Long Non‑Coding RNA PITPNA‑AS1 Silencing Suppresses Proliferation

MOLECULAR MEDICINE REPORTS 23: 212, 2021 Long non‑coding RNA PITPNA‑AS1 silencing suppresses proliferation, metastasis and epithelial‑mesenchymal transition in non‑small cell lung cancer cells by targeting microRNA‑32‑5p GANG CHEN1, ZHIFENG ZHENG2, JUNSHENG LI3, PEIGANG ZHANG4, ZHENJUN WANG5, SHIPING GUO1, JUN MA6, JIAN SHEN7 and HUIXIN LI8 1The Secondary Department of Thoracic Surgery, The Tumor Hospital Affiliated to Shanxi Medical University, Taiyuan, Shanxi 030013; 2Department of General Thoracic Surgery, Linfen People's Hospital, Linfen, Shanxi 041000; 3Department of Cardiothoracic Surgery, Taizhou Central Hospital (Taizhou University Hospital), Taizhou, Zhejiang 318000; 4Department of Cardio‑Thoracic Surgery, The People's Hospital of Lvliang, Lvliang, Shanxi 033000; 5Cardiothoracic Surgery Department of Shanxi Yangquan Coal Industry (Group) Co., Ltd., Yangquan, Shanxi 045000; 6Department of Thoracic Surgery, Heji Hospital, Changzhi Medical College, Changzhi, Shanxi 046011; 7Department of Cardiothoracic Surgery, Changzhi People's Hospital Affiliated to Shanxi Medical University, Changzhi, Shanxi 046000;8 Department of Cardiothoracic Surgery, Yuci District People's Hospital, Jinzhong, Shanxi 030600, P.R. China Received April 17, 2020; Accepted August 25, 2020 DOI: 10.3892/mmr.2021.11851 Abstract. Lung cancer is one of the most common types that PITPNA‑AS1 was highly expressed in NSCLC tissues of cancer and has a high mortality rate, worldwide. The and cell lines. It was found that PITPNA‑AS1 silencing major histopathological subtype is non‑small cell lung inhibited the proliferation, invasion and migration of NSCLC cancer (NSCLC). The aim of the present study was to cells. Furthermore, the protein expression of E‑cadherin was investigate the role of long non‑coding (lnc) RNA PITPNA upregulated, while the expression levels N‑cadherin and antisense RNA 1 (PITPNA‑AS1) in NSCLC and elucidate vimentin were downregulated. The luciferase reporter assay its potential mechanisms. The expression of PITPNA‑AS1 confirmed that miR‑32‑5p was a direct target of PITPNA‑AS1. was determined in several NSCLC cell lines. Following The rescue experiments suggested that a miR‑32‑5p inhibitor PITPNA‑AS1‑silencing, cell proliferation, invasion and significantly reversed the inhibitory effects of PITPNA‑AS1 migration were evaluated using Cell Counting Kit‑8, colony silencing on proliferation, invasion, migration and EMT in formation, Transwell assay and wound healing assays, respec‑ NSCLC cells. Collectively, the present results demonstrated tively. The expression levels of proliferation‑, migration‑ and that PITPNA‑AS1 silencing could suppress the progression epithelial‑mesenchymal transition (EMT)‑associated proteins of NSCLC by targeting miR‑32‑5p, suggesting a promising were examined using immunofluorescence assay or western biomarker in NSCLC diagnosis and treatment. blot analysis. A luciferase reporter assay was conducted to verify the potential interaction between PITPNA‑AS1 and Introduction microRNA(miR)‑32‑5p. Subsequently, rescue assays were performed to investigate the effects of PITPNA‑AS1 and Lung cancer is considered to be one of the most frequent miR‑32‑5p on NSCLC progression. The results demonstrated malignancies, with a high mortality rate, as there are 1.6 million tumor‑related mortalities annually worldwide (1,2). Non‑small cell lung cancer (NSCLC) is the predominant type of lung cancer and accounts for >80% of cases with a low 5‑year survival rate (3,4). Despite significant advances in the Correspondence to: Dr Gang Chen, The Secondary Department of diagnosis and treatment of lung cancer in recent decades, the Thoracic Surgery, The Tumor Hospital Affiliated to Shanxi Medical University, No. 3 of Workers' New Village, Xinghualing, Taiyuan, 5‑year survival rate still remains <20% (5). Poor prognosis in Shanxi 030013, P.R. China patients with NSCLC is associated with the lack of early diag‑ E‑mail: [email protected] nostic biomarkers, as well as high potentials of invasion and metastasis (6). Therefore, elucidating the detailed molecular Key words: non‑small cell lung cancer, proliferation, invasion, mechanism underlying the progression of NSCLC and identi‑ migration, PITPNA antisense RNA 1, microRNA‑32‑5p fying novel biomarkers is urgently required for early diagnosis, prevention and treatment. Long non‑coding (lnc) RNAs are functional non‑protein coding transcripts, with a length of >200 nucleotides (7). 