the previous study in pancreatic cancer [27]. Moreover, DLX6-AS1 reversed the inhibitory effect of miR-144 on PRR11 in NSCLC cells. In vivo experiments demonstrated that DLX6-AS1 knockdown suppressed tumor growth in NSCLC in vivo by upregulating miR-144 and downregulating PRR11.

It has been proposed that lncRNAs functioned as ceRNAs through suppressing the expressions and functions of miRNAs, which in turn regulate the expressions of specific gene targeted by miRNAs [21]. Accordingly, online databases TargetScan and miRcode were used to predict potential miRNAs that might interact with DLX6-AS1. As a result, DLX6-AS1 contained the complementary binding sites of miR-144 (Fig. 5A). To confirm the mutual effect between DLX6-AS1 and miR-144 in NSCLC cells, we cloned either wild-type or mutated DLX6-AS1 fragment into luciferase reporter vectors and performed luciferase reporter assay. The results implicated that miR-144 addition greatly reduced the luciferase activity of H1975 (Fig. 5B) and A549 (Fig. 5C) cells transfected with DLX6-AS1-WT, but not from cells transfected with DLX6-AS1-MUT, confirming the authentic binding between DLX6-AS1 and miR-144. Furthermore, qRT-PCR analysis demonstrated that miR144 expression was significantly increased in si-DLX6-AS1-introduced H1975 and A549 cells and evidently decreased in pcDNA-DLX6-AS1treated H1975 and A549 cells (Fig. 5G and 5 H). In addition, bioinformatics analysis which predicted the potential target genes of miR144 showed that PRR11 was one of the potential targets of miR-144 (Fig. 5D). Moreover, luciferase reporter assay proved that cotransfection with PRR11 3′UTR-WT and miR-144 strikingly limited the luciferase activity of H1975 (Fig. 5E) and A549 (Fig. 5F) cells, while cotransfection with PRR11 3′UTR-MUT and miR-144 exhibits no obvious effect on luciferase activity. Moreover, we found that PRR11 protein level was significantly blocked by transfection of miR-144 in H1975 (Fig. 5I) and A549 (Fig. 5J) cells, which was substantially recuperated by cotransfecction of miR-144 and pcDNA-DLX6-AS1. Collectively, these results revealed that DLX6-AS1 upregulated PRR11 expression by acting as a ceRNA of miR-144 in NSCLC cells. 3.6. DLX6-AS1 knockdown suppressed tumor growth in NSCLC in vivo by upregulating miR-144 and downregulating PRR11 To confirm the role of DLX6-AS1 in NSCLC in vivo, a xenograft nude mice model was constructed by subcutaneously injecting with A549 cells stably transfected with pcDNA-DLX6-AS1 or sh-DLX6-AS1. ShDLX6-AS1-injected mice displayed a remarkable reduction of tumor growth (Fig. 6A) and tumor weight (Fig. 6B) compared with control group, whereas pcDNA-DLX6-AS1-injected mice showed the opposite effects. DLX-AS1 expression was apparently impaired by introduction of si-DLX6-AS1 and conspicuously enhanced by treatment of pcDNADLX6-AS1 in the excised tumor tissues (Fig. 6C). Moreover, the expression of miR-144 was drastically increased in the si-DLX6-AS1 group but dramatically decreased in the pcDNA-DLX6-AS1 group (Fig. 6D). Meanwhile, the mRNA and protein levels of PRR11 were markedly declined in the si-DLX6-AS1 group and substantially elevated in the pcDNA-AS1 group (Fig. 6E and F). Together, these results demonstrated that DLX6-AS1 knockdown suppressed tumor growth in NSCLC in vivo by upregulating miR-144 and downregulating PRR11.

5. Conclusions In summary, our study demonstrated that DLX6-AS1 and PRR11 were enhanced in NSCLC tissues and cells. Depletion of DLX6-AS1 and PRR11 inhibited cell proliferation, migration and invasion while induced apoptosis in NSCLC cells, which was reversed by PRR11 overexpression. Moreover, DLX6-AS1 and PRR11 were shown to interact with miR-144 and DLX6-AS1 increased PRR11 expression by acting as a ceRNA of miR144 in NSCLC cells. Besides, DLX6-AS1 knockdown decreased tumor growth in NSCLC in vivo through increasing miR-144 and reducing PRR11. Collectively, inhibition of DLX6-AS1 attenuated cell proliferation, migration and invasion while promoted apoptosis by downregulating PRR11 expression and upregulating miR-144 in NSCLC, representing a novel prognostic biomarker and therapeutic target in NSCLC.

4. Discussion Accumulating evidence has suggested that lncRNAs play a pivotal role in tumorigenesis and have been considered as diagnostic and prognostic markers for various tumors including NSCLC [22]. In the present study, we confirmed that DLX6-AS1 and PRR11 were significantly upregulated in NSCLC tissues and cells. DLX6-AS1 and PRR11 silencing suppressed cell proliferation, migration and invasion and promoted apoptosis in NSCLC cells. We also found that DLX6-AS1 exerted its oncogenic role in NSCLC in large part due to its effect to act as a ceRNA for miR-144, and subsequently regulating its target PRR11 expression. To our knowledge, our present study first addressed the association

Conflicts of interests All authors declare that they have no conflicts of interest in this work. 1858

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Acknowledgement

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