Shen et al. Cell Death and Disease (2021) 12:349 https://doi.org/10.1038/s41419-021-03571-5 Cell Death & Disease ARTICLE Open Access ZEB1-induced LINC01559 expedites cell proliferation, migration and EMT process in gastric cancer through recruiting IGF2BP2 to stabilize ZEB1 expression Huojian Shen1, Hongyi Zhu1, Yuanwen Chen1, Zhiyong Shen1, Weiqing Qiu1, Changlin Qian1 and Jie Zhang1 Abstract Gastric cancer (GC) is a common type of tumor that is characterized with high metastatic rate. In recent years, increasing studies have indicated that lncRNAs are involved in the regulation on cancer cell proliferation and migration. However, the functional role of long intergenic non-protein coding RNA 1559 (LINC01559) in GC is still unclear. In this study, we applied quantitative real-time polymerase chain reaction (RT-qPCR) and examined that LINC01559 expression was significantly enhanced in GC cells. Functional assays such as EdU, colony formation, JC-1 and transwell assays displayed that silencing LINC01559 inhibited cell proliferation and migration while promoted cell apoptosis in GC. Besides, western blot analysis and immunofluorescence assays examined the expression of factors related to epithelial- mesenchymal transition (EMT) and indicated that EMT process was blocked by LINC01559 knockdown in GC cells. Besides, LINC01559 silencing inhibited tumor growth in vivo. In addition, Chromatin immunoprecipitation (ChIP) assays demonstrated that zinc finger E-box binding homeobox 1 (ZEB1) served as a transcription factor to combine with 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; LINC01559 promoter and activated the expression of LINC01559 in GC cells. In return, LINC01559 recruited insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) to stabilize ZEB1 mRNA to up-regulate ZEB1 in GC cells. In short, the findings in this research might provide a novel target for GC treatment. Introduction metastasis. Therefore, early diagnosis of GC is of sig- Nowadays, gastric cancer (GC) is one of the most nificant importance for prognosis5. common cancers worldwide1. In East Asia, especially in Long non-coding RNAs (lncRNAs) are a type of dis- Korea, Mongolia, Japan, and China, GC-associated covered RNAs with the length over 200 nucleotides and morbidity and mortality have been shown to be rela- without the ability of coding proteins6,7.LncRNAsare tively higher than in most western countries2,3. Despite involved in a wide range of biological processes in various advances in diagnostic techniques and improvements in cancers, such as cell proliferation, migration and apopto- chemotherapy and radiotherapy, patients with advanced sis8. Abnormally expressed lncRNAs can serves as onco- or metastatic GC still present a poor prognosis4.Oneof genes or tumor suppressors to affect the progression and the main causes of cancer-related mortality is tumor metastasis of cancers9,10. Mechanistically, lncRNAs exert their functions majorly through the interaction with RNAs or proteins in the regulation of gene expression11.Inrecent years, a large number of studies have shown that lncRNAs Correspondence: Jie Zhang ([email protected]) play vital roles in the progression of GC, such as MEG312, 1 fi Renji Hospital Af liated to Medical College of Shanghai Jiaotong University, HOXA11-AS13 and LINC0033714.However,therearestill Shanghai 200025, China These authors contributed equally: Huojian Shen, Hongyi Zhu Edited by G. Calin © 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 linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Shen et al. Cell Death and Disease (2021) 12:349 Page 2 of 13 some unknown lncRNAs whose mechanisms need to be medium (Invitrogen, Carlsbad, CA, USA). The culture further explored in GC. media were all supplemented with 10% FBS and 1% P/S. Zinc finger E-box binding homeobox 1 (ZEB1) is a All cells were grown in a humidified atmosphere of 5% transcription factor registered by some studies. In prostate CO2 at 37 °C. cancer cells, ZEB1 is a key promoter of tumor progres- sion15. In breast cancer cells, ZEB1 induces transcrip- Cell transfection tional regulation of genes involved in the cancer Specific shRNAs targeting LINC01559 (sh/ progression16. Thus, it is of great interest and importance LINC01559#1/#2) or IGF2BP2 (sh/IGF2BP2#1/#2) and to identify the association between lncRNAs and ZEB1 in control group (sh/Ctrl) were synthesized by GenePharma the progression of GC. (Shanghai, China). The pcDNA3.1 vectors from Invitrogen In addition to target and sequester specific microRNAs were utilized to construct pcDNA3.1/ZEB1, pcDNA3.1/ (miRNAs), lncRNAs can function in the progression of MAFK, pcDNA3.1/JUND, pcDNA3.1/JUN, pcDNA3.1/ cancers through