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YY1 Complex Promotes Quaking Expression Via Super-Enhancer Binding During EMT of Hepatocellular Carcinoma

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YY1 Complex Promotes Quaking Expression Via Super-Enhancer Binding During EMT of Hepatocellular Carcinoma Author Manuscript Published OnlineFirst on February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2238 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. YY1 complex promotes Quaking expression via super-enhancer binding during EMT of hepatocellular carcinoma Jingxia Han*, 1,2,3 Jing Meng*,1,2,3 Shuang Chen*,2,3 Xiaorui Wang*,4 Shan Yin*,5 Qiang Zhang, 2,3 Huijuan Liu,2,3,4 Rong Qin,1,3 Zhongwei Li,1,3 Weilong Zhong,1 Chao Zhang,1 Heng Zhang,1,3 Yuanhao Tang,1 Tingting Lin,6 Wanfeng Gao,1 Xiaoyun Zhang,1 Lan Yang, 2,3 Yanrong Liu,2,3 Hong-gang Zhou,1 Tao Sun#,1,2,3 Cheng Yang#,1, 3 1 State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China 2 Tianjin Key Laboratory for Evaluation of Pharmaceutical Property, Tianjin International Joint Academy of Biomedicine, Tianjin, China 3 Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China 4 College of Life Science, Nankai University, Tianjin, China 5 OBiO Technology (Shanghai) Corp.,Ltd. 6 Tianjin Medical University Eye Hospital, School of Optometry and Ophthalmology, TMU, Tianjin Medical University Eye Institute, Tianjin, China *These authors contributed equally to this work. #Correspondence to: Tao Sun, Nankai University, Haihe River Education Park, 38 Tongyan Road, Tianjin 300350, China. Phone: 8613-5129-22691; E-mail: [email protected]; Cheng Yang, Nankai University, Haihe River Education Park, 38 Tongyan Road, Tianjin 300350, China. Phone: 8615-9013-51388, [email protected] Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed. Quaking (QKI) is an alternative splicing factor that can regulate circRNA formation in the progression of epithelial-mesenchymal transition, but the mechanism remains unclear. High expression of QKI is correlated with short survival time, metastasis, and high clinical stage and pathology grade in hepatocellular carcinoma (HCC). Here we report that transcription of the QKI gene was activated by the Yin-Yang 1 (YY1)/p65/p300 complex, in which YY1 bound to the super-enhancer and promoter of QKI, p65 combined with the promoter, and p300 served as a mediator to maintain the stability of the complex. This YY1/p65/p300 complex increased QKI expression to promote the malignancy of HCC as well an increased circRNA formation in vitro and in vivo. Hyperoside is one of several plant-derived flavonol glycoside compounds. Through virtual screening and antitumor activity analysis, we found that hyperoside inhibited QKI expression by targeting the YY1/p65/p300 complex. Overall, our study suggests that the regulatory mechanism of QKI depends on the YY1/p65/p300 complex and that it may serve as a potential target for treatment of HCC. Key words: YY1/p65/p300 complex; QKI; super-enhancer; EMT; HCC Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2238 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Statement of significance Findings identify the YY1/p65/p300 complex as a regulator of QKI expression, identifying several potential therapeutic targets for the treatment of HCC. Hepatocellular carcinoma (HCC) is one of the most prevalent and malignant tumors with a high mortality rate worldwide (1,2). Metastasis is the most important cause of mortality in HCC (3,4). Epithelial mesenchymal transition (EMT) plays a critical role in tumor progression (5,6), and its pathological activation during tumor development can lead to the metastasis of primary tumors (3,7,8). CircRNAs are purposefully synthesized and implicated in specific biological roles in EMT. Quaking (QKI) is a distinct functional protein that mediate alternative splicing, which belongs to the STAR family of the KH domain, containing RNA binding proteins. QKI regulate a wide range of genes via alternative splicing and circRNA formation during EMT (9). Thus, it displays a potential role in tumorigenesis (10). QKI is also associated with the development and progression of human cancer (11,12). However, its potential role in HCC and its regulatory mechanism in EMT have yet to be described. Yin-Yang 1 (YY1) is a transcription factor involved in cancer progression (13). YY1 can act as a transcriptional activator or a repressor in the regulation of gene expression (14,15). Extensive evidence indicates that YY1 is inversely correlated with E-cadherin expression and crucial in EMT and tumor cell metastasis (16,17). YY1 is also carcinogenic in various cancer types, such as breast and prostate cancer (18). YY1 is significantly upregulated in HCC tissues (1). In the present