Increased expression of LINC01510 predicts poor prognosis and promotes malignant progression in human non-small cell lung cancer

Biomedicine & Pharmacotherapy 109 (2019) 519–529

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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Increased expression of LINC01510 predicts poor prognosis and promotes malignant progression in human non-small cell lung cancer

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Jiwei Li, Li Wei

Department of Thoracic Surgery, Henan Provincial People’s Hospital, Zhengzhou 450003, China

A R T I C LE I N FO

A B S T R A C T

Keywords: LINC01510 Poor prognosis Malignant progression Non-small cell lung cancer

Non-small cell lung cancer (NSCLC), the most prevalent type of lung cancer, is one of the most leading causes of cancer-related morbidity and mortality worldwide. Evidence is accumulating that long non-coding RNAs (lncRNAs) play vital regulatory roles in tumor development and progression. LINC01510, a novel tumor-related lncRNA, has been identified as an oncogene in colorectal cancer; however, its role in NSCLC remains poorly understood. This study aimed to characterize the biological role of LINC01510 in NSCLC and illuminate the molecular mechanisms. Here we found that LINC01510 was highly expressed in NSCLC tissues. Besides, Fisher’s exact test showed that high expression of LINC01510 was associated with larger tumor size, advanced TNM stage and lymph node metastasis. Kaplan-Meier survival analysis showed that patients with high LINC01510 expression had a much lower overall survival rate. Gain- and loss-of-function approaches were employed to investigate the effects of LINC01510 on NSCLC cell phenotypes. Functional studies demonstrated that LINC01510 over-expression promoted NSCLC cell proliferation, cell cycle progression, migration and invasion, but shRNAmediated LINC01510 depletion inhibited NSCLC cell proliferation, cell cycle progression, migration and invasion. Notably, LINC01510 ablation suppressed tumorigenicity of NSCLC cells in a murine xenograft model. Furthermore, mechanistic studies revealed that LINC01510 exerted its oncogenic functions in NSCLC through miR-339-5p-mediated regulation of CDK14. To sum up, our data indicate that increased expression of LINC01510 predicts poor prognosis and promotes tumorigenesis in NSCLC. Collectively, this study may provide a basis for LINC01510 as a candidate therapeutic target in NSCLC.

1. Introduction Lung cancer is one of the most frequently diagnosed human malignant tumors and remains the leading cause of cancer-related mortality worldwide, with approximately 1.2 million deaths occurring each year [1–3]. It was estimated that about 224,390 people were newly diagnosed with lung cancer and almost 158,080 patients died from this cancer in the United States in 2016 [4,5]. Non-small cell lung cancer (NSCLC) is the major pathological sub-type of lung cancer, accounting for 80∼85% of all the cases [6,7]. Despite encouraging advances in diagnosis and treatment over the past several decades, the therapeutic outcomes remain rather unfavorable. Hence, there is an urgent need to identify novel diagnostic bio-markers and develop effective therapeutic strategies. Long non-coding RNAs (lncRNAs) are a large group of non-proteincoding RNA transcripts longer than 200 nucleotides [8,9]. Up to now, thousands of lncRNAs have been discovered by virtue of chromatin signature analysis and large-scale sequencing [10,11]. A growing

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number of evidence has demonstrated that the ectopic expression of lncRNAs plays pivotal roles in diverse types of human malignancies and that their dys-regulation is closely related to cancer progression [12–14]. LINC01510, a novel tumor-associated lncRNA, is transcribed from an intergenic region of human chromosome 7 [15]. LINC01510 has been identified as an oncogene in human colorectal cancer [16]; however, to the best of our knowledge, its biological role in NSCLC remains poorly understood. It is widely acknowledged that cyclin-dependent kinases (CDKs) are a group of heterodimeric serine/threonine protein kinases and play critical roles in cell cycle progression, cell division and cell motility [17–19]. Recent studies have identified CDK14, a member of the CDKs family, as an oncogene in multiple types of human tumors. CDK14 has been reported to be highly expressed and contribute to tumor progression in NSCLC [20], breast cancer [21,22] and hepatocellular carcinoma [23]. In this study, we discovered that LINC01510 was significantly upregulated in NSCLC tissues compared with adjacent non-cancerous

