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• W2563909470 abstract "The expression of Ring1- and YY1-binding protein (RYBP) is reduced in several human cancers, but the molecular mechanism(s) have remained elusive. In this study, we used human hepatocellular carcinoma (HCC) cell lines and tissue specimens to study the mechanism and herein report several new findings. First, we cloned and characterized the basal promoter region of the human RYBP gene. We found that the decreased RYBP expression in HCC tissues was not due to promoter sequence variation/polymorphisms or CpG dinucleotide methylation. We identified two transcription factors, KLF4 and Sp1, which directly bind the promoter region of RYBP to induce and suppress RYBP transcription, respectively. We mapped the binding sites of KLF4 and Sp1 on the RYBP promoter. Studies in vitro showed that KLF4 suppresses whereas Sp1 promotes HCC cell growth through modulating RYBP expression. Deregulated KLF4 and Sp1 contributed to decreased expression of RYBP in HCC tumor tissues. Our studies of human HCC tissues indicated that a diminished RYBP level in the tumor (in association with altered KLF4 and Sp1 expression) was statistically associated with a larger tumor size, poorer differentiation, and an increased susceptibility to distant metastasis. These findings help to clarify why RYBP is decreased in HCC and indicate that deregulated KLF4, Sp1, and RYBP may lead to a poorer prognosis. Our findings support the idea that RYBP may represent a target for cancer therapy and suggest that it may be useful as a prognostic biomarker for HCC, either alone or in combination with KLF4 and Sp1. The expression of Ring1- and YY1-binding protein (RYBP) is reduced in several human cancers, but the molecular mechanism(s) have remained elusive. In this study, we used human hepatocellular carcinoma (HCC) cell lines and tissue specimens to study the mechanism and herein report several new findings. First, we cloned and characterized the basal promoter region of the human RYBP gene. We found that the decreased RYBP expression in HCC tissues was not due to promoter sequence variation/polymorphisms or CpG dinucleotide methylation. We identified two transcription factors, KLF4 and Sp1, which directly bind the promoter region of RYBP to induce and suppress RYBP transcription, respectively. We mapped the binding sites of KLF4 and Sp1 on the RYBP promoter. Studies in vitro showed that KLF4 suppresses whereas Sp1 promotes HCC cell growth through modulating RYBP expression. Deregulated KLF4 and Sp1 contributed to decreased expression of RYBP in HCC tumor tissues. Our studies of human HCC tissues indicated that a diminished RYBP level in the tumor (in association with altered KLF4 and Sp1 expression) was statistically associated with a larger tumor size, poorer differentiation, and an increased susceptibility to distant metastasis. These findings help to clarify why RYBP is decreased in HCC and indicate that deregulated KLF4, Sp1, and RYBP may lead to a poorer prognosis. Our findings support the idea that RYBP may represent a target for cancer therapy and suggest that it may be useful as a prognostic biomarker for HCC, either alone or in combination with KLF4 and Sp1. RYBP 4The abbreviations used are: RYBPRing1- and YY1-binding proteinHCChepatocellular carcinomaIHCimmunohistochemistryTMAtissue microarrayTSStranscription start siteIHHimmortalized human hepatocyteqRT-PCRquantitative RT-PCRMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. is becoming increasingly recognized as a central molecule involved in various processes. It interacts with Ring1A and Ring1B, making it a critical component of polycomb repressive complex 1 (1García E. Marcos-Gutiérrez C. del Mar Lorente M. Moreno J.C. Vidal M. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.EMBO J. 1999; 18: 3404-3418Crossref PubMed Scopus (191) Google Scholar, 2Tavares L. Dimitrova E. Oxley D. Webster J. Poot R. Demmers J. Bezstarosti K. Taylor S. Ura H. Koide H. Wutz A. Vidal M. Elderkin S. Brockdorff N. RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.Cell. 2012; 148: 664-678Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 3Gao Z. Zhang J. Bonasio R. Strino F. Sawai A. Parisi F. Kluger Y. Reinberg D. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.Mol. Cell. 2012; 45: 344-356Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). It promotes the monoubiquitination of Ring1B toward H2AK119, epigenetically regulating gene expression, and is involved in embryogenesis, stem cell self-renewal, cell differentiation, and X-chromosome inactivation (2Tavares L. Dimitrova E. Oxley D. Webster J. Poot R. Demmers J. Bezstarosti K. Taylor S. Ura H. Koide H. Wutz A. Vidal M. Elderkin S. Brockdorff N. RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.Cell. 2012; 148: 664-678Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Mouse embryos with homozygously deleted RYBP die around embryonic day 5.5–6.0, implying that RYBP plays a crucial role during embryonic development (4Pirity M.K. Locker J. Schreiber-Agus N. Rybp/DEDAF is required for early postimplantation and for central nervous system development.Mol. Cell. Biol. 2005; 25: 7193-7202Crossref PubMed Scopus (74) Google Scholar). RYBP also interacts with a multitude of transcription factors, including YY1, E2F2/3/6, and E4TF1/hGABP, acting as a bridging factor to mediate the formation of transcription factor complexes, and therefore modulates gene expression independent of its polycomb group functions (1García E. Marcos-Gutiérrez C. del Mar Lorente M. Moreno J.C. Vidal M. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.EMBO J. 1999; 18: 3404-3418Crossref PubMed Scopus (191) Google Scholar, 5Schlisio S. Halperin T. Vidal M. Nevins J.R. Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function.EMBO J. 2002; 21: 5775-5786Crossref PubMed Scopus (174) Google Scholar, 6Trimarchi J.M. Fairchild B. Wen J. Lees J.A. The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1519-1524Crossref PubMed Scopus (216) Google Scholar, 7Sawa C. Yoshikawa T. Matsuda-Suzuki F. Deléhouzée S. Goto M. Watanabe H. Sawada J. Kataoka K. Handa H. YEAF1/RYBP and YAF-2 are functionally distinct members of a cofactor family for the YY1 and E4TF1/hGABP transcription factors.J. Biol. Chem. 2002; 277: 22484-22490Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). RYBP has also been frequently reported to act as an adaptor protein to mediate interactions among death effector domain-containing proteins, such as caspase-8/10, FADD, and DEDD, as well as other apoptosis-associated proteins, including apoptin and Hippi, allowing it to induce apoptosis when localized in either the cytoplasm or nucleus. However, it did not show apparent cytotoxicity to non-tumorous cells (8Zheng L. Schickling O. Peter M.E. Lenardo M.J. The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm.J. Biol. Chem. 2001; 276: 31945-31952Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Schickling O. Stegh A.H. Byrd J. Peter M.E. Nuclear localization of DEDD leads to caspase-6 activation through its death effector domain and inhibition of RNA polymerase I dependent transcription.Cell Death Differ. 2001; 8: 1157-1168Crossref PubMed Scopus (55) Google Scholar, 10Danen-van Oorschot A.A. Voskamp P. Seelen M.C. van Miltenburg M.H. Bolk M.W. Tait S.W. Boesen-de Cock J.G. Rohn J.L. Borst J. Noteborn M.H. Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing.Cell Death Differ. 2004; 11: 564-573Crossref PubMed Scopus (76) Google Scholar, 11Stanton S.E. Blanck J.K. Locker J. Schreiber-Agus N. Rybp interacts with Hippi and enhances Hippi-mediated apoptosis.Apoptosis. 2007; 12: 2197-2206Crossref PubMed Scopus (23) Google Scholar, 12Novak R.L. Phillips A.C. Adenoviral-mediated Rybp expression promotes tumor cell-specific apoptosis.Cancer Gene Ther. 2008; 15: 713-722Crossref PubMed Scopus (26) Google Scholar, 13González I. Busturia A. High levels of dRYBP induce apoptosis in Drosophila imaginal cells through the activation of reaper and the requirement of trithorax, dredd and dFADD.Cell Res. 2009; 19: 747-757Crossref PubMed Scopus (20) Google Scholar). The genes and signaling pathways targeted by RYBP are still being elucidated. Ring1- and YY1-binding protein hepatocellular carcinoma immunohistochemistry tissue microarray transcription start site immortalized human hepatocyte quantitative RT-PCR 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. Our previous study (14Chen D. Zhang J. Li M. Rayburn E.R. Wang H. Zhang R. RYBP stabilizes p53 by modulating MDM2.EMBO Rep. 2009; 10: 166-172Crossref PubMed Scopus (67) Google Scholar) indicated that RYBP formed a complex with MDM2 and p53 and that it inhibited MDM2-mediated p53 proteasome degradation, leading to p53 activation. In agreement with its apoptosis-inducing capacity, the expression of RYBP has been reported to be reduced in a variety of human cancers, including lung, cervical, prostate, and liver cancers, and was recently shown to inhibit cancer growth, metastasis, and chemoresistance in vivo and in vitro (14Chen D. Zhang J. Li M. Rayburn E.R. Wang H. Zhang R. RYBP stabilizes p53 by modulating MDM2.EMBO Rep. 2009; 10: 166-172Crossref PubMed Scopus (67) Google Scholar, 15Lando M. Holden M. Bergersen L.C. Svendsrud D.H. Stokke T. Sundfør K. Glad I.K. Kristensen G.B. Lyng H. Gene dosage, expression, and ontology analysis identifies driver genes in the carcinogenesis and chemoradioresistance of cervical cancer.PLoS Genet. 2009; 5: e1000719Crossref PubMed Scopus (64) Google Scholar, 16Taylor B.S. Schultz N. Hieronymus H. Gopalan A. Xiao Y. Carver B.S. Arora V.K. Kaushik P. Cerami E. Reva B. Antipin Y. Mitsiades N. Landers T. Dolgalev I. Major J.E. et al.Integrative genomic profiling of human prostate cancer.Cancer Cell. 2010; 18: 11-22Abstract Full Text Full Text PDF PubMed Scopus (2724) Google Scholar, 17Wang W. Cheng J. Qin J.J. Voruganti S. Nag S. Fan J. Gao Q. Zhang R. RYBP expression is associated with better survival of patients with hepatocellular carcinoma (HCC) and responsiveness to chemotherapy of HCC cells in vitro and in vivo.Oncotarget. 2014; 5: 11604-11619Crossref PubMed Google Scholar), indicating that it is a potential candidate drug target for use against these tumors. However, little is currently known about the molecular mechanism(s) responsible for the down-regulation of RYBP in these tumors, and this has limited the understanding of its regulation and, consequently, the development of an optimal approach for targeting RYBP expression as a therapeutic strategy for human cancers. In this study, we investigated the molecular mechanism(s) underlying the down-regulation of RYBP using a normal liver cell line, tumor cell lines, and hepatocellular carcinoma (HCC) tissue samples as models. We herein report several important results, including the cloning and characterization of the previously uncharacterized promoter region of the human RYBP gene, the discovery