OBJECTIVE: To explore the effect of OCT4 over-expression on the expression of induced pluripotent stem cell (iPSC)-related transcription factors (cMYC，KLF4，LIN28，NANOG and SOX2) in human bone marrow derived mesenchymal stem cells (hBMMSCs), so as to provide fundamental basis for exploring the pathogenesis of hematological diseases (leukemia, aplastic anemia, etc.) from the perspective of hemopoietic microenvironment in the future. METHODS: Recombinant plasmid pcDNA3.1-OCT4 was constructed and transferred into the optimal generation P3-4 hBMMSCs by liposome transfection. The cells with stable and high expression of OCT4（hBMMSCs-OCT4）were screened by G418 resistance screening (limited dilution) and subcloning, the expression of OCT4 mRNA and OCT4 protein was verified by RT-PCR and FCM, respectively. The expression of iPSC-related transcription factors (cMYC, KLF4, LIN28, NANOG and SOX2) were also determined by FCM and RT-PCR, so as to evaluate the effect of ectopic high expression of OCT4 on the expression of iPSC related transcription factors in hBMMSCs. RESULTS: Recombinant plasmid pcDNA3.1-OCT4 was successfully constructed and cells with stable and high expression of OCT4 were successfully screened from hBMMSCs by limited dilution and subcloning. The result of flow cytometry showed that the mean expression level of OCT4 protein increased from (3.03±1.49)% to (95.46±1.40)% compared with the untransfected parental MSCs, which was also confirmed by RT-PCR analysis. At the same time, the expression levels of OCT4 protein and mRNA were compared between transient transfection (day 4) and stable expression cells (day 96), respectively, it was showed that the OCT4 protein level increased from (36.36±0.28)% at day 4 to (96.25±1.38)% at day 96, and the OCT4 mRNA level increased from 2.75-folds to 6.23-folds, respectively. Compared with the untransfected parental MSCs, the average expression levels of stemness transcription factors increased from (1.12±0.47)% (cMYC), (0.84±0.30)% (KLF4), (2.14±0.79)% (LIN28), (0.63±0.37)% (NANOG) and (14.34±2.44)% (SOX2) to (80.65±4.75)%, (73.03±4.70)%, (68.08±3.05)%, (39.39±1.85)%and (91.45±4.56)% in hBMMSCs-OCT4, respectively, which were consistent with results of RT-PCR analysis. Moreover, the expression levels of NANOG and SOX2 positively correlated with the mean expression of OCT4 (OCT4 vs NANOG: r=0.7802，OCT4 vs SOX2: r=0.4981；NANOG vs SOX2: r=0.7426). CONCLUSION Cells with stable and high expression of OCT4 have been successfully established from hBMMSCs. Ectopic high expression of transcription factor OCT4 in hBMMSCs can up-regulate the expression of other iPSC-related transcription factors such as cMYC, KLF4, LIN28, NANOG and SOX2.
Bone Marrow ; Humans ; Induced Pluripotent Stem Cells ; Mesenchymal Stem Cells ; Nanog Homeobox Protein ; genetics ; Octamer Transcription Factor-3 ; genetics ; Transcription Factors ; Up-Regulation

OBJECTIVETo study the relationship between Nanog-promoted metastasis of breast cancer and ezrin(T567) phosphorylation, and explore the possible mechanism by which Nanog regulates ezrin(T567) phosphorylation.

METHODSA siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrin(T567) were detected using Western blotting and immunofluorescent staining; the protein interaction between PKCε and ezrin was assayed by co-immunoprecipitation and Western blotting.

RESULTSNanog knockdown significantly decreased the expression of PKCε protein, phosphorylation level of ezrin(T567) and the invasion ability of breast cancer cells. PKCε knockdown obviously decreased the phosphorylation level of ezrin(T567) in the cells, and PKCε and ezrin were co-immunoprecipitated.

CONCLUDIONSNanogcan can upregulate the expression of PKCε to promote the phosphorylation of ezrin(T567), which can be a new mechanism by which Nanog promotes tumor metastasis.