Corresponding author at: Department of Thoracic Surgery, Henan Provincial People’s Hospital, No. 7 Weiwu Road, Jinshui District, Zhengzhou 450003, China. E-mail address: [email protected] (L. Wei).

https://doi.org/10.1016/j.biopha.2018.10.136 Received 27 August 2018; Received in revised form 21 October 2018; Accepted 21 October 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

Biomedicine & Pharmacotherapy 109 (2019) 519–529

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2.3. Cell lines and cell culture

Table 1 Relationship between LINC01510 expression and clinicopathological characteristics in 56 patients with NSCLC. Characteristics

Age (years) < 60 ≧60 Gender Female Male Tumor size (cm) <3 ≥3 TNM stage I II IIIa Histology Adenoma Squamous Lymph node metastasis Absent Present

Number of cases

LINC01510 expression High (n = 31)

Low (n = 25)

29 27

15 16

14 11

26 30

14 17

12 13

24 32

9 22

15 10

8 31 17

1 21 9

7 10 8

30 26

16 15

14 11

Normal human lung epithelial cell line BEAS-2B and four NSCLC cell lines (A549, H460, SPC-A1 and H23) were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in CO2 humidified atmosphere at 37 ℃.

P-value

0.571

0.832

2.4. Cell transfection 0.005

Human LINC01510 cDNA sequence was synthesized by Shanghai GeneChem (Shanghai, China) and then ligated into the pcDNA3.1 plasmids. The short hairpin RNA specially targeting LINC01510 (shRNA-LINC01510) and negative control shRNA (shRNA-NC) were designed, synthesized and ligated into the pLKO.1 plasmids (GeneChem), respectively. The cells were transfected with empty pcDNA3.1 plasmid, pcDNA3.1-LINC01510, shRNA-NC or shRNALINC01510. Cell transfection was performed using Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s instructions.

0.019

0.743

0.004 24 32

8 23

16 9

2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) tissues. Besides, increased expression of LINC01510 was found to correlate with poor prognosis of NSCLC patients. Functional studies demonstrated that LINC01510 promoted NSCLC cell proliferation, cell cycle progression, migration and invasion. In addition, mechanistic studies unveiled that LINC01510 exerted its oncogenic effects in NSCLC via miR-339-5p-mediated CDK14 regulation. Collectively, our findings may provide better understanding about the biological role of LINC01510 in NSCLC tumorigenesis and the molecular mechanisms involved.

Total RNAs were extracted using Trizol (Invitrogen) following the manufacturer’s instructions. The concentration and purity of total RNAs was determined using a NanoDrop ND-1000 spectrometer (NanoDrop Technologies, Wilmington, DE, USA). Total RNAs were reverse-transcribed using Reverse Transcription Kit (Takara, Dalian, China). RTPCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR systems (Applied Biosystems, Foster City, CA, USA). The specific primer sequences were synthesized by Sangon Biological Engineering Technology and Service (Shanghai, China). The sequences of primers were as followed：LINC01510, forward 5′-CTGTGGAAGTTTGAGT GAC-3′ and reverse 5′-TTCATCTATCCTCCTGCT-3′; CDK14, forward 5′-TTCCAAGCCAATTGGAAGAC-3′, reverse 5′-GACATGCCTGGTCCAC TTTT-3′; GAPDH, forward 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse 5′-GAAGATGGTGATGGGATTTC-3′. GAPDH served as an endogenous control to normalize LIN01510 and CDK14 expression. The relative expression of LINC01510 and CDK14 was calculated using the 2−△△Ct method.

2. Materials and methods 2.1. Clinical specimens Fifty-six patients who were pathologically diagnosed with NSCLC and underwent surgical resection were recruited from Henan Provincial People’s Hospital (Zhengzhou, China) between January 2008 and November 2014. All the participants gave their written informed consents. None of them had received any other anti-cancer treatments before operation. The clinicopathological characteristics of patients were listed in Table 1. All the tissues samples were frozen in liquid nitrogen at once after resection and stored at -80 for subsequent studies. Overall survival was defined as the time interval between the date of diagnosis and the date of death or last follow-up. This study was approved by the Ethics Committee of Henan Provincial People’s Hospital.

