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Twist1 Regulates Vimentin Through Cul2 Circular RNA to Promote EMT

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Published OnlineFirst May 29, 2018; DOI: 10.1158/0008-5472.CAN-17-3009 Cancer Molecular Cell Biology Research Twist1 Regulates Vimentin through Cul2 Circular RNA to Promote EMT in Hepatocellular Carcinoma Jing Meng1, Shuang Chen2, Jing-Xia Han1, Baoxin Qian3, Xiao-Rui Wang4, Wei-Long Zhong1, Yuan Qin1, Heng Zhang1, Wan-Feng Gao1,2, Yue-Yang Lei1,2, Wei Yang2, Lan Yang2, Chao Zhang1,2, Hui-Juan Liu2,4, Yan-Rong Liu2, Hong-Gang Zhou1, Tao Sun1,2, and Cheng Yang1,2 Abstract Twist is a critical epithelial–mesenchymal transition (EMT)–inducing transcription fac- Epithelial cells tor that increases expression of vimentin. How Twist1 regulates this expression remains unclear. Here, we report that Twist1 regulates Cullin2 (Cul2) circular RNA to increase E-cadherin expressionofvimentininEMT.Twist1bound Twist1 theCul2promotertoactivateitstranscription Cul2 pre-mRNA and to selectively promote expression of Cul2 circular RNA (circ-10720), but not mRNA. EMT circ-10720 positively correlated with Twist1, tumor malignance, and poor prognosis in circRNA-10720 hepatocellular carcinoma (HCC). Twist1 pro- moted vimentin expression by increasing Vimentin miRNA levels of circ-10720, which can absorb miR- NAs that target vimentin. circ-10720 knock- down counteracted the tumor-promoting Vimentin activity of Twist1 in vitro and in patient- derived xenograft and diethylnitrosamine- Mesenchymal cells induced TetOn-Twist1 transgenic mouse HCC Twist1 regulates vimentin by upregulating the expression of circ-10720, which absorbs miRNAs models. These data unveil a mechanism by targeting vimentin, thereby promoting epithelial-mesenchymal transition in hepatocellular which Twist1 regulates vimentin during EMT. carcinoma. They also provide potential therapeutic targets © 2018 American Association for Cancer Research for HCC treatment and provide new insight forcircularRNA(circRNA)-baseddiagnostic and therapeutic strategies. Significance: A circRNA-based mechanism drives Twist1-mediated regulation of vimentin during EMT and provides potential therapeutic targets for treatment of HCC. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4150/F1.large.jpg. Cancer Res; 78(15); 4150–62. Ó2018 AACR. Introduction exhibit increased motility and acquire a mesenchymal phenotype Epithelial–mesenchymal transition (EMT) is essential for (3–5). Twist1 is one of the EMT-inducing transcription factors, tumor metastasis (1, 2) and involves a cellular reprogramming which govern the transcription of EMT-associated genes via pro- process in which epithelial cells dramatically alter their shape, moter activation or repression, downregulating the epithelial 1State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, J. Meng, S. Chen, J.-X. Han, and B. Qian contributed equally to this article. Nankai University, Tianjin, China. 2Tianjin Key Laboratory of Molecular Drug Corresponding Authors: Tao Sun and Cheng Yang, Nankai University, Haihe Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China. River Education Park, 38 Tongyan Road, Tianjin 300450, China. Phone: 8613- 3Department of Gastroenterology and Hepatology, Tianjin Key Laboratory of 5129-22691; Fax: 022-6537-8009; E-mail: [email protected]; Artificial Cells, Tianjin Institute of Hepatobiliary Disease, Tianjin Third Central [email protected] Hospital, Tianjin, China. 4College of Life Science, Nankai University, Tianjin, China. doi: 10.1158/0008-5472.CAN-17-3009 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Ó2018 American Association for Cancer Research. 4150 Cancer Res; 78(15) August 1, 2018 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 29, 2018; DOI: 10.1158/0008-5472.CAN-17-3009 Twist1 Regulates Vimentin through circRNA-Cul2 in HCC phenotype-related genes, such as E-cadherin, and upregulating inspection and biomarkers detection of hepatocellular carcino- the mesenchymal cell phenotype-related genes, such as vimentin ma, growth curve analysis, and Mycoplasma testing. The plasmid (6). Vimentin is a type 3 intermediate filament protein that is information is provided in Supplementary Table S1 of the Sup- a critical EMT mesenchymal cell marker. Vimentin drives cell plementary Data. The cloning primers and circ-10720 siRNA are plasticity (7, 8) and is associated with poor prognosis and high provided in Supplementary Table S2. siRNA, miRNA mimic and frequency of metastasis (5, 9, 10). So far, several cis-elements and mutant miRNAs were synthesized by Genepharma. All the plas- associated factors were reported to be required to regulate vimen- mids or miRNA mimic were transfected into cells using Lipofec- tin expression (11). For example, Sp1/Sp3 