Caracterización de marcadores de células madre tumorales en cáncer escamocelular de cavidad oral

Introducción: El cáncer escamocelular de cavidad oral es una patología con bajas tasas de sobrevivencia. Cuando no es tratado adecuadamente es un tumor de alta recurrencia y resistente al tratamiento. Nuevas hipótesis plantean que las células tumorales progenitoras por sus propiedades de auto renovación, iniciación tumoral, migración y metástasis pueden ser responsables de la manutención y renovación de este tumor. Sin embargo, aún no existe un consenso sobre la verdadera participación de ellas, debido a que su identificación y caracterización es aún un reto experimental. Objetivo: En este trabajo se busca detectar células con expresión de marcadores de células tumorales Progenitoras en muestras cáncer escamocelular de cavidad oral y relacionarlo con los estadios de diferenciación del tumor. Metodología: En esta investigación se tomaron 32 muestras de pacientes con carcinoma escamocelular de cavidad oral. Se logró detectar in situ, mediante la técnica de inmunofluorescencia, cuatro reconocidos marcadores de células tumorales progenitoras. Resultados: Se identificaron los marcadores OCT4, SSEA4, NANOG y TRA-1-60 en los diferentes estadios de diferenciación tumoral, lo que sugiere la participación de las células progenitoras tumorales en la evolución de esta patología. Conclusiones: El establecimiento y correcta identificación de las células tumorales progenitoras abre nuevas vías terapéuticas para el abordaje de este tumor, en busca de mejorar el pronóstico, tasa de sobrevivencia y calidad de vida del paciente.

Introduction: Squamous cell carcinoma of the oral cavity is a pathology with poor survival rates. When it is not adequately treated, it is a tumor with high recurrence and resistance to treatment. According to new hypotheses, progenitor tumor cells, due to their properties of self-renewal, tumor initiation, migration, and metastasis, could be responsible for the maintenance and renewal of this tumor. However, there is still no consensus on their true participation, subsequent to difficult in their identification and characterization. Materials and methods: In this research, 32 samples provided from patients diagnosis with squamous cell carcinoma of the oral cavity were used. To detect specific markers progenitor tumor cells were used immunofluorescence microscopy. Results: The cells markers OCT4, SSEA4, NANOG and TRA-1-60 were identified in the different stages of the tumor samples, all these findings suggest the role of tumor progenitor cells in the evolution of this pathology. Conclusions: The establishment and correct identification of the progenitor tumor cells provide new therapeutic options for the approach of this tumor seeking to improve the prognosis, survival rate and quality of life of the patient.

Embryonic stem cell pluripotent factor NANOG mediates the activity and invasion of breast cancer cells via AMPK/mTOR pathway / 中华内分泌外科杂志