2.6. Cell proliferation Cell proliferation was evaluated using Cell Proliferation Kit (MTT, Roche, Indianapolis, IN, USA) according to the manufacturer’s guidelines. In brief, cells were placed in 96-well plates at a density of 2 × 103 cells per well. After being incubated for various periods of time (0, 24, 48 and 72 h) at 37 ℃, the medium was then removed and replaced with MTT solution. Following incubation for another 4 h at 37℃, MTT solution was removed and replaced with dimethyl sulfoxide (DMSO, Sigma-Aldrich). Absorbance was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, Grand Island, NY, USA).

2.2. In situ hybridization (ISH) Tissues samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Then tissue slides of 4 μm thickness were prepared. After being pre-hybridized in the hybridization buffer (Boster Bioengineering, Wuhan, China) at 37 ℃ for 2 h, slides were then hybridized with digoxigenin-labeled detection probes (Boster) complementary to LINC01510 overnight at 42℃. After stringent washes, an immunologic reaction was performed using biotinylated anti-mouse digoxigenin, followed by adding alkaline phosphatase-conjugated streptavidin dilution (BD Bioscience, Heidelberg, Germany) to detect streptavidin dilution probes. Tissue slides were mounted with aqueous mounting medium and then observed by microscopy.

2.7. Cell cycle analysis For cell cycle analysis, cells were trypsinized using 0.25% trypsin, washed twice with PBS, and then fixed in 70% ethanol overnight at 4℃. The cells were then stained with 10 μl propidium (PI; Sigma-Aldrich) containing 10 μg RNase A (Sigma-Aldrich) for 30 min at 4℃ in the dark. Cells were analyzed using a flow cytometer (Becton-Dickinson; San Jose, CA, USA). 520

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RIP assays according to the manufacturer’s instructions. In brief, cells were lysed in complete RIP lyiss buffer. Then cell lysate was incubated with RIP buffer containing magnetic beads coated with human antiAgo2 antibody (Millipore) or negative control IgG (Millipore) overnight at 4℃. Subsequently, the beads were washed and incubated with Proteinase K to remove the proteins. The co-precipitated RNAs were purified and subject to qRT-PCR analysis.

2.8. Cell apoptosis analysis Cell apoptosis was evaluated using Annexin-V FITC/PI Apoptosis Detection Kit (BD Bioscience, CA, USA). Cells were collected by lowspeed centrifugation and washed twice with ice-cold PBS. Subsequently, 300 μl binding buffer was added to cells. Afterward, cells were treated with 5 μl PI and 5 μl Annexin V-FITC at 4 ℃ for 20 min in the dark. Cell apoptosis was determined by flow cytometry (Becton Dickinson).

2.14. Tumor xenograft mouse models

2.9. Wound healing assay

Animal protocol was approved by Animal Care and Use Committee of Henan Provincial Hospital. Female BALB/c nude mice (5–6 weeks of age) were purchased from Beijing HFK Bioscience (Beijing, China). A549 cells treated with shRNA-LINC01510 or shRNA-NC (1 × 106 cells per mouse) were subcutaneously administrated into the flanks of the nude mice. The length and width of tumors were determined using a slide caliper every 3 days. At day 21 post-injection, all the mice were sacrificed, and the tumor nodules were removed and weighed. Tumor volume was calculated according to the following formula: tumor volume (mm3) = length (mm)×width (mm)2/2.

Wound healing assays were carried out to assess the migration abilities of cells. In brief, transfected cells were seeded in six-well plates to form a mono-layer at a density of 1 × 104 cells per well. The monolayer of cells was scratched using a sterile plastic micropipette tip and washed three times with medium to form a wound. Cells were cultured in the medium for 24 h. Closure of scratch was observed and photographed under an inverted microscope (Olympus IX50, Tokyo, Japan). 2.10. Transwell invasion assay

2.15. Immunohistochemistry (IHC)

Transwell invasion assays were performed to evaluate the invasion capabilities of cells. Briefly, transfected cells (1 × 105) were seeded into the upper chamber of the insert precoated with Matrigel (SigmaAldrich). Cells were placed in the medium without serum. Medium supplemented with 10% FBS (Sigma-Aldrich) was added to the lower chamber as chemoattractant. Cells were allowed to invade for 48 h at 37 °C. Subsequently, cell were fixed in 70% ethanol for 30 min and stained with 0.1% crystal violet (Sigma-Aldrich) for 10 min. Stained cells were counted in five randomly selected fields under an invert microscope (Olympus IX50).