2 CHEN et al: lncRNA PITPNA‑AS1 IN NON‑SMALL CELL LUNG CANCER Based on emerging research, lncRNAs exert a diverse set Thermo Fisher Scientific, Inc.) according to the manufacturer's of functions in numerous biological processes, such as cell instructions. Cells were harvested 48 h following transfection proliferation, invasion, differentiation, apoptosis and metas‑ and the transfection efficiency was determined using reverse tasis (8,9). In addition, the expression profiles of lncRNA have transcription‑quantitative PCR (RT‑qPCR). been associated with a number of diseases, including cancer, suggesting that they may serve as potential mediators in carci‑ Cell Counting Kit‑8 (CCK‑8) assay. A549 cells were main‑ nogenesis (10). PITPNA antisense RNA 1 (PITPNA‑AS1) is tained in the exponential phase and then seeded in 96‑well a newly reported lncRNA, which is located on chromosome plates (2x104 cells/well). Cell proliferation was detected using 17p13.3 (11). It has been reported that PITPNA‑AS1 facili‑ a CCK‑8 assay (Shanghai YiSheng Biotechnology Co., Ltd.) tates hepatocellular carcinoma progression via regulation of according to the manufacturer's instructions. At the indicated the microRNA(miRNA/miR)‑876‑5p/WNT5A signaling times of 24, 48 and 72 h, 10 µl CCK‑8 solution was added to pathway (11). Moreover, emerging evidence supports the each well and the cells were incubated for an additional 4 h hypothesis that PITPNA‑AS1 accelerates cervical cancer at 37˚C. The optical density was measured at 450 nm using a progression via regulating cellular proliferation and apoptosis microplate reader (Bio‑Rad Laboratories, Inc.). by targeting the miR‑876‑5p/c‑MET axis (12). Therefore, PITPNA‑AS1 may exert oncogenic effects. These findings Colony formation assay. Cells were exposed to the indicated indicated that PITPNA‑AS1 may serve as a potential diag‑ transfections accordingly and were plated into 6‑cm dishes nostic and therapeutic target for NSCLC. (1,000 cells per well), followed by incubation for 14 days at The aims of the present study were to investigate the role 37˚C, until the colonies could be observed under a fluores‑ of lncRNA PITPNA‑AS1 in NSCLC development and to cence microscope (magnification, x10; Olympus Corporation). identify the underlying molecular mechanisms. These data Subsequently, cells were fixed with 4% paraformaldehyde for may provide a novel diagnostic and therapeutic target for the 10 min at room temperature and stained with 0.2% crystal control of NSCLC progression. violet for 5 min at room temperature. Materials and methods Immunofluorescence assay.C ells (5x104 cells/well) were plated on coverslips in 24‑well plates following transfection and were The Cancer Genome Atlas (TCGA) database analysis. Human cultured until 80% confluence was reached. Subsequently, RNA‑sequencing data from NSCLC projects, which included cells were fixed in 4% paraformaldehyde at room tempera‑ 1,038 patients with NSCLC and 322 normal tissues were ture for 10 min and permeabilized with 0.05% Triton X‑100. obtained from TCGA data in the UALCAN database (ualcan. After blocking with 5% normal goat serum (Boster Biological path.uab.edu). A Mann‑Whitney test was used by UALCAN Technology) for 1 h at room temperature, cells were incu‑ to determine the statistical differences in the PITPNA‑AS1 bated with a primary antibody against Ki67 (cat. no. 12075S; expression levels between normal and tumor samples. 1:1,000; Cell Signaling Technology, Inc.) overnight at 4˚C. The cells were washed three times with PBS, incubated with Cells and cell culture. A total of four human NSCLC cell DyLight™ 488‑conjugated secondary antibody (cat. no. 35553; lines (HCC827, NCI‑H1299, A549 and NCI‑H1650) and one 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h at 37˚C normal human bronchial epithelioid cell line (BEAS‑2B), were and stained with DAPI (Sigma‑Aldrich; Merck KGaA) at room purchased from the Cell Bank of the Chinese Academy of temperature for 15 min. The stained cells were subsequently Sciences. The cell lines were cultured in RPMI‑1640 medium imaged under a fluorescence microscope (magnification, x200; (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Olympus Corporation). Thermo Fisher Scientific, Inc.), 100 µg/ml penicillin and 0.1 µg/ml streptomycin at 37˚C in a humidified incubator with Transwell assay. The invasive ability of A549 cells was 5% CO2. detected using an 8‑µm pore insert precoated with Matrigel (BD Biosciences) overnight at 37˚C. A total of 200 µl Cell transfection. Cells were plated into 6‑well plates serum‑free medium containing cells was added to the upper (1x106 cells per well), and transfection was performed when chamber following 48 h of transfection. RPMI‑1640 containing cells in the logarithmic growth phase reached 80% confluence. 10% FBS was added to the lower compartment as a chemoat‑ pIRES vectors containing (sh)RNAs targeting PITPNA‑AS1 tractant. After 24 h of incubation, cells invading the lower (shRNA‑PITPNA‑AS1‑1 or shRNA‑PITPNA‑AS1‑2; 1 µg) or a chamber were fixed with 4% formaldehyde for

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