Objective:To investigate the role of embryonic stem cell pluripotent factor NANOG in mediating the activity and invasion of breast cancer cells via AMPK/mTOR pathway.Methods:A total of 58 breast cancer patients were collected from Jul. 2019 to Aug. 2020, and the clinical data of each patient at admission were collected for comparative analysis. qRT-PCR was used to detect the expression level of NANOG in adjacent tissues and cancer tissues, and Western blot was used to verify the regulation of AMPK/mTOR pathway by NANOG. Cells were treated with NANOG specific plasmid or AMPK inhibitor Compound C. Cell viability was detected by MTT and invasion ability was detected by Transwell.Results:The expression of NANOG was increased in breast cancer tissues (adjacent to cancer tissue: 1.00±0.31, cancer tissue: 1.45±0.27, t=8.34, P<0.004) and cell lines (MCF-10A: 1.00±0.12, BT474: 2.64±0.25, t=10.24, P=0.001; MCF-7: 1.56±0.13, t=5.48, P=0.005; ZR-75-30:1.84±0.16, t=7.28, P=0.002), which could be used as a specific biomolecule for predicting breast cancer (all P<0.05). The expression level of NANOG may be related to lymph node metastasis, histological grade and pathological type. Compared with patients with non-lymph node metastasis (1.36±0.23) or non-invasive patients (1.35±0.25), patients with lymph node metastasis (1.54±0.27, t=2.61, P=0.012) or invasive patients (1.53±0.26, t=2.60, P=0.012) had higher expression of NANOG. After NANOG knockdown, AMPK protein and phosphorylation levels were increased, while mTOR and p70S6K protein and phosphorylation levels were decreased (all P<0.05). Knockdown of NANOG in cells inhibited the activity and invasion of breast cancer cells (activity: si-RNA: 100±8.65, si-NANOG: 58.36±4.58, t=7.37, P=0.002; invasion: si-RNA: 121.41±10.34, si-NANOG: 58.34±8.41, t=8.20, P=0.001), and the effect of knockdown of NANOG was relieved after AMPK inhibitor was used in cells (all P<0.05) . Conclusions:Embryonic stem cell pluripotent factor NANOG promotes the activity and invasion of breast cancer cells by inhibiting the activation of AMPK/mTOR pathway. NANOG can be used as an effective biomolecule for predicting breast cancer.

Utility of BMI-1 and NANOG expression levels in survival prediction of pediatric acute lymphoblastic leukemia

ABSTRACT Background Acute lymphoblastic leukemia (ALL) is the most common malignancy in children characterized by the overproduction and accumulation of immature lymphoid cells in the bone marrow and peripheral blood. The BMI-1 is an important component of the Polycomb Repressive Complex-1 (PRC1). It is an important molecule for the self-renewal of hematopoietic stem cells (HSCs). The BMI-1 expression is generally high in HSCs and decreases after cell differentiation. The BMI-1 is required for the maintenance of normal and cancer stem cells and has been reported as an oncogene in various tumors. The NANOG is a homeodomain transcription factor responsible for maintaining the stem cell compartment at the blastocyst stage of developing embryos. The NANOG gene has been proven to be transcribed in CD34+ cells and different leukemic cells. Methods The ribonucleic acid (RNA) was extracted from the peripheral blood mononuclear cells (PBMNCs) of 30 pediatric ALL patients (16 B-ALL and 14 T-ALL) and 14 healthy controls. The Bmi-1 and NANOG expression levels were determined using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results Compared to normal controls, patients with ALL exhibited upregulated levels of Bmi-1 (p = 0.03). Patients who overexpressed Bmi-1 and NANOG displayed a significantly worse survival than low-expressing patients (hazard ratio (HR) 5.74, 95% confidence interval (CI):1.48-22, p = 0.012 and HR 3.8, 95% CI:1.009-14.3, p = 0.048, respectively). Conclusions Taken together, these data suggest that the Bmi-1 and NANOG might serve as a novel survival predictor in ALL patients. Our observation also suggests that the Bmi-1 and NANOG could serve as new therapeutic targets for treatment of pediatric ALL.

Putative stemness markers octamer-binding transcription factor 4, sex-determining region Y-box 2, and NANOG in non-small cell lung carcinoma: A clinicopathological association