Paraffin-embedded tissues were sectioned at 4.5 μm thickness. After being dewaxed and hydrated, sections were incubated with 3% H2O2 for 30 min to block the endogenous peroxidase (POD) activity. Following antigen recovery by repeated cooling and heating, 5% bovine serum albumin (BSA) was applied to block non-specific binding. The sections were then incubated with primary antibodies overnight at 4℃. Anti-Ki67 (ab833) and anti-vimentin (ab45939) were purchased from Abcam (Cambridge, MA, USA) and used at the following dilutions: antiKi67 (1:200) and anti-vimentin (1:500). After being rinsed with PBS three times for 5 min each, sections were treated with biotinylated secondary antibody (Abcam) for 1 h, followed by incubation with streptavidin-horseradish peroxidase (HRP) for 20 min. Diaminobenzidine (DAB) substrate was used to visualize Ki67- and vimentin-positive cells. Slides were then observed under a microscope (Olympus BX51, Olympus Optical, Tokyo, Japan).

2.11. Western blotting Total proteins were extracted using RIPA buffer supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). The protein samples were separated using SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto the polyvinylidene fluoride PVDF membranes. After being blocked with 5% degrease milk in TBST buffer, the membranes were incubated with primary antibodies overnight at 4℃. Anti-CDK14 and anti-GAPDH were used at the following dilutions: anti-CDK14 (1:500) and anti-GAPDH (1:1000). After being washed three times, the membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibody at 37℃ for 1 h. The blots were developed using the enhanced chemiluminescence (ECL) Western Blotting Detection Kit (Amersham, Buckinghamshire, England) and visualized on a Gel Doc XR System (Bio-Rad Laboratories, Hercules, CA, USA).

2.16. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) TUNEL analysis was conducted to monitor the apoptosis using the In Situ Cell Death Detection Kit (Roche Diagnostic, Basel, Switzerland) in accordance with the manufacturer’s instructions. Briefly, transfected cells were pre-treated with 10 nmol/L DTX for 24 h. After being rinsed with PBS three times, cells were then labeled with dTd labeling reaction mix. Positively stained cells were subsequently observed under an EVOS FL microscope (Thermo Fisher Scientific, Waltham, MA, USA).

2.12. Luciferase reporter assay

2.17. Statistical analysis

Wild-type CDK14 3′UTR fragments were amplified from a human cDNA library. Wild-type CDK14 3′UTR and mutant CDK14 3′UTR fragments were inserted into pmirGLO reporter vectors (Promega, Madison, WI, USA), respectively. A dual-luciferase reporter assay system (Promega) was applied to determine the luciferase activity at 48 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. Similar experiments were conducted to validate the binding between miR-339-5p and LINC01510.

Data were presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 22.0 software (IBM, Chicago, IL, USA). Student’s t-test was applied to compare the difference between two groups. One-way analysis of variance (ANOVA) followed by Dunnett’s test was employed to compare the difference among three or more groups. The log-rank test and Kaplan-Meier survival curves were used to analyze the relationship between LINC01510 expression and overall survival. Fisher’s exact test was used to analyze the correlation between LINC01510 expression and clinicopathological characteristics. Pearson’s correlation analysis was applied to evaluate the relationship between LINC01510 expression and miR-339-5p or CDK14 mRNA expression in NSCLC tissues. P < 0.05 was considered as statistically significant.

2.13. RNA-binding protein immunoprecipitation (RIP) We used the EZ-Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) to carry out 521

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Fig. 1. Increased expression of LINC01510 is associated with poor prognosis in NSCLC patients. (A) Relative LINC01510 expression levels in 56 pairs of NSCLC and adjacent non-cancerous tissues were determined by qRT-PCR analysis. (B) LINC01510 expression patterns in NSCLC and matched non-tumorous tissues were visualized by ISH analysis. (C) NSCLC patients were assigned into high LINC01510 expression group and low LINC01510 expression group based on the average value of its expression levels. The log-rank test and Kaplan-Meier survival analysis was used to determine the relationship between LINC01510 expression and overall survival. (D) Relative LINC01510 expression levels in normal lung epithelial cell line BEAS-2B and four NSCLC cell lines (A549, H460, SPC-A1 and H23) were examined by qRT-PCR analysis. **P < 0.01. NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; ISH, in situ hybridization.

