In this study, we performed bioinformatics analysis and found that SP1 could also regulate ANRIL

In this study, we performed bioinformatics analysis and found that SP1 could also regulate ANRIL transcription in HCC cells. ChIP assay also showed that SP1 could directly bind to ANRIL promoter regions to silence ANRIL transcription. In addition, overexpression of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25112874 SP1 in HCC cells could up-regulate ANRIL expression, while knockdown of SP1 in HCC cells could down-regulate ANRIL expression. These data showed that ANRIL expression could also be regulated by SP1 in HCC cells, which suggests that one lncRNA may be simultaneously regulated by multiple different transcript factors. As is known, lncRNAs participated in cancer cells’ biological function, and we found that knockdown of ANRIL could impair HCC cell proliferation and invasion and induce cell apoptosis both in vitro and in vivo. These data suggests that lncRNA ANRIL contributes to HCC development via regulation of cell proliferation and apoptosis. A completely different mode of action is executed by thelncRNA ANRIL to block the activity of tumor suppressor genes. For example, ANRIL interacts with SUZ12 (a subunit of the PRC2) and recruits the complex to repress the expression of p15 (INK4B), a well-known tumor suppressor gene [13]. A similar study identified chromobox homolog 7 (CBX7), a subunit of the polycomb repressive complex 1 (PRC1) as molecular interaction partner of ANRIL, which results in the recruitment of PRC1 to the p16(INK4A)/p14(ARF) locus and silencing of this gene locus by H3K27 trimethylation [10]. However, we found that ANRIL could bind with both EZH2 and SUZ12 in HCC cells. Furthermore, bioinformatics analysis indicated that KLF2 could be a new ANRIL downstream target, and knockdown of ANRIL, EZH2 and SUZ12 expression indeed both up-regulated KLF2 expression levels in HCC cells. In addition, ChIP assays also demonstrated that EZH2 could directly bind to KLF2 promoter region and inhibition of ANRIL MS023 web decreased its binding ability. Our results indicated that ANRIL could repress KLF2 transcription by binding with EZH2 and SUZ12 and recruitment of PRC2 to the KLF2 gene locus in HCC cells. The Kruppel-like factor (KLF) family which consists of a set of transcription factors that have been identified in diverse organisms functions in cell differentiation and proliferation [27]. They have been identified as suppressors or activators of different genes in a cell type and promoter-dependent manner [28]. KLF2 is one of the critical members due to its tumor suppressor function in tumors [29, 30]. Moreover, previous study showed that EZH2 could directly bind to KLF2 promoter and silence of KLF2 expression result in blocking the tumor-Huang et al. Journal of Hematology Oncology (2015) 8:Page 6 ofFig. 2 (See legend on next page.)Huang et al. Journal of Hematology Oncology (2015) 8:Page 7 of(See figure on previous page.) Fig. 2 Effects of knockdown of ANRIL on HCC cell viability and apoptosis in vitro. a The ANRIL expression level was determined by qPCR when HepG2 and Hep3B cells transfected with si-ANRIL. b MTT assays were used to determine the cell viability for si-ANRIL-transfected HepG2 and Hep3B cells. Values represented the mean ?s.d. from three independent experiments. c Colony-forming assays were conducted to determine the proliferation of si-ANRIL-transfected HepG2 and Hep3B cells. d Flow cytometry assays were performed to analyze the cell cycle progression when HCC cells transfected with si-ANRIL 24 h later. The bar chart represented the percentage of cells in G0/G1,.