Background: The promising improvement in the clinical outcome of lung cancer can be possibly achieved by identification of the molecular events that underlie its pathogenesis. Cancer stem cell (CSC) being one of the subsets of tumor majorly participates in drug resistance and treatment failure because of the moderate cell cycle, lower proliferation, and increased expression of DNA repair and anti-apoptosis genes. Although many putative CSC markers exist, a precise characterization for non-small cell lung cancer is of utmost importance due to increased mortality rate and lack of targeted therapies. Hence, the article focuses on the expression of stemness-associated markers, namely octamer-binding transcription factor 4 (OCT4), NANOG, and sex-determining region Y-box 2 (SOX2) in non-small cell lung cancer (NSCLC) patients. Methods: The expression of OCT4, NANOG, and SOX2 were evaluated in 32 histopathologically confirmed NSCLC tissues using real-time polymerase chain reaction. The obtained expression was correlated with clinical and pathological manifestations using the statistical test such as Student's t-test and Pearson correlation in varied statistical software. Results: Results showed a significantly higher expression of OCT4 and NANOG compared to SOX2 in the tumor tissues. When the expression of these markers was correlated with the clinical parameters, higher expression was seen in males, patients with age above 60 years, and in adenocarcinoma subtype. In correlation with the habit, higher expression of OCT4 and SOX2 was observed in habituated patients. Expression of NANOG and OCT4 was higher even in patients with poor differentiation. Conclusion: The expression and prognostic significance of CSC markers obviously vary depending on histological NSCLC subtype. Importantly, our findings suggest that OCT4, SOX2, and NANOG network together may be promising for ongoing targeted therapies in specific NSCLC subgroups

Elevated expression of stemness genes in adipose-derived mesenchymal stem cells cultured on fibrin scaffold

In regenerative medicine, MSCs need to be pluripotent for better results. In this study, the effect of fibrinscaffold on expression of stemness genes was examined. Adipose-derived MSCs were cultured in tissue cultureplates (2D) and 3-dimensional (3D) fibrin scaffolds. The effect of fibrin scaffold on proliferation of adiposederived MSCs was evaluated by MTT assay. The expression of stemness genes (OCT4 and SOX2) wereevaluated by qRT-PCR, and flow cytometry was done for Nanog protein level. Cultured MSCs on fibrinscaffold were able to proliferate according to data obtained by MTT assay. Expression of OCT4 and SOX2 hada significant increase in cells were cultured in 3D condition compared to 2D condition (P \0.05). Also,increased expression of Nanog protein in 3D culture was observed (P \0.05). OCT4 and SOX2 in 3Dcondition increased two-fold and three-fold respectively in 2D and 3D conditions. Moreover, expression ofNanog increased 30% more than in 2D condition. Evaluation of important pluripotency regulators such asOCT4, SOX2, and Nanog showed that fibrin scaffolds are useful instruments to maintain stemness of MSCs,which is essential in field of stem cell therapy and regenerative medicine.

Far Beyond Cancer Immunotherapy: Reversion of Multi-Malignant Phenotypes of Immunotherapeutic-Resistant Cancer by Targeting the NANOG Signaling Axis

Cancer immunotherapy, in the form of vaccination, adoptive cellular transfer, or immune checkpoint inhibitors, has emerged as a promising practice within the field of oncology. However, despite the developing field's potential to revolutionize cancer treatment, the presence of immunotherapeutic-resistant tumor cells in many patients present a challenge and limitation to these immunotherapies. These cells not only indicate immunotherapeutic resistance, but also show multi-modal resistance to conventional therapies, abnormal metabolism, stemness, and metastasis. How can immunotherapeutic-resistant tumor cells render multi-malignant phenotypes? We reasoned that the immune-refractory phenotype could be associated with multi-malignant phenotypes and that these phenotypes are linked together by a factor that acts as the master regulator. In this review, we discussed the role of the embryonic transcription factor NANOG as a crucial master regulator we named “common factor” in multi-malignant phenotypes and presented strategies to overcome multi-malignancy in immunotherapeutic-resistant cancer by restraining the NANOG-mediated multi-malignant signaling axis. Strategies that blunt the NANOG axis could improve the clinical management of therapy-refractory cancer.