3. Results

with high LINC01510 expression had a significantly shorter overall survival (Fig. 1C). In addition, we evaluated the endogenous expression of LINC01510 in normal human lung epithelial cell line BEAS-2B and four NSCLC cell lines (A549, H460, SPC-A1 and H23) and discovered that LINC01510 was significantly up-regulated in NSCLC cell lines (Fig. 1D). Take together, these findings indicate that increased expression of LINC01510 correlates with poor prognosis in NSCLC patients.

3.1. Increased expression of LINC01510 is associated with poor prognosis in NSCLC patients Although LINC01510 has been identified as an oncogenic lncRNA in colorectal cancer, its biological role in NSCLC remains largely unknown. In this study, we firstly conducted qRT-PCR analysis to determine the expression of LINC01510 in 56 paired NSCLC tissues and adjacent noncancerous tissues. As exhibited in Fig. 1A, LINC01510 was significantly up-regulated in NSCLC tissues compared with matched non-tumorous tissues. Subsequently, ISH analysis was performed to visualize the expression patterns of LINC01510 in NSCLC tissues and matched normal lung tissues. As evident from Fig. 1B, NSCLC tissues exhibited higher LINC01510 expression level than adjacent normal lung tissues. To investigate the correlation between LINC01510 expression and clinicopathological parameters in 56 patients, NSCLC tissues were categorized into high LINC01510 expression group (n = 31) and low LINC01510 expression group (n = 25) on the basis of the mean value of LINC01510 expression levels. Fisher’s exact test demonstrated that high LINC01510 expression was associated with larger tumor size, advanced TNM stage and lymph node metastasis; but no significant correlation existed between LINC01510 expression and other clinicopathological features, including age, gender and histology (Table 1). The log-rank test and Kaplan-Meier survival analysis showed that the NSCLC patients

3.2. LINC01510 promotes NSCLC cell proliferation and inhibits apoptosis Given the findings mentioned above, we speculated that LINC01510 was implicated in the tumorigenesis and progression of NSCLC. Gainand loss-of-function approaches were applied to investigate the effects of LINC01510 on NSCLC cell phenotypes. Over-expression and knockdown studies were carried out in H23 cells (lowest endogenous LINC01510 expression) and A549 cells (highest endogenous LINC01510 expression), respectively. Transfection efficacy was evaluated using qRT-PCR analysis (Fig. 2A). As evident from MTT assays, LINC01510 over-expression significantly accelerated H23 cell proliferation compared with control group, whereas shRNA-mediated LINC01510 depletion dramatically slowed down A549 cell proliferation (Fig. 2B). As displayed in Fig. 2C, LINC01510 over-expression markedly expedited H23 cell cycle progression compared with control group, whereas shRNA-mediated LINC01510 depletion induced A549 cell cycle arrest. 522

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Fig. 2. LINC01510 promotes NSCLC cell proliferation and inhibits apoptosis. (A) Transfection efficiency was evaluated by qRT-PCR analysis. (B) Cell proliferation was examined by MTT assays after transfection with LINC0150 expression vectors or shRNA-LINC01510. (C) Cell cycle was analyzed by flow cytometry after transfection with LINC01510 expression vectors or shRNALINC01510. (D) Cell apoptosis was evaluated by flow cytometry after transfection with LINC01510 expression vectors or shRNALINC01510. **P < 0.01. NSCLC, non-small cell lung cancer; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; shRNA, short hairpin RNA.

3.3. LINC01510 facilitates NSCLC migration and invasion

As shown in Fig. 2D, apoptosis of H23 cells was remarkably inhibited by LINC01510 over-expression in comparison with control treatment, while shRNA-mediated LINC01510 depletion significantly promoted A549 cell apoptosis. To sum up, these results suggest that LINC01510 facilitates NSCLC cell proliferation, accelerates cell cycle progression and inhibits apoptosis.