study, YY1 is highly expressed and positively correlated with QKI in HCC patients with a short survival time. YY1 binds to the super-enhancer and the promoter of QKI, p65 combines with the promoter, and p300 serves as a mediator, causing the formation of DNA loops and leading to the abnormal activation of QKI. Abnormally activated QKI causes the formation of circular RNA and the occurrence of EMT and tumor metastasis in HCC. Cell culture All the cells were bought from the American Type Culture Collection and KeyGen medium supplemented with 10% FBS and 1% penicillin streptomycin solution at 37 °C in 5% CO2 atmosphere. The HCC cell lines were used within less a year and tested for mycoplasma before thawing original stocks. All the cells were identified and periodically authenticated by morphologic inspection and biomarkers detection of hepatocellular carcinoma, growth curve analysis. Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2238 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Plasmid construction and transfection pcDNA3.1-3×Flag-YY1, pcDNA3.1-3×Flag-p65, pDONR223-EP300, pLKD-U6-shRNA and pCD2.1-ciR plasmids were obtained from Youbio (Hunan, China), Obio technology (Shanghai, China), and Geneseed (Guangzhou, China). The QKI sequence was constructed into the pGL3 luciferase vectors (Promega, USA) containing the luciferase gene under the control of the SV40 promoter. siRNA was obtained from SANTA Genepharma (Shanghai, China). All of the plasmids and control vectors were transfected into the cells by using Lipofectamine 2000 (Invitrogen, USA). All of the constructs were prepared through PCR by using the appropriate primers (Table S1). The primers of the overexpression vector are provided in Table S2. qRT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen, USA) from cells and tumor tissues. cDNA synthesis was performed with Oligo(dT) or random primers by using a Quantscript RT kit (Tiangen, China). A SYBR RT PCR kit (Tiangen, China) was used for transcript quantification with specific primers. Expression levels were quantified using the 2 Ct -actin as an internal control. The primers are provided in Table S3 of the supplementary data. Dual-luciferase reporter gene assay A pGL3 promoter vector containing different fragments of YY1 or p65 binding sites and YY1 or p65-overexpressed or QKI knockdown vectors were cotransfected into the HCC cells. After 48 h of transfection, luciferase activities were detected using a dual-luciferase reporter gene assay kit (Promega, USA) in accordance with the Renilla luciferase activity. All of the experiments were performed in triplicates. Immunopurification and silver staining Lysates from PLC-PRF-5 cells expressing Flag-YY1 were prepared using 0.3% NP-40 lysis buffer (0.2 mM EDTA, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.3% Nonidet P-40) containing the protease inhibitor cocktail (Roche). Anti- Flag Tag (L5) Affinity beads (Biolegend, USA) were incubated with the cell extracts for 12 h at 4 °C. After binding was completed, the beads were washed with cold 0.1% NP-40 lysis buffer (0.2 mM EDTA, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% NP-40). Afterward, the Flag peptide (Sigma, USA) was applied to the beads to elute the Flag protein complex. The eluents were collected and visualized through 10% SDS-PAGE. Subsequently, silver staining was performed using a Fast Silver Stain kit (Beyotime, China). Distinct protein bands were retrieved and analyzed through LC MS/MS. Immunoprecipitation and Western blot analysis In immunoprecipitation, 50 µL of 50% protein A/G agarose (Pierce, USA) was incubated with control or specific antibodies at 4 °C with constant rotation for 8 h. PLC-PRF-5 cell lysates were prepared by incubating the cells in 0.3% NP-40 lysis Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2238 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. buffer in the presence of protease inhibitor cocktails. Lysates were centrifuged at 12,000 rpm for 10 min at 4 °C and incubated with antibody-conjugated beads for additional 12 h. After incubation was performed, the beads were washed 5-6 times by using cold 0.1% NP-40 lysis buffer. The precipitated proteins were eluted from the beads by resuspending the beads in 2 × SDS-PAGE loading buffer and boiling for 10 min at 99 °C. The boiled immune complexes were subjected to SDS-PAGE and subsequent immunoblotting. Antibody against p65 (1:100, Cell Signaling Technology, USA), YY1 (1:100, Cell Signaling Technology, USA), p300 (1:100, Cell Signaling Technology, USA). The antibodies for western blot: QKI (1:1000, Affinity, USA), p300 (1:1000, Affinity, USA), E-cadherin (1:1000, Affinity, USA), Vimentin (1:1000, Affinity, USA) and GAPDH (1:5000, Affinity, USA).
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