Long non-coding RNA-H19 promotes proliferation and migration of non-small cell lung cancer cells through sponging microRNA-760 to regulate nanog gene expression / 第二军医大学学报

ObjectiveTo explore the role of long non-coding RNA (lncRNA)-H19 in the proliferation and migration of non-small cell lung cancer (NSCLC) cells and the molecular mechanisms. MethodsThe expressions of lncRNA-H19 in 20 NSCLC tissues and paired non-tumor tissues, which were collected from Changhai Hospital of Naval Medical University (Second Military Medical University) from Oct. 2015 to May 2016, were detected by real-time quantitative PCR (qPCR). We also examined lncRNA-H19 expressions in NSCLC cell lines A549 and NCI-H1299 and normal lung epithelial cell line BEAS-2B by qPCR. The proliferation and migration of A549 cells overexpressing lncRNA-H19 were detected by CCK-8 assay and Transwell assay, respectively. Bioinformatics analysis and duel-luciferase reporter assay were conducted to predict and confirm the interaction between microRNA (miRNA)-760 and lncRNA-H19. Western blotting analysis and RT-qPCR were performed to observe the influence of lncRNA-H19 overexpression on the expression of miRNA-760 and target gene nanog. MiRNA-760 was overexpressed in A549 cells, and its role in lncRNA-H19 promoting proliferation and migration of NSCLC cells was observed. Results The expressions of lncRNA-H19 in NSCLC tissues and A549 and NCI-H1299 cells were significantly upregulated compared with those in normal tissues and BEAS-2B cells (all P<0.01). Overexpression of lncRNA-H19 significantly improved the proliferation ability (P<0.05) and migration ability (P<0.01) of A549 cells compared with the negative control group. The results of starBase v3.0 showed that lncRNA-H19 could specifically adsorb miRNA-760, and duel-luciferase reporter assay showed that lncRNA-H19 directly bound to miRNA-760. Compared with the negative control group, overexpression of lncRNA-H19 significantly inhibited miRNA-760 expression in A549 cells (P<0.05) and promoted the expression of the downstream gene nanog at mRNA and protein levels (all P<0.01). Overexpression of miRNA-760 significantly inhibited lncRNA-H19-induced proliferation and migration of A549 cells (all P <0.05). ConclusionLncRNA-H19 can promote the proliferation and migration of NSCLC cells through sponging miRNA-760 to regulate nanog gene expression.

NANOG Alleviates the Damage of Human Hair Follicle Mesenchymal Stem Cells Caused by H2O2 through Activation of AKT Pathway / 生物医学与环境科学（英文）

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.

Correlation of Cancer Stem-Cell Markers OCT4, SOX2, and NANOG with Clinicopathological Features and Prognosis in Operative Patients with Rectal Cancer

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.

Expression of CD44 and NANOG in epithelial ovarian cancer and its clin-ical significance / 中国病理生理杂志

AIM:To investigate the expression of CD44 and transcription factor NANOG in epithelial ovarian cancer(EOC)and its clinical significance.METHODS:The expression of CD44 and NANOG in EOC and benign ovarian tumor tissues were detected by immunohistochemical method.The correlation between the expression of CD 44 and NANOG in EOC was analyzed.The expression of CD44 and NANOG in ovarian cancer cell line SKOV3 after treatment with cisplatin at different concentrations were detected by Western blot.RESULTS:The positive expression rates of CD44 and NANOG in EOC were significantly higher than those in benign ovarian tumor tissues(P<0.01).In EOC,positive correlation was observed between the expression of CD 44 and NANOG(r=0.346,P<0.01).The positive expression rate of CD44 was associated with clinical stage and lymphatic metastasis(P<0.05), and had no relationship with age, histological grade, pathological types and the location of tumors.The positive expression rate of NANOG was associated with clinical stage and histological grade(P<0.05),and had no relationship with age,lymphatic metastasis,pathological types and the location of tumors.Increased expression of CD44 and NANOG were detected in ovarian cancer cell line SKOV 3 after treatment with cisplatin(P<0.05).CONCLUSION:The over-expression of CD44 and NANOG are associated with the occurrence and development of EOC.The expression of cancer stem cells marker CD 44 and the pluripotent gene product pertaining to em-bryonic stem cells NANOG are increased in cisplatin-induced SKOV3 cells.Cancer stem cells and the expression of stem-ness-related gene may well be the underlying pathogenesis that promotes the onset, progression, and chemotherapy resist-ance of EOC.