To investigate whether LINC01510 contributes to the motility of NSCLC cells, we then evaluated their migration and invasion capabilities. As evident from wound healing assays, LINC01510 over-expression notably strengthened the migration abilities of H23 cells 523

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Fig. 3. LINC01510 facilitates NSCLC cell migration and invasion. (A) Cell migration was evaluated by wound healing assays after transfection with LINC01510 expression vectors or shRNA-LINC01510. (B) Cell invasion was assessed by Transwell invasion assays after transfection with LINC01510 expression vectors or shRNALINC01510. **P < 0.01. NSCLC, non-small cell lung cancer; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; shRNA, short hairpin RNA.

targets of LINC01510. MiR-339-5p drew our attention for its importance in the occurrence and development of several types of human tumors, and was selected as a candidate target of LINC01510 (Fig. 4A). As presented in Fig. 4B, transfection of miR-339-5p mimics decreased luciferase activity of the reporter vectors carrying wild-type LINC01510 fragments compared with control group, whereas transfection of miR339-5p mimics failed to induce a significant decrease in luciferase activity of the reporter vectors carrying mutant LINC01510 fragments. As shown in Fig. 4C, more miR-339-5p was notably enriched by LINC01510 over-expression treatment in comparison with control treatment. Moreover, H23 cells transfected with LINC01510 expression vectors exhibited lower miR-339-5p expression levels than control group, whereas higher miR-339-5p expression levels were observed in the A549 cells treated with shRNA-LINC01510 (Fig. 4D). These findings suggest that LINC01510 interacts with miR-339-5p in NSCLC cells.

compared with control group, whereas shRNA-mediated LINC01510 depletion markedly weakened the migration abilities of A549 cells (Fig. 3A). As shown in Fig. 3B, LINC01510 over-expression significantly contributed to H23 cell invasion in comparison with control group, while shRNA-mediated LINC01510 depletion dramatically suppressed A549 cell invasion. These data suggest that LINC01510 facilitates NSCLC cell migration and invasion. 3.4. LINC01510 positively regulates CDK14 expression through sponging miR-339-5p in NSCLC cells Growing evidence has demonstrated that lncRNAs serve as competing endogenous RNAs to exert their functions in the oncogenesis and development of a wide range of human malignancies. To clarify the molecular mechanisms by which LINC01510 exerts its oncogenic effects in NSCLC, we used miRDB online software to predict the potential 524

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Fig. 4. LINC01510 interacts with miR-339-5p in NSCLC cells. (A) A putative binding site of LINC01510 in miR-339-5p was predicted by mRDB online software. (B) Luciferase activity of the reporter vectors carrying wild-type or mutant LINC01510 fragments was determined after transfection of miR-339-5p. (C) Anti-Ago2 RIP assays were performed to enrich the miRNAs interacting with LINC01510 in H23 cells after transfection with LINC01510 expression vectors or empty vectors. (D) Relative miR-339-5p expression levels were determined by qRT-PCR analysis after transfection with LINC01510 expression vectors or shRNA-LINC01510. ** P < 0.01. NSCLC, non-small cell lung cancer; NC, negative control; shRNA, short hairpin RNA.

LINC01510 expression was positively correlated with CDK14 mRNA expression in NSCLC tissues (Fig. 5F). To sum up, our findings indicate that LINC01510 positively regulates CDK14 expression through sponging miR-339-5p in NSCLC cells.

3.5. LINC01510 positively regulates CDK14 expression through sponging miR-339-5p in NSCLC cells To further elucidate the potential molecular mechanisms, we applied TargetScan online database to predict the potential targets of miR339-5p. Among all the putative targets of miR-339-5p, CDK14 aroused our interest for its involvement in multiple types of human cancers, and was selected as a candidate target of miR-339-3p (Fig. 5A). To verify the binding of miR-339-5p and CDK14, luciferase reporter assays were performed. As shown in Fig. 5B, transfection of miR-339-5p dramatically decreased the luciferase activity of wild-type CDK14 3′UTR reporter vectors, whereas transfection of miR-339-5p failed to alter the luciferase activity of mutant CDK14 3′UTR reporter vectors. Moreover, miR-339-5p mimics significantly inhibited CDK14 protein expression compared with negative control treatment (Fig. 5C). In addition, transfection of shRNA-LINC01510 notably reduced the luciferase activity of wild-type CDK14 3′UTR reporter vectors, while transfection of shRNA-LINC01510 did not significantly change the luciferase activity of mutant CDK14 3′UTR reporter vectors (Fig. 5D). As obvious from Fig. 5E, LINC01510 knockdown remarkably suppressed CDK14 protein expression in comparison with negative control group, suggesting that LINC01510 knockdown has a similar effect as miR-339-5p mimics treatment in modulating CDK14 expression. Notably, Pearson’s correlation analysis showed that LINC01510 expression was inversely correlated with miR-339-5p expression in NSCLC tissues; whereas