of the direct binding of two transcription factors (Krüppel-like factor 4 (KLF4) and specificity protein 1 (Sp1)) to this region of RYBP as well as the specific binding sites of these transcription factors, and the involvement of RYBP in KLF4- and Sp1-modulated liver cancer cell growth. We also demonstrate that the deregulation of KLF4, Sp1, and RYBP is related to a more malignant phenotype of HCC. A total of 77 liver cancer patients who underwent curative surgery between January 2012 and May 2013 at Nantong Third Hospital were recruited for this study. This study was approved by the ethics board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Science, and Nantong Third Hospital, and informed consent was provided by the patients. All of the patients were pathologically diagnosed to have HCC, and their detailed clinicopathological characteristics are described below. TMA was constructed from tumor and adjacent normal tissues from each patient as described previously (18Fowler C.B. Man Y.G. Zhang S. O'Leary T.J. Mason J.T. Cunningham R.E. Tissue microarrays: construction and uses.Methods Mol. Biol. 2011; 724: 23-35Crossref PubMed Scopus (13) Google Scholar). Then 4-μm sections were obtained and incubated with antibodies from Sigma against RYBP, KLF4, or Sp1 at a 1:200 dilution and then washed and incubated with a goat anti-rabbit or anti-mouse secondary antibody labeled with biotin. After the washing step, the sections were incubated with streptavidin-biotin complex and diaminobenzidine and finally counterstained with hematoxylin, dehydrated, and mounted. The expression levels of the three target proteins in each tissue specimen were evaluated as described previously with minor modifications (19Yang J. Zhao L. Tian W. Liao Z. Zheng H. Wang G. Chen K. Correlation of WWOX, RUNX2 and VEGFA protein expression in human osteosarcoma.BMC Med. Genomics. 2013; 6: 56Crossref PubMed Scopus (25) Google Scholar). The immortalized human hepatocyte (IHH) cell line was a kind gift from Dr. Jerome Torrisani (Cancer Research Center of Toulouse, France). HEK293T, Hep3B, and HepG2 cell lines were from the Cell Resource Center, PUMC (Beijing, China), and Huh7, PLC/PRF/5, and SK-Hep-1 cell lines were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All of the cell lines were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum. PrimeStar HS DNA polymerase and SYBR® Premix Ex TaqTM II were purchased from TaKaRa (Dalian, China). Lipofectamine 2000 transfection reagent and the TRIzol reagent were from Invitrogen. The CellTiter 96® AQueous One solution cell proliferation assay kit, GoScriptTM reverse transcription system, and Dual-Luciferase reporter assay kit were from Promega (Madison, WI). The control siRNA and siRNAs against KLF4 or Sp1 were from Ribobio (Guangzhou, China). Genomic DNA extraction and gel extraction kits were from Sangon (Shanghai, China). The EZ DNA Methylation-GoldTM kit and ZymoTaqTM DNA polymerase were from Zymo Research (Irvine, CA). Protein A-Sepharose beads were purchased from GE Healthcare. Propidium iodide, control rabbit IgG, anti-FLAG M2 (F1804), anti-β-actin (A5441), anti-RYBP (PRS2227), anti-KLF4 (SAB5300069), and anti-Sp1 (SAB1404397) antibodies were from Sigma. Anti-KLF4 (H-180) and anti-Sp1 (D4C3) antibodies for chromatin immunoprecipitation (ChIP) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX) and CST (Beverly, MA), respectively. The full-length open reading frames (ORFs) for Ets-1(441), MZF-1(734), NKX2-5(112), NKX2-5(324), and YY-1 were amplified by proofreading PCR from cDNAs of HEK293T cells and were cloned into the pFLAG-CMV-2 vector (numbers in parentheses represent the