Effects of NANOG/deleted in breast cancer 1 pathway on biological behavior of gastric cancer cells / 中华消化杂志

Objective To investigate the effects of NANOG/deleted in breast cancer 1 (DBC1)pathway on biological behavior of gastric cancer cells.Methods From May 2014 to May 2015,25 patients who underwent gastric cancer resection were selected.The expression of NANOG and DBC1 was detected by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting in N-tera,SGC-7901,HGC-27,MKN-45,MGC803,NCI-N87,BGC823 cell lines,normal gastric epithelial cell line GES-1 and gastric cancer tissues.The proliferation,apoptosis and colony formation ability of MKN-45 cells in short hairpin (sh)NANOG-1,shNANOG-2,sh-control and shDBC1 groups were determined by MTT assay,flow cytometry and colony forming assay.The effects on the expression of the two genes in MKN-45 cells were verified by polymerase chain reaction (PCR) in shNANOG,sh-control,shDBC1 and shDBC1+NANOG groups and the effects of down regulation of DBC1 on cell biological behavior were further investigated.The differences in gene expression profile after interference which were screened by gene chips and bioinformatics were analyzed.The mechanism of NANOG regulating DBC1 was explored by Dual-luciferase assay.T test was used for two groups comparison while one-way analysis of variance was for multiple groups.Results NANOG and NANOG mRNA were highly expressed in N-tera cells,which were 1.02±0.08 and 0.95 ±0.03,respectively,and the expressions in SGC-7901,HGC-27,MKN-45 and NCI-N87 cell lines were 0.67±0.03 and 0.64±0.04,0.58±0.02 and 0.28±0.02,0.83±0.03 and 1.04 ± 0.05,and 0.61 ± 0.02 and 0.64 ± 0.08,respectively;no expression was detected in normal gastric epithelial cell line GES-1,and the expressions in MKN-45 cells were the highest in gastric cancer cells (F=21.51 and 85.53,both P＜0.01).The expression of DBC1 in HGC-27,MGC803,NCIN87,SGC-7901,BGC823 and MKN-45 cells were 0.37±0.02,0.33±0.02,0.42±0.01,0.58±0.04,0.33±0.05,and 0.87±0.02,respectively;while there was no expression of NANOG,NANOG mRNA and DBC1 in GES-1 cells.The expression of NANOG mRNA and DBC1 was detected in gastric cancer tissues of 24.0％ (6/25) patients.Compared with that of the sh control group,the apoptosis rates of MKN-45 cells in the shNANOG-1,shNANOG-2 groups were increased ((2.24±0.17)％ vs (6.03±0.24) ％ and (6.95 ± 0.38) ％),and the difference was statistically significant (F =81.18,P ＜ 0.01).Compared with that of the sh-eontrol group,the colony forming abilities of MKN-45 cells in the shNANOG-1 and shNANOG-2 groups were significantly decreased (172.03±6.35 vs 74.32±5.32 and 53.08±3.82),and the difference was statistically significant(F=171.61,P＜0.01).The results of PCR showed that compared with that of sh-eontrol group,the expression levels of NANOG mRNA and DBC1 mRNA in shNANOG group were lower (1.04±0.05 vs 0.54±0.03,1.08±0.08 vs 0.42±0.03),the level of DBC1 mRNA in shDBC1 group was lower (1.08±0.08 vs 0.50±0.04),and the differences were statistically significant (t=9.15,7.37 and 6.06,all P＜0.01).The expression level of NANOG mRNA in shDBC1 + NANOG group was higher (1.04 ± 0.05 vs 3.01 ± 0.08),while the expression level of DBC1 mRNA was lower (1.08 ± 0.08 vs 0.71 ± 0.06),and the difference was statistically significant (t=-20.22 and 3.74,both P＜0.05).The expression level of DBC1 mRNA in shDBC1±NANOG group was higher than that in shDBC1 group (0.71±0.06 vs 0.50±0.04),and the difference was statistically significant (t=4.00,P＜0.05).Bioinformatic analysis showed that DBC1 gene promoter region had the potential NANOG protein binding sites.Dual-luciferase assay indicated NANOG played the role in transcription activation in DBC1 promoter regions.Conclusion NANOG and DBC1 are highly expressed in various gastric cancer cell lines.NANOG may affect the proliferation,apoptosis and colony formation of MKN-45 cells by regulating the expression of DBC1.NANOG/DBC1 pathway may be a promising new target of gastric cancer treatment.