3.6. MiR-339-5p mediates the effects of LINC01510 on NSCLC cell malignant phenotypes To determine whether the effects of LINC01510 on NSCLC cell phenotypes are mediated by miR-339-5p, we down-regulated the expression of miR-339-5p in A549 cells treated with shRNA-LINC01519. As presented in Fig. 6A–E, miR-339-5p inhibitor reversed the inhibitory effects of LINC01510 depletion on proliferation, cell cycle progression, migration and invasion, and alleviated the promoting effect of LINC01510 depletion on cell apoptosis. Besides, miR-339-5p inhibitor was found to reverse the suppressing effect of LINC01510 knockdown on CDK14 expression (Fig. 6F). Taken together, these results indicate that miR-339-5p mediates the effects of LINC01510 on NSCLC cell proliferation, apoptosis, migration and invasion. 3.7. LINC01510 ablation suppresses tumorigenicity of NSCLC cells in a murine xenograft model To validate the findings in vitro, we established murine xenograft models by subcutaneous injection of A549 cells treated with shRNA525

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Fig. 5. LINC01510 positively regulates CDK14 expression through sponging miR-339-5p in NSCLC cells. (A) A putative binding site of miR-33p-5p in CDK14 3′UTR was predicted by TargetScan online software. (B) Luciferase activity of the reporter vectors carrying wild-type or mutant CDK14 3′UTR fragments was determined after transfection with miR-339-5p. (C) CDK14 protein expression was determined by western blotting after transfection with miR-339-5p. (D) Luciferase activity of the reporter vectors carrying wild-type or mutant 3′UTR fragments was examined after transfection with shRNA-LINC01510. (E) CDK14 protein expression was detected by western blotting after transfection with shRNA-LINC01510. (F) Pearson’s correlation analysis was performed to detect the relationship between LINC01510 expression and miR-339-5p or CDK14 mRNA expression in the tumorous tissues. **P < 0.01. NSCLC, non-small cell lung cancer; NC, negative control; CDK14, cyclin dependent kinase 14; RIP, RNA-binding protein immunoprecipitation; shRNA, short hairpin RNA; 3′UTR, 3′ untranslated region.

long-term prognosis remains so poor. Accumulating evidence has revealed that the aberrant expression of lncRNAs was implicated in NSCLC tumorigenesis and tumor progression [26–28]. Up to now, increasing attention has been paid to identify novel diagnostic bio-markers and develop effective therapeutic strategies for NSCLC. Nonetheless, available literature about NSCLC-associated lncRNAs with clinical prognostic values is still limited compared to the vast number of lncRNAs in the human genome. Even though LINC01510 has been identified as an oncogenic lncRNA in human colorectal cancer [16], its biological functions and clinical implications remain elusive. To the best of our knowledge, this is the first report to characterize the expression pattern and biological role of LINC01510 in NSCLC. In the current study, we discovered that LINC01510 was significantly upregulated in NSCLC tissues compared with normal lung tissues, which was in agreement with the observations in human colorectal cancer [16]. In addition, our data uncovered that high expression of LINC01510 was associated with tumor progression and poor prognosis, suggesting that LINC01510 was implicated in NSCLC. To gain a better understanding of the biological functions of

LINC01510 or negative control shRNA. As demonstrated in Fig. 7A, the tumors formed by the shRNA-LINC01510-treated A549 cells grew notably slower than those formed by the negative control shRNA-treated A549 cells; furthermore, the tumors collected from the shRNALINC01510 group weighed significantly lighter than those harvested from the negative control group. In addition, IHC staining showed that the tumors collected from the shRNA-LINC01510 group displayed higher Ki67 and vimentin expression levels than those harvested from the negative control group (Fig. 7B). Furthermore, a significant increase in TUNEL-positive cells was observed in the tumors harvested from the shRNA-LINC01510 group in comparison with the negative control group (Fig. 7C). Taken together, our data suggest that LINC01510 ablation suppresses NSCLC tumor growth in vivo.