amino acid numbers of different protein isoforms). The human GV227-KLF4 cloning vector was purchased from Genechem (Shanghai, China) and was subcloned into the pFLAG-CMV-2 vector. The human pcDNA3.1-His-Sp1 template was a generous gift from Dr. Xiaozhong Peng (Institute of Basic Medical Sciences, Chinese Academy of Medical Science, Beijing, China), and its full-length ORF was resubcloned into the pFLAG-CMV-2 vector. pcDNA3.1-HNF1A was a kind gift from Dr. Xiaoming Yang (Beijing Institute of Radiation Medicine). The plasmids for GFP-RYBP, shCtrl, and shRYBP were described previously (20Ma W. Zhang X. Li M. Ma X. Huang B. Chen H. Chen D. Proapoptotic RYBP interacts with FANK1 and induces tumor cell apoptosis through the AP-1 signaling pathway.Cell. Signal. 2016; 28: 779-787Crossref PubMed Scopus (22) Google Scholar). All of the constructed clones were confirmed by sequencing, and detailed cloning primer information is provided in Table 1.TABLE 1Primers used to construct candidate transcription factor expression vectorsVector namePrimerspFLAG-CMV-2-KLF4Forward5′-GAAGATCTTATGAGGCAGCCACCTGGCGAGTCTGACATGGCTGTCAGCGACG-3′ (BglII)Reverse5′-ACGCGTCGACTTAAAAATGCCTCTTCATGTGTAAGG-3′ (SalI)pFLAG-CMV-2-Ets1Forward5′-GAAGATCTTATGAAGGCGGCCGTC-3′ (BglII)Reverse5′-ACGCGTCGACTCACTCGTCGGCATCTGG-3′ (SalI)pFLAG-CMV-2-Mzf1Forward5′-GGAATTCCATGAGGCCTGCGGTGC-3′ (EcoRI)Reverse5′-ACGCGTCGACCTACTCGGCGCTGTGGAC-3′ (SalI)pFLAG-CMV-2-YY1Forward5′-GGAATTCCATGGCCTCGGGCGAC-3′ (EcoRI)Reverse5′-ACGCGTCGACTCACTGGTTGTTTTTGGCC-3′ (SalI)pFLAG-CMV-2-Nkx2-5Forward5′-GGAATTCCATGTTCCCCAGCCCTGC-3′ (EcoRI)Reverse5′-ATATATGCTAGCGCCGTGCGCGCC-3′ (SalI)/112Reverse5′-ACGCGTCGACCTACCAGGCTCGGATACCAT-3′ (SalI)/324 Open table in a new tab PCR was used to amplify the RYBP promoter region from the genomic DNAs prepared from the six liver cell lines (see Table 2 for primer information). The products were cloned into pGL3-Basic vector. pGL3-P(I-R) and pGL3-P(P-R) represent the origin of the genomic DNA from IHH and PLC/PRF/5 cells, respectively.TABLE 2Primers used to construct truncated RYBP promoter reporter vectorsVector namePrimer(s)pGL3-P(I-R)Forward5′-ATATATGCTAGCAAGGAAACGCCCTATTTAGACTCT-3′ (NheI)pGL3-P(−507/+1040)Forward5′-ATATATGCTAGCAGCTCCGCATGGCAGAG-3′ (NheI)pGL3-P(−294/+1040)Forward5′-ATATATGCTAGCGAAGCCAGTTGCCAGCTC-3′ (NheI)pGL3-P(−91/+1040)Forward5′-ATATATGCTAGCCCGCCCTCCCATTGG-3′ (NheI)pGL3-P(+122/+1040)Forward5′-ATATATGCTAGCACGGCGTTTCTCCTCCG-3′ (NheI)pGL3-P(+312/+1040)Forward5′-ATATATGCTAGCGAGGCGCTGCTAAGATGGA-3′ (NheI)pGL3-P(+513/+1040)Forward5′-ATATATGCTAGCAGATTTAACCAGGTGGGGAGG-3′ (NheI)pGL3-P(+820/+1040)Forward5′-ATATATGCTAGCTCCCCGGAAAGGGTGG-3′ (NheI)Reverse5′-ATATATAAGCTTGCAGACGCTACAATCCCAA A-3′ (HindIII)pGL3-P(+312/+512)Forward5′-ATATATGCTAGCGAGGCGCTGCTAAGATGGA-3′ (NheI)pGL3-P(+413/+512)Forward5′-ATATATGCTAGCGCCGTGCGCGCC-3′ (NheI)Reverse5′-ATATATAAGCTTCCCTTTGTCTGGGAGGAGTG-3′ (HindIII)pGL3-P(+513/+819)Forward5′-ATATATGCTAGCAGATTTAACCAGGTGGGGAGG-3′ (NheI)pGL3-P(+614/+819)Forward5′-ATATATGCTAGCCCCCCTCCAGATCCGG-3′ (NheI)pGL3-P(+715/+819)Reverse5′-ATATATAAGCTTCTGGAAGAGAGAGCGAGGAAG-3′ (HindIII) Open table in a new tab To generate serial truncated constructs of the RYBP promoter region, the pGL3-P(I-R) vector was used as a template to amplify a series of RYBP promoter truncated fragments (−507/+1040, −294/+1040, −91/+1040, +122/+1040, +312/+1040, +513/+1040, +820/+1040, +312/+512, +413/+512, +513/+819, +614/+819, and +715/+819), using the fragment-specific primers listed in Table 2, and cloned into pGL3-Basic vector. For site-directed mutagenesis, truncated constructs (pGL3-P-M1, pGL3-P-M2, pGL3-P-M3, and pGL3-P-M4) and corresponding full-length constructs (pGL3-M1, pGL3-M2, pGL3-M3, and pGL3-M4) were generated from the pGL3-P(I-R) plasmid using specific mutant primer sets (Table 3). All of the constructs were confirmed by DNA sequencing.TABLE 3Primers used to construct the site-directed RYBP promoter mutant reporter vectorsVector namePrimerspGL3-P-M1Forward5′-TAAGATGGAGATCCGAAATAGTTGGGAGGCGGCGGG-3′Reverse5′-CCCGCCGCCTCCCAACTATTTCGGATCTCCATCTTA-3′pGL3-P-M2Forward5′-TGGCGGCGGCGGCGGTACGGTATCTCGCTCCCGCTCGGG-3′Reverse5′-CCCGAGCGGGAGCGAGATACCGTACCGCCGCCGCCGCCA-3′pGL3-P-M3Forward5′-GCGGCTCCCCTCCATTAAATTTCAGCCCCACGCTCA-3′Reverse5′-TGAGCGTGGGGCTGAAATTTAATGGAGGGGAGCCGC-3′pGL3-P-M4Forward5′-TCCCCTCCCGGCCCCCCTATAATTAAGCTCAAGTCCACA-3′Reverse5′-TGTGGACTTGAGCTTAATTATAGGGGGGCCGGGAGGGGA-3′ Open table in a new tab Assayed cells were grown to about 90% confluence in 24-well plates and then were co-transfected with 1 μg of promoter reporter vectors and 50–100 ng of Renilla luciferase expression vector (pRL-TK) together with either the empty vector or a KLF4 or Sp1 expression vector using the Lipofectamine 2000 reagent. After 24 h, the cells were lysed, and the promoter activities were assessed as described by the manufacturer. Assayed cells were grown to about 40–50% confluence in 6-well plates and then were transfected with either control siRNA or siRNAs against KLF4 or Sp1 using Lipofectamine 2000. The cells were harvested 36 or 48 h after transfection and were used for quantitative real-time reverse transcription-PCR (qRT-PCR) or Western blotting. Total RNA was extracted from cultured cells using the TRIzol reagent. cDNA was synthesized using a GoScriptTM reverse transcription system with oligo(dT)12–18 primers. qRT-PCR was performed using SYBR® Premix Ex TaqTM II on a CFX-96 system (Bio-Rad). The SYBR signal was normalized to that of endogenous glyceraldehyde-3-phosphate dehydrogenase. All primers used for qRT-PCR are shown in Table 4.TABLE 4Primers used for quantitative real-time RT-PCRGene namePrimersGAPDHForward5′-AAGGTCGGAGTCAACGGATT-3′Reverse5′-CTCCTGGAAGATGGTGATGG-3′KLF4Forward5′-ATTACCAAGAGCTCATGCCAC-3′Reverse5′-AGTGGTAAGGTTTCTCACCTG-3′RYBPForward5′-TCAGAGAGCACAGACAAGGG-3′Reverse5′-GCAGCATCACTAAGAGGTCG-3′Sp1Forward5′-AATTTGCCTGCCCTGAGTGC-3′Reverse5′-TTGGACCCATGCTACCTTGC-3′ Open table in a new tab Genomic DNA was isolated using a genomic DNA extraction kit according to the manufacturer's protocol. Subsequently, bisulfate conversion of the extracted DNA was performed using the EZ DNA Methylation-GoldTM kit according to the manufacturer's instructions. The modified DNA was used as a template and amplified by ZymoTaqTM DNA polymerase following the manufacturer's manual. The amplified PCR products were gel-extracted and sequenced directly using PCR primers and internal oligonucleotide sequencing primers. The primer sequences are listed in Table 5.TABLE 5Primers used for methylation analysisPrimer namePrimer sequencesRYBP-Me-primer-1Forward (−584/−564)5′-ATAGGGGTTTGATAGGGAGTG-3′Reverse (−388/−407)5′-AAAACTTACAACAACCTCAC-3′RYBP-Me-primer-2Forward (−412/−392)5′-GAGYGGTGAGGTTGTTGTAAG-3′Reverse (−272/−291)5′-CCCRAAAACTAACAACTAAC-3′RYBP-Me-primer-3Forward (−301/−280)5′-GGGGTTYGAAGTTAGTTGTTAG-3′Reverse (+262/+241)5′-CCCCCAACCCTCCCTCCCCTTC-3′Forward (−69/−48)5′-GTGTGTGATGTGTGGTGTTAGG-3′aInternal sequencing primers.Forward (+35/+54)5′-TGGTGGTTGGAGTTTGAGTT-3′aInternal sequencing primers.Reverse (−51/−73)5′-AACACCACACATCACACACCATA-3′aInternal sequencing primers.RYBP-Me-primer-4Forward (+202/+225)5′-AAGAGTTYGATTAGGTATTTGTTT-3′Reverse (+526/+507)5′-ACCTAATTAAATCTCCCTTT-3′RYBP-Me-primer-5Forward (+502/+527)5′-TAGATAAAGGGAGATTTAATTAGGTG-3′Reverse (+786/+767)5′-CCCCACTAACACCCTACAAA-3′a