recruiting RNA-binding proteins ESR1 and pcDNA3.1/RFX5, with the empty vector as the (RBPs)17. Abundant studies have demonstrated the par- negative control. Lipofectamine 3000 (Invitrogen) was used ticipation of RBPs in the maintenance of mRNA stability to execute transfection experiments for 48 h. by lncRNAs in cancer cells. For instance, CERS6-AS1 promotes the progression and metastasis of breast cancer Quantitative real-time polymerase chain reaction (RT- by recruiting with IGF2BP3 to increase the stability of qPCR) CERS6 mRNA18. Besides, LIN28B-AS1 interacts with by Total RNA was extracted from GC cells using the IGF2BP1 to activate LIN28B expression in lung adeno- TRIzol reagent according to manufacturer’s requirements carcinoma cells19. In our study, we also investigated (Thermo Fisher Scientific), and then cDNA was obtained whether LINC01559 could recruit certain protein partner after reverse transcription. RT-qPCR was performed with to exert its function in GC cells. SYBR Green Master Mixture (Takara, Dalian, China). Epithelial-to-mesenchymal transition (EMT) is a cel- GAPDH was used as the control. Relative gene expression −ΔΔ lular process related to metastasis20. EMT is essential for levels were calculated using the 2 Ct method. The embryonic development and has been associated with a experiment was repeated for three times. variety of diseases. EMT-related transcription factors (Snail, Twist, and ZEB1) can induce EMT process in vitro 5-ethynyl-2’-deoxyuridine (EdU) assay and in vivo21. However, whether and how EMT is After transfection, GC cells were seeded onto 96-well modulated by LINC01559 in GC cells remain to be fur- plates. The EdU (5-ethynyl-20-deoxyuridine) Apollo- ther specified. 567 Kit (RIBOBIO, Shanghai, China) was used to In conclusion, our study expounded that LINC01559 quantify the proliferation ability of indicated GC cells. expression was up-regulated in GC cells. Knockdown of EdU and DAPI dyes were exploited to treat GC cells. LINC01559 inhibited cell proliferation, migration and A fluorescent microscope was used for cell visualization EMT process in GC. Moreover, ZEB1 could activate the (Olympus, Tokyo, Japan). The experiment was repeated expression of LINC01559, and LINC01559 recruited for three times. insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) to stabilize ZEB1 mRNA in GC cells. In a Colony formation assay word, this study might be helpful to identify effective GC cells were seeded into 6-well plate at the density of treatments for GC. 500 cells per well. After 2 weeks, colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet Materials and methods (DINGGUO) and then counted manually. The experi- Cell culture ment was repeated for three times. Human GC cell lines (SNU-1 and AGS) were purchased from American Type Culture Collection (ATCC) cell Flow cytometry analysis bank (Manassas, VA); another two kinds of human GC The apoptosis rate of SNU-1 and AGS cells was cell lines (MKN45 and HGC-27) were gotten from the analyzed with propidium iodide (PI)/Annexin V Cell Institute of Biochemistry and Cell Biology, Chinese Apoptosis Kit (Invitrogen, V13245) in reference to man- Academy of Sciences (Shanghai, China); the normal ufacturer’s suggestions. In short, after 48 h transfection, human gastric epithelial cell line (GES-1) was obtained SNU-1 and AGS cells were stained by FITC-Annexin V from Procell Life Science & Technology Co., Ltd. and PI. Finally, cell apoptosis rate was examined by (Wuhan, China). SNU-1, MKN45, HGC-27 and GES-1 flow cytometry (FACSCantoII, 338960; BD Biosciences, cells were cultivated in RPMI-1640 medium (Invitrogen, San Jose, CA, USA). The experiment was repeated for Carlsbad, CA, USA). AGS cells was cultivated in F-12K three times. Official journal of the Cell Death Differentiation Association Shen et al. Cell Death and Disease (2021) 12:349 Page 3 of 13 JC-1 assay groups of nude mice. The tumor volume was tested every Cells were incubated with 500 μl JC-1 staining working four days and tumor growth lasted for 4 weeks. After that, solution for 20 min at 37 °C (Beyotime, China). The the mice were mercy killing and their tumors were taken fluorescence labeled cells were washed using phosphate out. The tumor weight was calculated and the tumors buffered saline (PBS) and analyzed by an EnSpire Reader. were collected for following analysis. The data and images were processed by GraphPad Prism 6.0 statistical software. The experiment was repeated for Immunohistochemical (IHC) assay three times. The collected tumor tissues from xenograft assay were dehydrated after fixing in 4% paraformaldehyde, and then Transwell assay embedded in paraffin and cut into sections of 4 μm thick.