Immunohistochemical expression of NANOG in urothelial carcinoma of the bladder

Urothelial carcinoma is a common malignant neoplasm that has a poor prognosis and a high frequencyof recurrence and metastasis. Constant disease surveillance with periodic and long term cystoscopyexamination is necessary for management of the disease. However, the monitoring and therapyregimen is expensive, incurring a massive burden to patients and the government. Therefore, thedevelopment of specific biomarkers for urothelial carcinoma at an early stage and recurrence detectionbecomes a priority. Homeobox genes are a family of genes that are involved in tumourigenesis.They might be potential prognostic markers for urothelial carcinoma. The study investigated theexpression pattern of NANOG which is one of a homeobox gene in different stages and grades ofurothelial carcinoma. NANOG expressions were also correlated with patient demographic factors andclinicopathological parameters. The expression of NANOG in 100 formalin-fixed paraffin-embeddedurothelial carcinoma tissues was determined by immunohistochemistry. Immunohistochemistryshowed positive expression of NANOG in all specimens with detection in the cytoplasm, nucleiand the nuclear membrane of the cancer cells. The immunohistochemical expression of NANOGincreased across stages and grades of the tumour. The expression of NANOG was not significantlyassociated with demographic factors; gender (p = 0.376), race (p = 0.718) and age (p = 0.058) aswell as with most of the clinicopathological parameters; pathological stage (p = 0.144), grade (p =0.625), lymph node involvement (p = 0.174) and distant metastasis (p = 0.228). However, NANOGexpression showed significant correlation with tumour invasion (p = 0.019). We concluded thatNANOG might be a potential biomarker for early diagnosis of urothelial carcinoma of the bladder.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

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.

Nanog influences the proliferative ability of HepG2 cells through the modulation of the cell cycle related proteins / 实用肿瘤学杂志

Objective To study the effect of pluripotency factor Nanog on the expression of the cell cy-cle related proteins,and then to explore its effect on the proliferative ability of HepG 2 cells.Methods TALENs gene editing tool was employed to induce mutation and downregulation expression of Nanog .T7 endonuclease 1 and genomic sequencing was used to analyze the mutation efficiency of Nanog .RT-PCR and western blot were used to determine the expression of mRNA and protein of Nanog ,respectively .Real-time cell based assay system was used to measure the proliferative ability of wild -type HepG2 cells and monoclonal HepG 2 cells with Nanog mutation.Results TALENs successfully induced mutation of Nanog gene .The targeting efficiency of mixed cells was analyzed by T7 endonuclease 1 approached 40%after two transfection with plasmid of Nanog -TALENs.Ad-ditionally,the Nanog mRNA expression level of monoclonal HepG 2 with Nanog mutation was downregulated by 3.4 times compared to the wild type HepG 2 cells,and the Nanog protein expression level was downregulated by 3.6 times.The cell cycle related proteins CyclinD1/D3,CyclinE1 and CDK2 expression were downregulated in monoclonal HepG2 with Nanog mutation in comparison to the wild type HepG 2 cells.Conclusion Nanog plays a role in influencing the proliferative ability of HepG 2 cells through modulating the expression of the cell cycle re-lated proteins CyclinD1/D3,CyclinE1 and CDK2.The downregulation expression of Nanog can inhibit the prolif-erative capacity of HepG 2 cells via the regulation of the cell cycle related proteins .