Blotting, Western ; Breast Neoplasms ; metabolism ; Cytoskeletal Proteins ; metabolism ; Gene Knockdown Techniques ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplasm Invasiveness ; Phosphorylation ; Protein Kinase C-epsilon ; metabolism ; RNA, Small Interfering ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

The aim of this study was to detect the expression of NANOG gene in acute lymphoblastic leukemia (ALL) cells, and to construct the lentiviral vector carrying NANOG specific shRNA. The expression of NANOG was detected by RT-PCR and Western blot in MOLT-4, CCRF-HSB2, Jurkat cells and bone marrow cells from 15 patients with ALL in our hospital. The lentiviral vector carrying NANOG specific shRNA was constructed. After infection of MOLT-4 cells with the lentivirus constructs, GFP (+) cells were harvested by flow cytometry. The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot. The results showed that the expression of NANOG mRNA and protein was detected in MOLT-4, CCRF-HSB2 cells and 33.3% samples of bone marrow from patients with ALL. The sequencing results demonstrated that the mRNAs amplified from these leukemic cells showed higher homology to NANOGP8 than NANOG1. The lentiviral vector pLB-shNANOG-1, pLB-shNANOG-2 and pLB-shcontrol were constructed. The viral particles were harvested and concentrated by ultracentrifugation. The virus titers were (1.83-3.12) ×10(8) IU/ml. After infection of MOLT-4 cells with the lentivirus, flow cytometry detection indicated that the GFP(+) cells were harvested by real-time quantitative PCR and Western blot, the assays showed that the 2 designed shRNA could significantly down-regulate expression of NANOG gene and protein. It is concluded that NANOGP8 is expressed in various types of ALL cells and in 33.3% of marrow cell samples obtained from ALL patients. After infection with the lentivirus constructs, MOLT-4 cells which stably down-regulate the expression of NANOG mRNA are obtained.
Adult ; Case-Control Studies ; Cell Line, Tumor ; Female ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Lentivirus ; genetics ; Male ; Middle Aged ; Nanog Homeobox Protein ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Young Adult

OBJECTIVETo explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells.

METHODSReal-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected.

RESULTSBoth NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06 ± 1.61)%]and shNANOG-2[(5.67 ± 1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13 ± 0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05).

CONCLUSIONNANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.

Apoptosis ; genetics ; Cell Line, Tumor ; Down-Regulation ; Gene Expression ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Nanog Homeobox Protein ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

Cancer stem cells (CSCs) are thought to drive uncontrolled tumor growth, and the existence of CSCs has recently been proven by direct experimental evidence, including tracing cell lineages within a growing tumor. However, CSCs must be analyzed in additional cancer types. Cancer stem cell-like cells (CSCLCs) are a good alternative system for the study of CSCs, which hold great promise for clinical applications. OCT4, NANOG, and SOX2 are three basic transcription factors that are expressed in both CSCLCs and embryonic stem cells (ESCs). These transcription factors play critical roles in maintaining the pluripotence and self-renewal characteristics of CSCLCs and ESCs. In this review, we discuss the aberrant expression, isoforms, and pseudogenes of OCT4, NANOG, and SOX2 in the CSCLC niche, which contribute to the major differences between CSCLCs and ESCs. We also highlight an anticancer therapy that involves killing specific cancer cells directly by repressing the expression of OCT4, NANOG, or SOX2. Importantly, OCT4, NANOG, and SOX2 provide great promise for clinical applications because reducing their expression or blocking the pathways in which they function may inhibit tumor growth and turn-off the cancer "switch." In the future, a clear understanding of transcription factor regulation will be essential for elucidating the roles of OCT4, NANOG, and SOX2 in tumorigenesis, as well as exploring their use for diagnostic and therapeutic purposes.
Animals ; Embryonic Stem Cells ; metabolism ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; metabolism ; Octamer Transcription Factor-3 ; metabolism ; SOXB1 Transcription Factors ; metabolism ; Signal Transduction

