CDKN3 Promotes Cell Proliferation, Invasion and Migration by Activating the AKT Signaling Pathway in Esophageal Squamous Cell Carcinoma

542 ONCOLOGY LETTERS 19: 542-548, 2020 CDKN3 promotes cell proliferation, invasion and migration by activating the AKT signaling pathway in esophageal squamous cell carcinoma HANXU YU1,2*, JUN YAO3*, MINGYU DU1*, JINJUN YE1, XIA HE1 and LI YIN1 1Department of Radiotherapy, The Affiliated Cancer Hospital of Nanjing Medical University, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing, Jiangsu 210000; 2Department of Radiotherapy, Lianshui County People's Hospital, Lianshui, Jiangsu 223001; 3Department of Radiotherapy, Yancheng Second People's Hospital, Yancheng, Jiangsu 22400, P.R. China Received April 8, 2019; Accepted September 26, 2019 DOI: 10.3892/ol.2019.11077 Abstract. In China, esophageal squamous cell carcinoma promoted cell proliferation and invasion by activating the (ESCC), capable of direct invasion and early metastasis, AKT signaling pathway in ESCC cells. To the best of our exhibits high mortality. Identification of the molecular basis knowledge, the present study is the first to identify the func- driving ESCC progression and development of new diag- tions of CDKN3 in ESCC and provide evidence that CDKN3 nostic biomarkers are urgently needed. Cyclin-dependent regulates tumor progression by activating the AKT signaling kinase inhibitor 3 (CDKN3) performs crucial roles in the pathway. Therefore, CDKN3 may serve as a potential effective modulation of tumor development. The present study aimed therapeutic target for ESCC treatment. to explore the functions and underlying mechanism of CDKN3 in regulating ESCC cell proliferation and invasion. Introduction The expression levels of CDKN3 in ESCC cells were evalu- ated by reverse transcription-quantitative PCR. Cell counting Esophageal cancer (EC) is one of the most common malig- kit-8 and colony forming assays were used to evaluate cell nancies that leads to high mortality and poor prognosis viability. Wound-healing assay was performed to explore worldwide (1). Approximately 572,034 new diagnosed cases cell migration. Transwell invasion analysis was conducted to and 508,585 deaths occurred in 2018 worldwide (2). In investigate the invasive capacity of ESCC cells. Protein levels China, esophageal squamous cell carcinoma (ESCC), which were detected by western blot assay. The results demonstrated is capable of direct invasion and early metastasis, is the most that the expression of CDKN3 was significantly upregulated prevalent pathological type of EC; ESCC accounted for in ESCC tissues, as predicted using the UALCAN and Gene >90% of EC cases in 2011 in China (3,4). Despite advances in Expression Omnibus databases. PCR and western blot assays diagnostic technologies and treatment, the overall survival of confirmed that CDKN3 was upregulated in ESCC cell lines. patients with ESCC remains poor (5-7). Thus, identifying the Functional assays revealed that CDKN3 knockdown with mechanistic basis of ESCC progression and developing novel small interfering RNA decreased the ability of ESCC cells to therapeutic targets for patients with ESCC is urgently needed. proliferate, invade and migrate and suppressed G1/S transi- Cyclin-dependent kinase inhibitor 3 (CDKN3) serves tion. Further mechanistic analyses demonstrated that CDKN3 crucial roles in the cell cycle and proliferation (8-10). CDKN3 performs its functions by binding to cyclin proteins, which results in dephosphorylation of CDK1 and CDK2 proteins and inhibition of cell cycle progression (11-13). The dynamic expression of CDKN3 and its oncogenic role have been widely Correspondence to: Dr Xia He or Dr Li Yin, Department of explored in various types of cancer (14-16). A previous study Radiotherapy, The Affiliated Cancer Hospital of Nanjing Medical has reported that CDKN3 is upregulated in cervical cancer and University, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer associated with reduced survival time, indicating that it may Research, 42 Bai Zi Ting Road, Nanjing, Jiangsu 210000, P.R. China be a potential biomarker for patients with cervical cancer (15). E-mail: [email protected] E-mail: [email protected] Zang et al (16) have demonstrated that CDKN3 is expressed at high levels in lung adenocarcinoma and is associated with *Contributed equally poor survival outcomes. Silencing CDKN3 suppresses cell proliferation and tumorigenesis in nasopharyngeal carcinoma Key words: esophageal squamous cell carcinoma, cyclin-dependent by regulating the expression of p27 (17). Deng et al (18) kinase inhibitor 3, proliferation, invasion demonstrated that CDKN3 exhibits a high expression in breast cancer cell lines, thus promoting apoptosis and inhibiting cell migration. Xu et al (19) used pathway analysis to explore the YU et al: CDKN3 promotes ESCC cell tumorigenesis Via AKT pathway 543 differentially expressed genes in ESCC; the results indicated solution (10 µl) was added to each well for 1 h at 37˚C and that CDKN3 is upregulated