binds GC-Box 1 (12), tamine 2000 (Invitrogen) or nanoparticle (Micropoly Biotech) Stat1/Stat3 bind the ASE (13, 14) and c-Jun can form either homo- transfection reagents. The lentiviral vector pLCDH-ciR carrying or heterodimers with other AP-1 family members and bind the circ-10720 and two assistant vectors-psPAX2, pMD2.G were tran- AP-1 sites (15), all of which can increase vimentin transcription. siently transfected into HEK293T cells. Viral supernatants were In addition, phosphorylated Slug does not directly bind the collected 48 hours later, clarified by filtration and concentrated by vimentin promoter, but it is likely to recruit additional transcrip- ultracentrifugation. tion factors to upregulate vimentin (16). There is evidence that Twist1 upregulates vimentin expression in EMT (17), but the qRT-PCR fi regulatory mechanism remains unclear, and there is no de nitive Total RNA was isolated using TRIzol reagent (Invitrogen). Twist1-binding site on the vimentin promoter. cDNA synthesis was performed with Oligo(dT), random or Cullin2 (Cul2) is a core component of multiple ECS miRNA-specific stem-loop primers using a Quantscript RT Kit – (ElonginB/C-CUL2/5-SOCS-box protein) E3 ubiquitin protein (Tiangen). A SYBR RT-PCR kit (Tiangen) was used for transcript ligase complexes, which mediate the ubiquitination of target quantification with specific primers. The expression levels were ÀDD proteins. Cul2 was predicted to be a tumor-suppressor protein quantified using the 2 Ct method, with U6 or b-actin as the a that degrades ubiquitinated HIF (18, 19). However, it has also internal control. The primers were provided in Supplementary been reported that Cul2 is required for normal vasculogenesis Table S3 of the Supplementary Data. and is involved in cell-cycle regulation (20). In recent years, circular RNAs (circRNA) have been identified as important Dual-luciferase reporter gene assay molecules in gene-expression regulation at the post-transcrip- The recombinant luciferase reporter plasmids contained Cul2 tional level (21). circRNAs result from a noncanonical form of promoter (pEZX-PG04-Cul2) and the potential miRNA-binding alternative splicing, most commonly in which the splice donor 0 site sequences of the vimentin 3 -untranslated region (UTR; site of one exon is ligated to the splice acceptor site of an 0 psiCHEK-2-vimentin-3 UTR) and circ-10720 (psiCHEK-2-circ- upstream exon (22–24). There is evidence that some circRNAs 10720). Cul2 promoter and Twist1 overexpressed or knockdown might regulate miRNA functions, and roles in transcriptional vectors cotransfected into PLC-PRF-5 or SMMC-7721 cells, respec- control have also been suggested (25, 26). circRNAs were tively. After 48 hours of transfection, gaussia luciferase and reported to function in gene regulation by competing with secreted alkaline phosphatase were measured by dual-luciferase linear splicing (27). However, the role of Cul2 and its circRNA reporter gene assay kit (GeneCopoeia). HEK293T cells were in tumor progression is poorly understood 0 transfected with vimentin 3 -UTR and circ-10720 luciferase report- In this study, we demonstrate that Twist1 promotes Cul2 er vector and miRNA mimics. Renilla luciferase acted as an internal transcription through binding to the Cul2 promoter and upre- control to normalize transfection efficiencies. After 48 hours of gulates Cul2 circRNA expression but represses Cul2 expression. transfection, luciferase activities were detected with a dual-lucif- We next demonstrated that circ-10720 was positively corre- erase reporter gene assay kit according to the manufacturer's lated with Twist1 and promoted EMT progression. circ-10720 instructions. upregulated vimentin expression by adsorbing a series of miRNAs targeting vimentin. We further showed that circ- Cell invasion assays 10720 knockdown blocked the promotive effect on vimentin Cell invasion assay was performed using a 24-well Transwell in Twist1-overexpressing HCC cells and in vivo.Thisstudy chamber. At 48 hours after transfection, PLC-PFR-5 and SMMC- describes a novel Twist1–circRNA–vimentin regulation mech- 7721 cells were trypsinized and transferred to the Matrigel-coated anism and provides a potential therapeutic target for mesen- top chamber containing 100 mL of serum-free medium. FBS was chymal tumors. added to the bottom chamber as chemoattractant. After 24 hours, cells on the bottom of the chambers were fixed in 4% parafor- Materials and Methods maldehyde and stained with 0.1% crystal violet. Cells that invad- ed into the bottom surface were counted in at least five random Cell culture and plasmid transfection fields (magnification, Â200; Nikon). Each experiment was per- PLC-PRF-5 and SMMC-7721 cells were cultured
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