Internal sequencing primers. Open table in a new tab Cells were harvested and lysed in ice-cold lysis buffer (50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1% Nonidet P-40, 10% (v/v) glycerol, 1 mm EDTA), supplemented with a protease inhibitor mixture, and were rotated at 4 °C for 20 min followed by centrifugation at 12,000 × g at 4 °C for 15 min. The protein concentration was measured using a BCA kit. 40 μg of proteins were separated by SDS-PAGE, and the targeted proteins were probed with corresponding antibodies. Huh7 cells were seeded into 96-well plates at a density of 3 × 103 cells/well and transfected the next day with the indicated plasmids for 72 h. The viable cells were assayed using the MTS reagent according to the manufacturer's protocol. HepG2 cells were seeded into 60-mm dishes at a density of 4 × 105/dish. On the next day, cells were transfected with different combinations of the indicated plasmids for 48 h. The cells were collected and fixed in 75% alcohol overnight, and the cell pellets were digested with RNase A at 37 °C for 20 min and stained with propidium iodide. Then the cell cycle distribution was analyzed by Coulter Epics XL Flow Cytometer (Coulter Corp.). Cells were seeded into 6-well plates at 300 cells/well and were transfected with pFLAG-CMV-2 or pFLAG-Sp1 together with either shCtrl or shRYBP expression vector for 24 h. The medium was replaced every 3 days. After 2 weeks of culture, the medium was removed, and cell colonies were stained with crystal violet (0.1% in 20% methanol). Pictures were taken using a digital camera. Cells were seeded into 10-cm dishes and cultured to about 90% confluence. The cells were fixed with 1% formaldehyde, and the cross-linking reaction was quenched by the addition of glycine. Cellular lysates were collected, and genomic DNAs were sonicated to lengths around 500 bp. The DNA solution was clarified by centrifugation, and 15 μl of the supernatant was decross-linked by heating at 65 °C for 5 h and used as an input. Equal amounts of the rest of the supernatants were immunoprecipitated with control rabbit IgG, anti-KLF4, or anti-Sp1 antibodies and decross-linked by heat. The DNA samples were purified and resuspended in TE buffer. The primers used for ordinary PCR and real-time quantitative PCR were as follows: forward, 5′-CCACGCTCAAGTCCACAA-3′; reverse, 5′-ATTTCGCAGGAATCCAGTG-3′. The GraphPad Prism version 5 software program for Windows (GraphPad Software, La Jolla, CA) was used to analyze the promoter activity and mRNA levels. Data were expressed as the means ± S.D. of triplicate samples, and all experiments were repeated at least two times. Pearson's χ2 test was used to compare qualitative variables, and Student's t test was applied to analyze quantitative variables. The IHC results were analyzed utilizing the SPSS version 19.0 software program for Windows (IBM Corp, Armonk, NY). All statistical tests were two-sided, and p < 0.05 was considered statistically significant. Previous studies have shown that the expression of RYBP mRNA and protein is reduced in HCC tumor tissues (14Chen D. Zhang J. Li M. Rayburn E.R. Wang H. Zhang R. RYBP stabilizes p53 by modulating MDM2.EMBO Rep. 2009; 10: 166-172Crossref PubMed Scopus (67) Google Scholar, 17Wang W. Cheng J. Qin J.J. Voruganti S. Nag S. Fan J. Gao Q. Zhang R. RYBP expression is associated with better survival of patients with hepatocellular carcinoma (HCC) and responsiveness to chemotherapy of HCC cells in vitro and in vivo.Oncotarget. 