in ESCC and functions as a key absorbance was recorded at 450 nm on days 1, 2, 3 and 4. For gene in signal transduction networks (including PI3K-Akt colony formation assay, transfected ESCC cells (100 cells/well) signaling pathway, and cell cycle). Although a number of were seeded in a 6‑well plate and incubated at 37˚C with 5% studies have demonstrated that CDKN3 expression is upregu- CO2 for 10 days. The colonies were fixed with 4% formalde- lated in ESCC, limited information is available regarding the hyde for 30 min and stained with 0.2% crystal violet for 2 h at function and mechanism of CDKN3 in ESCC development. room at room temperature. Colonies containing >50 cells were In the present study, database search was used to determine counted using a light microscope (magnification, x200) using the levels of CDKN3 expression in ESCC tissues and cells. the ImageJ software (National Institutes of Health). Functional experiments were also employed to explore the functions of CDKN3 in ESCC cells. Wound‑healing assay. Wound-healing assay was used to detect the migration of Eca-109 and TE-1 cells. ESCC cells Materials and methods were transfected with siCDKN3 or si-NC in six-well plates and cultured for 48 h until 90% conﬂuency was reached. A 200‑µl Cell lines. ESCC cell lines EC-1 (cat. no. BNCC339894, l), pipette tip was used to create an artificial wound. The cells EC-7 (named KYSE510; cat. no. BNCC342111), Eca-109 were washed with PBS and cultured in serum-free RPMI-1640 (cat. no. BNCC337687) and TE-1 (cat. no. BNCC100151) were medium. Wound closure was visualized at 0 and 24 h using an obtained from the BeNa Culture Collection. An epithelial cell inverted light microscope (magnification, x200). The distance line Het1A (cat. no. ATCC CRL-2692) was obtained from the between the edges of the wound was calculated as previously American Type Culture Collection. ESCC cells were cultured described. in RPMI‑1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum at 37˚C with 5% CO2. Invasion assay. Matrigel assay was used to determine cell invasiveness. Transfected Eca-109 and TE-1 cells (~2x104) Cell transfection. Small interfering (si)RNAs si-CDKN3 and were harvested, resuspended in 200-µl serum-free RPMI-1640 si-NC were obtained from Guangzhou RiboBio Co., Ltd. The medium and added into the upper Transwell chamber sequences were as follows: CDKN3 siRNA 1, 5'-GTG GAA pre-coated with Matrigel. The lower part of the chamber was TTA TCA CCC ATC A-3'; CDKN3 siRNA 2, 5'-CTG CTT GTC filled with medium containing 20% FBS. Following 12 or 24 h TCC TAC TAT A-3'; si-NC, 5'-GGC UCU AGA AAA GCC UAU of incubation, the cells on the top surface of the filters were GC-3'. For transfection, Eca-109 and TE-1 cells were cultured removed from the upper chamber using a cotton swab. The in 6-well plates (1.5x105 cells/well) and transfected with cells that invaded through the Matrigel membrane were fixed 2 µg si-CDKN3 or 2 µg si-NC using Lipofectamine® iMAX with 4% paraformaldehyde and stained with 0.5% crystal (Invitrogen; Thermo Fisher Scientific, Inc.). Following trans- violet at 37˚C for 2 h at room temperature. Cell images from fection, Eca‑109 and TE‑1 cells were cultured at 37˚C with five randomly fields were selected and counted using a light 5% CO2 for 48 h prior to subsequent experiments. Reverse microscope with x200 magnification. transcription-quantitative PCR (RT-qPCR) and western blot- ting were used to determine transfection efficiency. Flow cytometric analysis. Following transfection, 1x105 cells (Eca-109 and TE-1 cells) were seeded onto 6-well plates and RT‑qPCR assay. RNA was extracted from Eca-109 and TE-1 cultured for 24 h at 37˚C. The cells were harvested and fixed cells using TRIzol® reagent (Invitrogen; Thermo Fisher with 70% ethanol for 24 h at ‑20˚C. The processed cells were Scientific, Inc.). cDNA was reverse‑transcribed from RNA stained with propidium iodide (PI) staining solution (BD using the PrimeScript™ High Fidelity RT-PCR kit (Takara Pharmingen) at 37˚C for 10 min. Cell cycle distribution was Biotechnology Co., Ltd.) for mRNA expression analysis. The analyzed by FACScalibur ﬂow cytometer (BD Biosciences) reaction conditions for reverse transcription were 37˚C for and the ModFit LT (version 3.2) software. All experiments 15 min and 85˚C for 5 sec. RT‑qPCR was conducted using were repeated three times. SYBR®‑Green (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI7500 real-time PCR instrument (Applied Western blot analysis. Transfected Eca-109 and TE-1 cells Biosystems; Thermo Fisher Scientific, Inc.). The thermocy- were suspended and lysed with RIPA buffer (Beyotime cling conditions were as follows: 95˚C for 30 sec, followed by Institute of Biotechnology). Protein was extracted from the 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. The specific cells and quantified using a Bicinchoninic Acid kit (Beyotime primers for CDKN3 and β-actin used were: CDKN3 forward, Institute of Biotechnology).

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