Expression and significance of Nanog and Oct4 in endometriosis and adenomyosis / 实用医学杂志

Objective To investigate the expressions of Nanog and Oct4 (stem cell transcription factors) in endometriosis and adenomyosis, and to explore their potential functions in the development of endometriosis and adenomyosis. Methods The expressions of Nanog and Oct4 in the ectopic and eutopic endometrium of 50 patients with endometriosis and/or adenomyosis (ectopic endometrium group and eutopic endometrium group), and 21 patients free from endometriosis and adenomyosis (control group) were detected by immunohistochemical SABC methods. Statistical analysis was conducted for the correlation between the expressions of Nanog and Oct4 based on patients′ clinical pathological parameters. Results Nanog and Oct4 protein expressions in ectopic endometrium group were higher than that in control group (P 0.05); there was positive correlation between the expressions of Nanog and Oct4 in ectopic endometrium group (r = 0.590, P < 0.01). Conclusion Nanog and Oct4 present high expression in eutopic and ectopic endometrium , which may play a important role in the development of endometriosis and adenomyosis.

STAT3 correlates with stem cell-related transcription factors in cervical cancer / 华中科技大学学报（医学）（英德文版）

Cancer stem cells (CSCs) are considered responsible for the high recurrence rate in cervical carcinoma. It has been demonstrated that the signal transducer and activator of transcription 3 (STAT3) is involved in the oncogenesis and takes part in mediating the effects of maintaining stem cell phenotype and pluripotency by regulating the expression of stem cell-related transcription factors. However, the correlation between STAT3 and stem cell-related transcription factors in cervical cancer has not been elucidated. In this study, we established overexpressing plasmid (GV316-STAT3) and siRNA-STAT3 for transfecting Siha cells. Cells negative or positive for Nanog, Oct4, or Sox2 were selected by flow cytometry. Proliferation and differentiation rate of Siha cells was determined by detecting the efficiency of tumor sphere formation. The expression of Nanog, Oct4 and Sox2 (cancer stem cell markers) and STAT3 was detected by quantitative real-time PCR and immunoblotting for Siha cells and by immunohistochemistry (IHC) for cervical tissues, respectively. The results showed that Nanog+, Oct4+, and Sox2+ Siha-STAT3 over-expressing cells displayed the typical non-adherent spheres. The sphere formation efficiency was significantly different between Siha-STAT3 overexpressing cells and siRNA-STAT3 cells (P<0.05). Meanwhile, the expression levels of Oct4, Nanog and Sox2 mRNA and protein were significantly higher in Siha-STAT3 overexprssing cells than in siRNA-STAT3 cells (P<0.05). In addition, the positive rate of STAT3, Nanog, Oct4 and Sox2 in cervical cancer tissues was higher than that in chronic cervicitis group (P<0.05). There was a significantly positive relationship between STAT3 and Nanog or Oct4 or Sox2 expression (all P<0.001). These results suggested that Oct4+, Sox2+, and Nanog+ cell population possesses stem cell properties in cervical cancer, which may contribute to cervical carcinogenesis and be regulated by STAT3.