PURPOSE: To investigate the association of cancer stem-cell markers [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homebox (NANOG)] expression with clinicopathological properties and overall survival (OS) in operative rectal cancer (RC) patients receiving adjuvant therapy. MATERIALS AND METHODS: 153 patients with primary RC receiving surgery were enrolled. Tumor tissue and paired adjacent normal tissue sample were collected, and OCT4, SOX2, and NANOG expressions were assessed by immunofluorescent staining. The median follow-up duration was 5.2 years, and the last follow-up date was August 2016. RESULTS: Tumor tissue OCT4 (p < 0.001), SOX2 (p=0.003), and NANOG (p < 0.001) expressions were higher than those in adjacent tissue. OCT4 expression was positively correlated with pathological grade (R=0.185, p=0.022), tumor size (R=0.224, p=0.005), and N stage (R=0.170, p=0.036). NANOG expression was positively associated with tumor size (R=0.169, p=0.036). Kaplan-Meier suggested that OCT4+ was associated with worse OS compared with OCT4− (p < 0.001), while no association of SOX2 (p=0.121) and NANOG expressions (p=0.195) with OS was uncovered. Compared with one or no positive marker, at least two positive markers were associated with shorter OS (p < 0.001), while all three positive markers were correlated with worse OS compared with two or less positive markers (p < 0.001). Multivariate Cox's analysis revealed that OCT4+ (p < 0.001) and N stage (p=0.046) were independent factors for shorter OS. CONCLUSION: Tumor tissue OCT4 expression was correlated with poor differentiation, tumor size, and N stage, and it can serve as an independent prognostic biomarker in operative patients with RC receiving adjuvant therapy.
Aged ; Biomarkers, Tumor/metabolism ; Female ; Humans ; Male ; Multivariate Analysis ; Nanog Homeobox Protein/metabolism ; Neoplastic Stem Cells/metabolism ; Octamer Transcription Factor-3/metabolism ; Prognosis ; Rectal Neoplasms/metabolism ; Rectal Neoplasms/pathology ; Rectal Neoplasms/surgery ; SOXB1 Transcription Factors/metabolism ; Survival Analysis

OBJECTIVE: To explore the protective effect of NANOG against hydrogen peroxide (H2O2) -induced cell damage in the human hair follicle mesenchymal stem cells (hHF-MSCs). METHODS: NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 μmol/L hydrogen peroxide (H2O2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared. RESULTS: Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 μmol/L H2O2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT. CONCLUSION Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H2O2 through activating AKT signaling pathway.
Cell Survival ; Drug Evaluation, Preclinical ; Hair Follicle ; cytology ; Humans ; Hydrogen Peroxide ; Lentivirus ; Mesenchymal Stem Cells ; drug effects ; metabolism ; Nanog Homeobox Protein ; metabolism ; pharmacology ; Oxidative Stress ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.
Animals ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Doxycycline ; pharmacology ; Gene Expression Regulation ; drug effects ; Human Embryonic Stem Cells ; metabolism ; Humans ; Mice ; Nanog Homeobox Protein ; biosynthesis ; genetics ; Pluripotent Stem Cells ; metabolism

OBJECTIVE: To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism. METHODS: P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined. RESULTS: P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells. CONCLUSION Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.
Animals ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; Dimethyl Sulfoxide ; pharmacology ; Embryonal Carcinoma Stem Cells ; pathology ; Hedgehog Proteins ; metabolism ; Hepatocyte Nuclear Factor 3-beta ; physiology ; Homeodomain Proteins ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; Nanog Homeobox Protein ; Proteins ; metabolism ; Transfection

BACKGROUNDHuman amniotic epithelial cells (HAECs), which have characteristics of both embryonic and pluripotent stem cells, are therefore a candidate in cell therapy without creating legal or ethical problems. In the present study, we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer's disease (AD) coexpressing presenilin-1 (PS1) and mutant Sweden amyloid precursor protein (APPswe) genes.

METHODSThe offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene. The doubly transgenic (TG) mice (n = 20) and wild-type (WT) mice (n = 20) were randomly divided into two groups respectively: the transplantation group treated with HAECs and the control group with phosphate buffered saline. Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice. Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively. Immunofluorescence cytochemistry was used to track the survival of HAECs. Immunohistochemistry was used to determine the expression of octamer-binding protein 4 (Oct-4) and Nanog in the HAECs. High performance liquid chromatography was used to measure acetylcholine in hippocampus. The density of cholinergic neurons in basal forebrain and nerve fibers in hippocampus was measured using acetylcholinesterase staining.

RESULTSAmyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months, but not in WT mice. The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection. The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell. Compared with the control group, transplantation of HAECs can not only significantly improve the spatial memory of the TG mice, but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites.

CONCLUSIONSThese results demonstrate that intracerebroventricular transplantation of HAECs can improve the spatial memory of the double TG mice. The higher content of acetylcholine in hippocampus released by more survived cholinergic neurites is one of the causes of this improvement.

Acetylcholine ; metabolism ; Alzheimer Disease ; genetics ; metabolism ; therapy ; Amnion ; cytology ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Chromatography, High Pressure Liquid ; Epithelial Cells ; cytology ; transplantation ; Genotype ; Hippocampus ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Memory Disorders ; genetics ; metabolism ; therapy ; Mice ; Mice, Transgenic ; Nanog Homeobox Protein ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Polymerase Chain Reaction ; Presenilin-1 ; genetics ; metabolism