4. Discussion NSCLC, one of the most common and fetal human malignancies, has posed great threats to the public health [24,25]. Although significant progress has been made in the diagnosis and treatment of NSCLC, the 526

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Fig. 6. LINC01510 mediates the effects of LINC01510 on NSCLC cell phenotypes. (A) Proliferation was examined by MTT assays after transfection with miR-339-5p inhibitor in shRNA-LINC01510-treated A549 cells. (B) Cell cycle was evaluated via FCM after transfection with miR-339-5p inhibitor in shRNA-LINC01510-treated A549 cells. (C) Apoptosis was assessed by FCM after transfection with miR-339-5p inhibitor in shRNA-LINC01510-treated A549 cells. (D) Migration was analyzed using wound healing assays after transfection with miR-339-5p inhibitor in shRNA-LINC01510-treated A549 cells. (E) Invasion was detected by Transwell invasion assays after transfection with miR-339-5p inhibitor in shRNA-LINC01510-treated A549 cells. (F) CDK14 protein protein was examined by western blotting after transfection with miR-339-5p inhibitor in shRNA-LINC01510-treated A549 cells. **P < 0.01 vs shRNA-NC group, ##P < 0.01 vs shRNA-LINC01510 group. NSCLC, non-small cell lung cancer; NC, negative control; shRNA, short hairpin RNA; CDK14, cyclin-dependent kinase 14.

carcinogenesis and progression of diverse type of human neoplasms [29–31]. To elucidate the potential molecular mechanisms by which LINC01510 promotes NSCLC cell proliferation, cell cycle progression, migration and invasion, we carried out mechanistic studies. Mechanistic studies revealed that LINC01510 positively regulated CDK14 to promote cell proliferation, migration and invasion through sponging miR-339-5p. Previous studies have identified miR-339-5p as a tumor suppressor in various types of human malignant tumors, such as nonsmall cell lung cancer [32], breast cancer [33], colorectal cancer [34], hepatocellular carcinoma [35], and ovarian cancer [36]. It is well documented that CDKs are a group of ATP-dependent serine/threonine protein kinases, which exert their regulatory roles in cell cycle progression and cell motility [18,19]. CDK14 belongs to the CDKs family

LINC01510 in NSCLC, gain- and loss-of-function methods were employed to manipulate its expression in the present study. In the current study, we discovered that over-expression of LINC01510 significantly promoted NSCLC cell proliferation, accelerated cell cycle progression, facilitated cell migration and invasion, and inhibited apoptosis; on the contrary, shRNA-mediated depletion of LINC01510 suppressed NSCLC cell proliferation, cell cycle progression, migration and invasion, and promoted apoptosis. Additionally, LINC01510 depletion was found to inhibit tumor growth in murine xenograft models. Taken together, these results indicate that LINC01510 may serve as an oncogene and promote malignant progression in NSCLC. A growing number of studies have proposed that lncRNAs function as competing endogenous RNAs to exert their regulatory roles in the 527

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Fig. 7. LINC01510 ablation suppresses tumorigenicity of NSCLC cells in a murine xenograft model. (A) shRNA-NC- or shRNA-LINC01510-treated A549 cells were subcutaneously injected into the flanks of nude mice (n = 5). Volumes of tumors were measured every 3 days using a slide caliper; mice were sacrificed under anesthesia at day 21 post-implantation, and tumors were collected. (B) Ki67 and vimentin protein expression was visualized by IHC staining in the collected tumors. (C) Apoptotic cells were determined by TUNEL analysis in the collected tumors. **P < 0.01. NSCLC, non-small cell lung cancer; shRNA, short hairpin RNA; IHC, immunohistochemistry; TUNEL, terminal-deoxynucleotidyl transferase mediated nick end labeling.

and its increased expression has been reported to accelerate cell proliferation in NSCLC [20] and hepatocellular carcinoma [21], and contribute to cell motility in breast cancer [22,23], ovarian cancer [37], and glioma [38]. In conclusion, the current study demonstrated for the first time that LINC01510 was highly expressed in NSCLC tissues and that its increased expression correlated with poor prognosis of NSCLC patients. Furthermore, our data revealed that LINC01510 exerted its oncogenic roles in NSCLC through miR-339-5p-mediated modulation of CDK14. Taken together, our findings may provide some new insights into the molecular mechanisms underlying NSCLC tumorigenesis and implicate LINC01510 as a novel prognostic bio-marker and a potential therapeutic target for NSCLC.

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