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Silencing of FTX Suppresses Pancreatic Cancer Cell Proliferation and Invasion by Upregulating Mir-513B-5P Shan Li†, Qian Zhang†, Wen Liu and Chunbo Zhao*

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Silencing of FTX Suppresses Pancreatic Cancer Cell Proliferation and Invasion by Upregulating Mir-513B-5P Shan Li†, Qian Zhang†, Wen Liu and Chunbo Zhao* Li et al. BMC Cancer (2021) 21:290 https://doi.org/10.1186/s12885-021-07975-6 RESEARCH ARTICLE Open Access Silencing of FTX suppresses pancreatic cancer cell proliferation and invasion by upregulating miR-513b-5p Shan Li†, Qian Zhang†, Wen Liu and Chunbo Zhao* Abstract Background: Abnormal expression of long non-coding RNA (lncRNA) FTX (five prime to Xist), which is involved in X chromosome inactivation, has been reported in various tumors. However, the effect of FTX on the development of pancreatic cancer (PC) has not been elucidated. The purpose of this study was to explore the possible molecular mechanism of FTX in PC. Methods: Quantitative real-time PCR (qRT-PCR) was used to measure the expression levels of FTX and miR-513b-5p in PC cell lines. Proliferation and apoptosis of PC cells were determined by CCK-8, Edu assay, and flow cytometry. Invasion and migration ability of PC cells were detected by Transwell assay and scratch test. Bioinformatics analysis, luciferase reporter gene assay, and RNA immunoprecipitation (RIP) assay were used to verify the direct binding between FTX and miR-513b-5p. The xenotransplantation mouse model was established to explore the effect of FTX and miR-513b-5p on the PC tumor growth in vivo. Results: The expression levels of FTX were increased in PC cell lines, and silencing of FTX remarkably suppressed the invasion ability and cell viability. Besides, FTX could bind to miR-513b-5p as a competitive endogenous RNA, thus promoting the invasion and proliferation ability of PC cells. Moreover, knockdown of FTX inhibited the tumor growth and increased the expression levels of miR-513b-5p and apoptosis-related proteins in vivo. Conclusions: FTX could directly combine with miR-513b-5p as a competitive endogenous RNA, thus promoting the occurrence and development of PC in vitro and in vivo. Keywords: Pancreatic cancer, Long non-coding RNA FTX, miR-513b-5p, Xenotransplantation mouse model Background and metastasis are important biological characteristics of Pancreatic cancer (PC) is a malignancy of the digestive PC and the main reasons for poor prognosis of PC pa- tract, which is characterized by high mortality rate, poor tients [7]. However, due to the inconspicuous early prognosis, and extremely low cure rate [1–4]. Currently, symptoms and the lack of effective biomarkers for early the main treatment for PC is traditional surgery, radio- diagnosis, most PC patients have shown local invasion therapy and chemotherapy [5]. When clinical symptoms or even distant metastasis when seeking treatment, thus appear, it is often in the middle or late stage, and the 5- missing the best time for treatment [8, 9]. Extensive year survival rate of patients is less than 5% [6]. Invasion studies have identified various genes and proteins in- volved in the regulation of PC invasion and metastasis * Correspondence: [email protected] [10, 11], and the expression and activation of these genes †Shan Li and Qian Zhang contributed equally to this work. Department of Gastrointestinal Radiation Oncology, Cancer Hospital of and proteins are regulated by a variety of molecular Harbin Medical University, 150 Haping Road, Nangang District, Harbin City, pathways [12]. However, the pathogenesis of PC remains Heilongjiang Province 150081, P. R. China © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. BMC Cancer (2021) 21:290 Page 2 of 12 unclear. Therefore, the key to improve the prognosis of Methods PC is to further study the biological characteristics of PC Cell culture and the molecular regulatory mechanism of its occur- HPDE6-C7, HS-766 T, SW1990, AsPC-1P, BxPC-3, and rence and development, and to search for reliable PANC-1 cells were purchased from ATCC (Manassas, markers for early diagnosis and prognosis evaluation. VA). RPMI 1640 medium (GIBCO, Carlsbad, CA) sup- The development of molecular biology provides plemented with 10% Fetal Bovine Serum (FBS, Gibco, new strategies for the early diagnosis and treatment Thermo Fisher Scientific) and 1% streptomycin- of PC [13–15]. Long non-coding RNAs (lncRNAs) penicillin (Gibco, Thermo Fisher Scientific) was used to are non-coding RNAs with a length of more than incubate the