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Effects of Lncrna ANRIL‑Knockdown on the Proliferation, Apoptosis and Cell Cycle of Gastric Cancer Cells

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Effects of Lncrna ANRIL‑Knockdown on the Proliferation, Apoptosis and Cell Cycle of Gastric Cancer Cells ONCOLOGY LETTERS 22: 621, 2021 Effects of lncRNA ANRIL‑knockdown on the proliferation, apoptosis and cell cycle of gastric cancer cells XUEQIAN HU, TING LOU, CHUNYING YUAN, YONGSHENG WANG, XIAOLONG TU, YI WANG and TINGSU ZHANG Department of Oncology, Ningbo Municipal Hospital of Traditional Chinese Medicine, Affiliated Hospital of Zhejiang Chinese Medical University, Ningbo, Zhejiang 315000, P.R. China Received February 22, 2021; Accepted June 2, 2021 DOI: 10.3892/ol.2021.12882 Abstract. Gastric cancer is one of the most common types miR‑181a‑5p, inhibited the proliferation of drug‑resistant cells of malignant tumor of the gastrointestinal tract worldwide. and enhanced their sensitivity to DDP. Cisplatin (DDP) is a commonly used chemotherapeutic drug in the clinic; however, the resistance of gastric cancer cells Introduction to DDP limits its efficacy. In the present study, drug‑resistant gastric cancer cell lines were constructed using the stepwise Gastric cancer is one of the most common types of malignant continuous selection method, and the relative expression levels tumor in the gastrointestinal tract worldwide. Its morbidity and of long non‑coding RNA (lncRNA) CDKN2B antisense RNA 1 mortality are ranked fourth and fifth, respectively, amongst all (ANRIL) and microRNA (miR)‑181a‑5p were detected using cancer types globally (1,2). The development of gastroscopy reverse transcription‑quantitative PCR. The knockdown of technology has significantly improved the diagnostic and lncRNA ANRIL and miR‑181a‑5p expression was performed cure rates of patients with early gastric cancer. Except for a by transfection with shRNA‑ANRIL and an miR‑181a‑5p small number of early gastric cancer cases with small lesion inhibitor, respectively. Cellular proliferation and sensitivity to areas, gastroscopy is suitable for endoscopic treatment. For DDP were assessed using Cell Counting Kit‑8 analysis. Cell other cases of gastric cancer, surgery and chemotherapy apoptosis and cell cycle distribution were assessed using flow remain the primary treatment options (3,4). Cisplatin (DDP) cytometry and western blotting. The binding relationships is a commonly used chemotherapeutic drug in the clinic; between ANRIL, miR‑181a‑5p and cyclin G1 (CCNG1) were however, the insensitivity of gastric cancer cells to DDP limits verified using a dual luciferase reporter assay. The results its efficacy (5). Therefore, further in‑depth studies into the revealed that the expression levels of miR‑181a‑5p were down‑ molecular mechanisms of gastric cancer and cellular resistance regulated in all drug‑resistant cell lines. ANRIL‑knockdown are required to identify novel therapeutic targets and implement inhibited cellular proliferation, and promoted apoptosis individualized treatment strategies for gastric cancer. and cell cycle arrest; however, following the knockdown of Long non‑coding RNAs (lncRNA) are a type of non‑coding miR‑181a‑5p, the inhibition of cell cycle arrest was alleviated. RNA of >200 nucleotides in length, which contain neither a Notably, miR‑181a‑5p, ANRIL and CCNG1 were found to start nor stop codon (6). LncRNAs have been revealed to play have targeting relationships. In conclusion, the findings of the vital roles in multiple cellular processes, such as gene expression present study suggested that knocking down the expression of regulation, genome imprinting and chromatin packaging, and ANRIL inhibited cellular proliferation, and promoted apop‑ during various stages, including cellular differentiation and tosis and cell cycle arrest. Furthermore, its downstream target, embryonic development (7). lncRNA CDKN2B antisense RNA 1 (ANRIL) is an antisense non‑coding RNA (8), and a 403‑kb deletion in the gene was first identified in patients with melanoma and tumors of the nervous system (9). ANRIL is expressed in a variety of normal tissues, with the highest expression levels found in ovarian tissue and the lowest in muscle Correspondence to: Dr Tingsu Zhang, Department of Oncology, tissue (10). In addition, previous studies have reported that the Ningbo Municipal Hospital of Traditional Chinese Medicine, Affiliated Hospital of Zhejiang Chinese Medical University, expression levels of ANRIL were significantly upregulated 819 North Liyuan Road, Haishu, Ningbo, Zhejiang 315000, in lung cancer, liver cancer and esophageal squamous cell P. R. Ch i na carcinoma, which suggests that ANRIL may function as an E‑mail: [email protected] oncogene (11,12). It is well established that lncRNAs can regulate microRNA Key words: gastric cancer, resistance, long non‑coding RNA (miRNA/miR) expression and thereby influence tumor CDKN2B antisense RNA 1, microRNA‑181a‑5p, cyclin G1 progression (13). A large number of studies have reported that miRNAs bind to target genes to regulate the occurrence and development of numerous tumor types (14). Previous research 2 HU et al: EFFECTS OF lncRNA ANRIL ON GASTRIC CANCER CELLS has also shown that the expression of miR‑181a‑5p was down‑ provided by GenePharma Co., Ltd. Following transfection for regulated in gastric cancer and that the overexpression of 24 h, the cells were utilized for subsequent experiments. miR‑181a‑5p inhibited cellular proliferation (15). Drug resistance in gastric cancer cells limits the efficacy Cell Counting Kit‑8 (CCK‑8) assay. Transfected cells in the of drugs in the clinic. The present study aimed to determine logarithmic growth phase were digested with 0.25% trypsin, the effects of ANRIL and miR‑181‑5p on DDP‑sensitive seeded into a 96‑well plate at a density of 5x104 cells/well, and and ‑resistant gastric cancer cells, as well as the underlying incubated at 37˚C for 24 h. Following incubation, 10 µl CCK‑8 mechanisms of action. The results may provide novel targets reagent (Beyotime Institute of Biotechnology) was added to each to overcome drug resistance in gastric cancer. well and the plate was incubated for a further hour. The optical density of each well was measured at a wavelength of 450 nm Materials and methods using a microplate reader (Thermo Fisher Scientific, Inc.). Cell lines and culture. Normal gastric epithelial cells (GES1) Flow cytometry. For apoptosis analysis, cells were digested with and gastric cancer cell lines (HGC‑27, AGS, HSC‑39 and 0.25% trypsin and washed twice with PBS. Cells (1x105 per FU97) were purchased from the American Type Culture sample) were subsequently centrifuged at 150 x g, resuspended Collection. DDP (purity >99.99%) was purchased from Merck in binding buffer, and then incubated with Annexin V‑FITC in KGaA. Cells were cultured in DMEM (Gibco; Thermo Fisher the dark at room temperature for 30 min. The cells were then Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo incubated at room temperature with propidium iodide (PI) in Fisher Scientific, Inc.), and maintained at 37˚C with 5% CO2. the dark for 5 min. For cell cycle distribution analysis, following Cells were exposed to increasing concentrations of DDP to centrifugation at 150 x g and resuspension in PBS, the cells were establish DDP‑resistant gastric cancer cells. Briefly, cells in the incubated with PI staining solution (50 mg/l P1, 1 g/l Triton X logarithmic growth phase were first cultured with 0.05 µg/ml and 100 g/l RNase) in the dark at 4˚C for 30 min. Data were DDP. Following a week, the surviving cells were subsequently analyzed using FlowJo (v7.6.1; FlowJo, LLC) following flow cultured with increasing concentrations of DDP until the cells cytometry on a FACSCalibur flow cytometer (BD Biosciences). could be stably subcultured in medium containing 0.5 µg/ml DDP. Each time the concentration of DDP was increased Western blotting. Total protein was extracted from cells using by 1.2 or 1.5 times, the process experienced 9 times of R I PA lysis (Beyotime I nstit ute of Biotech nology), a nd qua ntified concentration increase and six months in total. using a BCA assay kit (Beyotime Institute of Biotechnology). Protein samples (25 µg) were separated via 10% SDS‑PAGE, Reverse transcription‑quantitative PCR (RT‑qPCR). For transferred onto PVDF membranes, and then blocked with mRNA expression analysis, total RNA was extracted from 5% BSA (Thermo Fisher Scientific, Inc.) at room temperature cultured cells using TRIzol® reagent (Invitrogen; Thermo for 1 h. The membranes were subsequently incubated with Fisher Scientific, Inc.). Total mRNA was reverse transcribed the following primary antibodies at 4˚C overnight: Anti‑p53 into cDNA using the FSQ‑101 reverse transcription system (cat. no. MA5‑16387; 1:1,000), anti‑Bax (cat. no. PA5‑11378; kit (Toyobo Life Science), and qPCR was subsequently 1:2,000), anti‑Bcl‑2 (cat. no. PA5‑27094; 1:1,000), anti‑cleaved performed using the QuantiTect SYBR‑Green PCR kit caspase‑3 (cat. no. PA5‑114687; 1:1,000), anti‑cyclin G1 (Qiagen, Inc.). For miRNA expression analysis, total RNA (CCNG1; cat. no. PA5‑36050; 1:1,000), anti‑cyclin D1 was extracted from cells using the fast microRNA isolation (CCND1; cat. no. PA5‑32373; 1:1,000), anti‑CDK4 (cat. kit (BioTeke Corporation). Reverse transcription and qPCR no. PA5‑27827; 1:1,000) and anti‑GAPDH (cat. no. MA1‑16757; were subsequently performed using the miScript RT kit 1:2,000) (all Thermo Fisher Scientific, Inc.). Following primary and miScript SYBR Green qPCR kit (GeneCopoeia, Inc.), antibody incubation, the membranes were incubated with an respectively, according to the manufacturers' protocols. HRP‑conjugated goat anti‑rabbit antibody (cat. no. G‑21234; The qPCR thermocycling conditions were as follows: Thermo Fisher Scientific, Inc.; 1:100,000) at room temperature Pre‑denaturation at 95˚C for 30 sec, followed by 40 cycles of for 1 h. Protein bands were visualized using chemiluminescent denaturation at 95˚C for 5 sec, annealing at 60˚C for 30 sec, reagents and densitometric analysis was performed using and extension at 72˚C for 10 sec. The primers used for qPCR ImageJ version 1.52 software (National Institutes of Health). are listed in Table I.
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