Lncrna GAS5 Inhibits Cellular Proliferation by Targeting P27kip1
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Published OnlineFirst April 10, 2017; DOI: 10.1158/1541-7786.MCR-16-0331 Cell Cycle and Senescence Molecular Cancer Research LncRNA GAS5 Inhibits Cellular Proliferation by Targeting P27Kip1 Gang Luo1,2, Dong Liu1, Chao Huang1, Miao Wang1, Xingyuan Xiao1, Fuqing Zeng1, Liang Wang1, and Guosong Jiang1 Abstract Recent studies have demonstrated that long noncoding RNAs regulator of cell cycle, was positively regulated by GAS5 and (lncRNA) have important roles in cancer biology, and that the upregulation of GAS5 increased its promoter activity. E2F1, an downregulation of lncRNA growth arrest–specifictranscript5 important transcription factor, was shown to bind directly to (GAS5) has been reported in a variety of human cancers. and activate the P27Kip1 promoter. In addition, GAS5 interacted However, its role in prostate cancer is largely unknown. This with E2F1 and enhanced the binding of E2F1 to the P27Kip1 study aims to investigate the biological role and underlying promoter. Collectively, these findings determine that GAS5 mechanism of GAS5 on proliferation in prostate cancer. The functions as a tumor suppressor in prostate cancer develop- results demonstrate that GAS5 expression is significantly ment and progression via targeting P27Kip1. decreased in prostate cancer cells compared with prostate epithelial cells. Ectopic expression of GAS5 inhibited cell pro- Implications: This study reveals a molecular pathway involving Kip1 liferation and induced a cell-cycle arrest in G0–G1 phase, lncRNA GAS5/E2F1/P27 which regulates cell proliferation whereas GAS5 knockdown promoted the G1–S phase transi- and could be a potential therapeutic target in prostate cancer. tion. Subsequent analysis demonstrated that P27Kip1,aknown Mol Cancer Res; 15(7); 789–99. Ó2017 AACR. Introduction identifying potential tumor-suppressor genes enriched during growth arrest (13). Previous studies have revealed that the expres- Prostate cancer is the second most frequently diagnosed cancer sion level of GAS5 is decreased in many kinds of cancers (14–16). in men worldwide, accounting for 15% of all male cancers (1). In Furthermore, overexpression of GAS5 could lead to cell-cycle 2015, there were 220,800 estimated new cases of prostate cancer arrest or cell apoptosis in several human cancers including lung, and 27,540 deaths by prostate cancer, making this disease the bladder, kidney, and other cancers (17–20). A recent study second leading cause of cancer-related death for North American showed that GAS5 gene expression was reduced in prostate cell men (2). Androgen deprivation therapy is an effective initial lines derived from metastases compared with those derived from treatment for prostate cancer, but almost all these patients even- normal prostate or primary prostate cancer (14). In addition, tually develop into castration-resistant prostate cancer, which is a further research revealed that high level of GAS5 promoted basal major cause of death (3–6). In consequence, better understanding apoptosis and enhanced the action of apoptotic stimuli in pros- of the tumorigenesis is essential for the development of diagnostic tate cancer cells (21). Despite these findings, the effects of GAS5 markers and novel effective therapies for prostate cancer patients. on prostate cell proliferation and the underlying mechanism Long noncoding RNAs (LncRNA), defined as transcripts con- remain largely unknown. taining >200 nucleotides without evident protein coding func- In the present study, we assessed the expression level of GAS5 in tion, were once considered to be transcriptional "noise" (7, 8). prostate cancer cells, investigated its effect on cell proliferation in Growing evidence indicates that lncRNAs participate in diverse vitro and in vivo, and explored the underlying mechanism. Our cellular processes, including cell differentiation, proliferation, results demonstrate that GAS5 plays an important role in the and apoptosis (9–12). The growth arrest–specific transcript 5 genesis and development of prostate cancer and may be a poten- (GAS5) was originally isolated from a subtraction cDNA library tial therapeutic target. 1Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 2Department of Urology, Materials and Methods The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Plasmid and RNA interference Science and Technology, Wuhan, China. The GAS5 sequence was synthesized according to the full- G. Luo, D. Liu, and C. Huang contributed equally to this article and should be length GAS5 sequence lacking a poly A tail (based on the GAS5 considered cofirst authors. sequence, NR_002578, in NCBI) and then subcloned into GV144 Corresponding Authors: Guosong Jiang, Union Hospital, Tongji Medical College, vector (GV144-GAS5; Genechem Co. Ltd.). Empty vector was Huazhong University of Science and Technology, NO. 1277, Jiefang Road, Wuhan used as a control. The shRNA targeting GAS5 (sh-GAS5) was 430022, Hubei Province, China. Phone: 86-27-8535-1624; Fax: 86-27-8365-1606; designed and synthesized by Genechem. The sequence was: sense, E-mail: [email protected]; and Liang Wang, 50-ccggGGACCAGCTTAATGGTTCTttcaagagaAGAACCATTAAGC- [email protected] TGGTCCtttttg-30; anti-sense, 50-aattcaaaaaGGACCAGCTTAATG- doi: 10.1158/1541-7786.MCR-16-0331 GTTCTtctcttgaaAGAACCATTAAGCTGGTCC-30. It was scrambled Ó2017 American Association for Cancer Research. to generate a negative control. GV122 vector expressing the shRNA www.aacrjournals.org 789 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst April 10, 2017; DOI: 10.1158/1541-7786.MCR-16-0331 Luo et al. was constructed by Genechem. The siRNA targeting E2F1 (siE2F1) iodide (2 mg/mL, Keygen biotech) for 30 minutes at room and P27Kip1 (siP27) were also obtained from Genechem. si-E2F1 temperature, followed by flow cytometry analysis (FACScan, sequence: 50-AACUCCUCGCAGAUCGUCAUC-30. siP27 sequence: Becton Dickinson). 50-GGAGCAATGCGCAGGAAUATT-30. Tumor xenograft models Cell culture and transfection Male BALB/c-nude mice (4 weeks old) were purchased from The human prostate cancer cell lines PC3 and DU145 as well as Hunan SJA Laboratory Animal Co., Ltd. A total of 5 Â 106 stably benign prostate epithelial cells PNT2C2 were purchased from transfected PC3 cells in 200 mL of sterile PBS were injected American Type Culture Collection. Cells were cultured in RPMI- subcutaneously into the right flanks of the mice. The tumors were 1640 (Gibco) supplemented with 10% FBS (HyClone), 100 units/ measured every 3 days. Three weeks after inoculation, the mice mL penicillin (Gibco), and 100 mg/mL streptomycin (Gibco) at were sacrificed by cervical dislocation, and the tumors were 37 C in a humid atmosphere with 5% CO2. GV144-GAS5, resected, measured, and weighed. The tumor volume was calcu- GV144, sh-GAS5, or sh-NC were transfected into PC3 and DU145 lated according to the following formula: volume ¼ 0.5 Â W2 Â L cells using Lipofectamine2000 Transfection Reagent (Invitrogen) (W, width; L, length). Animal care and protocols were approved according to the manufacturer's instructions. Stable cell lines were by the Institutional Animal Care and Use Committee of Huaz- screened by the treatment with G418 (Invitrogen). hong University of Science and Technology. RNA extraction and RT-PCR analysis Immunohistochemistry analysis Total RNA was isolated from cultured cells with TRIzol reagent The primary antibody used to detect P27Kip1 was purchased according to the manufacturer's protocol (Invitrogen). The iso- from Santa Cruz Biotechnology. Tissue sections were deparaf- lated RNA was reverse transcribed into cDNA using a reverse finized in xylene and rehydrated with ethanol. Then, tissue transcription kit (Takara Bio Inc.). Quantitative real-time PCR sections were incubated with 10% normal goat serum followed was performed using a standard protocol from the SYBR Green with incubation with primary antibody overnight at 4C. After PCR Kit (Toyobo) on the StepOnePlus Real-Time PCR System being washed with PBS, the sections were incubated with (Applied Biosystems). The PCR primers were as follows: biotinylated secondary antibody, followed by further incuba- tion with streptavidin–horseradish peroxidase (HRP) at 37C 50-GAAGGTGAAGGTCGGAGTC-30(GAPDH-forward), for 30 minutes. Color was developed using 3,3-diaminobenzi- 50-GAAGATGGTGATGGGATTTC-30(GAPDH-reverse), dine for 10 minutes, and then the sections were counterstained 50-ACAGGCATTAGACAGAAAGC-30(GAS5-forward), with hematoxylin. 50-TACCCAAGCAAGTCATCCA-30(GAS5-reverse), 0 0 Kip1 5 -AGACGGGGTTAGCGGAGCAA-3 (P27 -forward), Western blot assay 0 0 Kip1 5 -TCTTGGGCGTCTGCTCCACA-3 (P27 - reverse). Transfected cells were resuspended in RIPA lysis buffer fi m The PCR reaction was conducted at 95C for 30 seconds (Thermo Scienti c). Note that 20 g of total protein was followed by 40 cycles of 95C for 5 seconds and 60C for 30 separated by electrophoresis, transferred onto PVDF mem- branes, and incubated with primary antibodies for GAPDH, seconds. Each sample was analyzed in triplicate, and the relative ÀDD Kip1 quantification of GAS5 expression was calculated using the 2 Ct P27 , P21, cyclin E, cyclin D1, and E2F1 overnight at 4 C. The fi method relative to GAPDH. speci c antibodies were purchased from Cell Signaling Tech- nology. Subsequent to being washed with Tris-buffered saline MTT assay containing Tween-20, the membranes were incubated with the After transfection, prostate