BackgroundAccumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the key regulator responsible for stemness, epithelial-mesenchymal transition (EMT), metastasis, chemoresistance and other malignant phenotypes. Here we try to explore these issues with gastric adenocarcinoma cell lines in vitro using variety of molecular and cellular techniques.MethodsSpecial primers were designed to distinguish PCR products from NANOG1 and NANOGP8. Sphere-forming cells were cultured with serum-free and selective medium. A stable cell line was established with infection of lentivirus containing NANOGP8. qPCR was performed to measure NANOGP8 expression and its association with stemness, EMT and CSC markers in adherent cells and sphere-forming cells. Western blot analysis was deployed to confirm results of the transcript analysis. Experiments of cell proliferation, migration, invasion, clonogenic assay, sphere cell growth assays, cell cycle analysis, β-catenin accumulation and translocation in nucleus, and drug resistance were conducted to measure the impact of NANOGP8 on malignant statuses of gastric cancer cells. Immunofluorescence staining was used to analyze cell subpopulations with different markers.ResultsNANOGP8 is mainly responsible for NANOG expression in sphere-forming (stem cell-like) cells derived from gastric cancer cell lines regardless their differentiation status. Ectopic expression of NANOGP8 significantly up-regulates stemness transcription factors, EMT inducers, and cancer stem cell markers (CSC) including Lgr5. NANOGP8 also promotes expression of the signature genes vimentin and N-caderin for mesenchymal cells and down-regulates the signature gene E-caderin for epithelial cells whereby confer the cells with mesenchymal cell phenotype. In NANOGP8 over-expressed adherent and sphere-forming cells, Lgr5+ cells are significantly increased. Ectopic expression of NANOGP8 endows gastric cells with enhanced proliferation, migration, invasion, sphere-forming and clonogenic capacity, and chemoresistance. NANOGP8 expression also enhances β-catenin accumulation in nucleus and strengthens Wnt signal transduction.ConclusionNANOGP8 is the main regulator of gastric cancer stem cells. It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway. NANOGP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, β-catenin accumulation in nucleus, and chemoresistance in gastric cancer. NANOGP8 is a promising molecular target for clinical intervention of gastric cancer.

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Uncontrolled proliferation is the hallmark of cancer cells. Previous studies mainly focused on the role of protein-coding genes in cancer cell proliferation. Emerging evidence showed that long non-coding RNAs (lncRNAs) also play critical roles in cancer cell proliferation and growth. LncRNA KCNQ1OT1 is found to contribute to carcinogenesis, but its role in acute promyelocytic leukemia (APL) is unclear. In this study, by analyzing data from Gene Expression Omnibus, The Cancer Genome Atlas database and our clinical samples, we found that KCNQ1OT1 was selectively highly expressed in APL. Functional assays demonstrated that knockdown of KCNQ1OT1 reduced APL cell proliferation and increased apoptosis. Further evidence showed that KCNQ1OT1 was mainly located in the cytoplasm of APL patient-derived NB4 cells and APL patient bone marrow samples. Mechanistically, KCNQ1OT1 bound to RNA binding protein FUS, and silencing either KCNQ1OT1 or FUS reduced the expression level and stability of MAP3K1 mRNA. Whereas KCNQ1OT1 and FUS did not affect each other. Importantly, knockdown of MAP3K1 impaired APL cell proliferation. Finally, c-Myc transactivated KCNQ1OT1 in APL cells through binding to its promoter while knockdown of c-Myc decreased KCNQ1OT1 expression. Our results not only revealed that c-Myc transactivated KCNQ1OT1 and upregulated KCNQ1OT1 promoted APL cell proliferation, but also demonstrated that KCNQ1OT1 bound to FUS to synergistically stabilize MAP3K1 mRNA, thus facilitating APL cell proliferation. This study established a previously unidentified role of KCNQ1OT1 in the development of APL, and KCNQ1OT1 may serve as a potential therapeutic target for APL.