Effects of small interfering RNA(siRNA) silencing Nanog gene on migration and invasion of human lung adenocarcinoma A549 cells / 实用医学杂志

Objective To investigate the effects of small interfering RNA (siRNA) silencing Nanog gene on the ability of migration and invasion of the human lung adenocarcinoma A549 cells. Methods The human lung adenocarcinoma A549 cells were transfected with siRNA targeting Nanog gene , and three experiment groups were set up. The expression level of Nanog was detected using reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Cell migration was examined by wound healing assay and cell invasion was detected by Transwell assay. Results The Nanog silencing cell group (A549-siNanog) showed much lower level of Nanog mRNA and protein (0.40 ± 0.06, 0.50 ± 0.03) than A549-siNC cell group (0.97 ± 0.03, 0.85 ± 0.02; P < 0.05) under RT-PCR and Western blot analysis. Meanwhile, the wounded area filled rate and the number of invaded cells of A549-siNanog cell group (57% ± 0.04, 69.60 ± 17.14) were decreased significantly compared to A549-siNC cell group (95% ± 0.02, 209.60 ± 15.40; P < 0.05). Conclusion siRNA targeting human Nanog could specially suppress the expression of Nanog gene in lung adenocarcinoma A549 cells. In this way, it couldsignificantly reduce the capability of migration and invasion of A549 cells.

The expression and significance of Nanog and CD44 in spheroid body-forming cells of gastric cancer cell line MKN-45 / 天津医药

Objective To detect the expression of stem-cell related factors Nanog and CD44 in spheroid body-form?ing cells of gastric cancer cell line MKN-45. Methods The gastric cancer cell line MKN-45 was used to culture spheroid bodies in non-adherent condition in a serum-free medium supplemented with epidermal growth factor (EGF) and basic fibro?blast growth factor (bFGF). Using Western blot analysis, immunofluorescence staining and quantitative real time polymerase chain reaction(qRT-PCR), the expression levels of stem cell-related genes Nanog and CD44 were studied. Results In this study, we observed that MKN45 cells formed spheroid bodies in non-adherent condition in a serum-free medium, and the levels of Nanog and CD44 mRNA expression in spheroid body-forming cells were 2.34 ± 0.22 and 1.18 ± 0.04,respectively, which were higher than those in parental cells (1.00±0.00 and 1.00±0.05). The levels of Nanog and CD44 protein expression in spheroid body-forming cells were 0.18±0.02 and 0.24±0.04, respectively, which were significantly higher than those in pa?rental cells (0.07±0.02 and 0.18±0.01, P<0.05). Nanog protein was positively stained within the perinuclear and cytoplasm of the spheroid body-forming cells, and CD44 was positively stained mainly in the membrane. Dual staining of Nanog/CD44 indicated that the embryonal protein Nanog was co-localized with CD44 in the spheroid body-forming cells. Conclusion Spheroid body-forming cells developed from human gastric cancer cell line MKN-45 in serum-free medium supplemented with EGF and bFGF show characteristics of cancer stem cell (CSC). The cells co-expressed of CD44 and Nanog maybe a phe?notype of gastric CSCs.

Expressions and clinical significance of Nanog and CD44 protein in lung cancer / 实用医学杂志

Objective To detect the expressions and clinical significance of Nanog and CD44 protein in lung cancer. Methods The expressions of Nanog and CD44 were detected by immunohistochemistry in 50 cases of lung cancer, 32 cases of benign lesion lung tissue and 18 cases of paraneoplastic normal lung tissue. Then their relationships with clinicopathological factors were analyzed. Results The expression of Nanog in lung cancer was significantly higher than those in benign lesion lung tissue and paraneoplastic normal lung tissue (P 0.05). The expressions of Nanog and CD44 in squamous cell carcinomas were higher than those in adenocarcinomas and small cell lung carcinomas (P 0.05). The positive correlation was also noted between the expressions of Nanog and CD44 in lung cancer (r = 0.564, P < 0.05). Conclusion Nanog and CD44 proteins may participate in the genesis and progression of lung cancer. Nanog protein is a potential diagnostic marker and therapeutic target for lung cancer.

Growth Properties and Pluripotency Marker Expression of Spontaneously Formed Three-dimensional Aggregates of Human Adipose-derived Stem Cells

BACKGROUND AND OBJECTIVES: Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor. METHODS AND RESULTS: Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected. CONCLUSIONS: Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.