cells. All cell lines were cultured in a hu- 200 nucleotides and play important regulatory roles midified incubator at 37 °C with 5% CO2. in the physiological and pathological processes of the body through epigenetic, transcriptional, and Cell transfection post-transcriptional regulations [16]. LncRNAs are Negative control (NC) vector and LV-FTX vector were abnormally expressed in various tumor tissues constructed using lenti-pac HIV expression package showing tissue specificity, making them ideal diag- mixture and lentiviral vector (Gene Copoeia, Rockville, nostic markers and potential therapeutic targets [17, Md) following the manufacturer’s instructions. All cell 18]. Studies have shown that a variety of lncRNAs lines were seeded into 6-well plates and incubated over- played important regulatory roles in the invasion night to reach 4 × 105 cells per well. The original culture and metastasis of PC [19, 20]. For example, HOXA medium was replaced with 2 ml fresh culture medium terminal transcriptional antisense RNA (HOTTIP) is containing 6 g/ml polybrene, and appropriate viral sus- highly expressed in pancreatic ductal adenocarcin- pension was added for incubation at 37 °C. Transfected oma, and silencing of HOTTIP can suppress the cells were incubated for 48 h for subsequent growth and metastasis of PC cells and promote the experiments. apoptosis of cancer cells [21]. LncRNA-ROR can trigger the occurrence of epithelial-mesenchymal transition (EMT) and promote the invasion and me- Cell proliferation and apoptosis assay tastasis of PC [22]. LncRNA-MALAT can maintain Cell viability was determined by CCK-8 kit (Dojindo, the stability of PC stem cells by upregulating SOX2 Japan). The proliferation capacity was detected using ™ [19]. Studies have found that lncRNA FTX (five Cell Light EdU kit (RiboBio, Nanjing, China). After pre- μ prime to Xist) was abnormally expressed in various treatment, 50 M EdU was added to the medium to cul- cancers, such as hepatocellular carcinoma, renal cell ture cells for another 2 h. After fixed with 4% carcinoma and colorectal cancer [23–25], but the paraformaldehyde, the cells were staining with Hoechst 33342 (1:10,000, Sigma, USA) at room temperature for regulation of lncRNA FTX in PC remains unclear. 5 MicroRNAs (miRNAs) are non-coding miRNAs with 10 min. EdU positive cells (1 × 10 ) were counted by about 18–25 nucleotide in length and can inhibit the ex- fluorescence microscope (Nikon, Tokyo, Japan). More- pression of target mRNAs by targeting their 3′-UTR re- over, cell apoptosis was measured using Annexin V- gion [26]. Altered expression of miRNAs have been FITC/PI kit (KeyGEN, Nanjing, China) for 15 min in dark. The Negative control (NC) and LV-FTX cells (1 × shown to play important roles in the invasion and me- 5 tastasis of PC [27]. It was reported that miR-513b-5p 10 ) were washed twice with PBS and re-suspended in had low expression levels in gastric cancer, lung cancer PBS, then stained with Annexin V (1:1000) and PI for and colon cancer, and acted as an oncogene [28–30]. 15 min in dark. Apoptosis rates were analyzed using However, the expression of lncRNA FTX in PC and its FACSAria flow cytometry (BD Biosciences, Franklin biological mechanism are unclear. Lakes, NJ). In this study, the expression levels of lncRNA FTX in PC cell lines were detected, and appropriate PC Cell cycle detection cells were selected to verify the interaction between Cells were inoculated into a 6-well plate with a cell lncRNA FTX and miR-513b-5p. In addition, changes density of 1 × 106 cells per well. After 24 h, the cells were in the biological behaviors of PC cells such as prolif- digested by trypsin and fine cells in the supernatant were eration, migration, invasion and apoptosis were de- collected and washed with PBS for 3 times. Cells were tected by inhibiting the expression of lncRNA FTX. then fixed with 70% ethanol, and each cell sample was The aim of this study was to explore the role of FTX resuspended and added with 500 L pre-mixed PI dye. and miR-513b-5p in PC and their possible mecha- Cells were resuspended and incubated in dark for 30 nisms, hoping to provide insights into the early diag- min. GO/G1 cell rations were analyzed using FACSAria nosis and treatment of PC. flow cytometry (BD Biosciences, Franklin Lakes, NJ). Li et al. BMC Cancer (2021) 21:290 Page 3 of 12 Cell migration and invasion assay and the relative expression levels of protein were quanti- For cell migration assay, all cell lines after transfection tatively analyzed by ImageJ software.
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