2014; 5: 11604-11619Crossref PubMed Google Scholar). To elucidate the molecular mechanism underlying the reduced expression, we first examined the mRNA levels of RYBP in IHHs and five HCC cell lines (Huh7, HepG2, Hep3B, PLC/PRF/5, and SK-Hep-1), which we intended to use as cellular models. In comparison with the IHHs, three of the HCC cell lines (Huh7, PLC/PRF/5, and SK-Hep-1) showed significantly decreased RYBP mRNA expression, whereas one cell line (HepG2) had highly elevated RYBP mRNA expression (Fig. 1A). Because there was a gap starting from +19 nt downstream of the transcription start site (TSS) of the human RYBP gene in the GenBankTM sequence (accession numbers NT_022459, NC_000003, and NC_018914) when we started this project, we first filled the gap (located from +19 to +513 bp; 495 bp in total). After obtaining the complete promoter sequence of the human RYBP gene, we analyzed the area around the TSS (ranging from −3000 to +3000 bp). As shown in Fig. 1, B and C, we could see that there was a large CpG island between −557 and +901 bp predicted by CpGFinder, and this region contained 202 CpG dinucleotides. This had a (G + C) content of 77.0%, and the ratio of observed to expected CpGs was 0.935. In this region, three GC boxes (−145/−125, −123/−102, and −50/−35) and one CAAT box (−83/−70) were predicted to cluster together in close proximity to the TSS, but no TATA box was predicted near the TSS. However, a canonical initiator element exists around the TSS (−2/+5). The above analysis indicated that the RYBP promoter is a GC box-rich but TATA box-less promoter. To determine whether there were variations around the 5′-flanking sequence of RYBP, we first amplified the DNA fragments around the TSS of the gene (−711 to +1040 relative to the IHH sequence), which encompassed the predicted CpG island, using genomic DNA samples from the six liver cell lines as templates. Our results showed that there were no detectable deletion/insertion variations around the TSS sites in any of these cell lines based on an agarose gel electrophoresis assay, suggesting that the differential expression of RYBP among these cells was not due to major sequence variations that led to a difference in length (data not shown). Next, we subcloned these promoter fragments into the pGL3-Basic vector and sequenced them. The sequence alignments showed that although there were several minor variations/polymorphisms, no consistent alterations were present that were associated with the RYBP expression levels in the six cell lines (Fig. 1D), implying that sequence variations/polymorphisms were also unlikely to explain the altered RYBP expression in the HCC cells. Because CpG dinucleotides are rich in the promoter region of RYBP, we explored whether CpG methylation was associated with reduced RYBP expression. Although a number of methylated CpG sites upstream of the predicted CpG island were detected by bisulfite sequencing in all six cell lines, none of these sites were specifically related to altered RYBP expression (Fig. 1E). We subsequently treated Huh7, PLC/PRF/5, and SK-Hep-1 cells with 5-azacytidine, a reagent that inhibits CpG methylation, but no obvious induction of RYBP was observed. Taken together, these results indicated that CpG methylation in the RYBP promoter was not a major contributor to the reduced RYBP expression in HCC cells. We then wondered whether the reduced RYBP level resulted from factors outside the DNA sequence itself. To test this hypot" @default.
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• W2563909470 date "2017-02-01" @default.
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• W2563909470 title "RYBP Expression Is Regulated by KLF4 and Sp1 and Is Related to Hepatocellular Carcinoma Prognosis" @default.
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