Octamer-binding Transcription Factor Research Articles

Epigenetic regulation of pluripotency inducer genes NANOG and SOX2 in human prostate cancer.

The cells of multicellular organisms are genetically homogeneous but heterogenous in structure and function by virtue of differential gene expression. During embryonic development, differential gene expression by modification of chromatin (DNA and histone complex) regulates the developmental proceedings before and after the germ layers are formed. Post-replicative DNA modification, where the fifth carbon atom of the cytosine gets methylated (hereafter, DNA methylation), does not incorporate mutations within the DNA. In the past few years, a boom has been observed in the field of research related to various epigenetic regulation models, which includes DNA methylation, post-translational modification of histone tails, control of chromatin structure by non-coding RNAs, and remodeling of nucleosome. Epigenetic effects like DNA methylation or histone modification play a cardinal role in development but also be able to arise stochastically, as observed during aging, in tumor development and cancer progression. Over the past few decades, researchers allured toward the involvement of pluripotency inducer genes in cancer progression and apparent for prostate cancer (PCa); also, PCa is the most diagnosed tumor worldwide and comes to the second position in causing mortality in men. The anomalous articulation of pluripotency-inducing transcription factor; SRY-related HMG box-containing transcription factor-2 (SOX2), Octamer-binding transcription factor 4 (OCT4) or POU domain, class 5, transcription factor 1 (POU5F1), and NANOG have been reported in different cancers which includes breast cancer, tongue cancer, and lung cancer, etc. Although there is a variety in gene expression signatures demonstrated by cancer cells, the epigenetic mode of regulation at the pluripotency-associated genes in PCa has been recently explored. This chapter focuses on the epigenetic control of NANOG and SOX2 genes in human PCa and the precise role thereof executed by the two transcription factors.

Oct-4 induces cisplatin resistance and tumor stem cell-like properties in endometrial carcinoma cells

ObjectiveResearch has suggested that tumor-initiating tumor stem cells are derived from normal stem cells and that tumor cells undergo progressive de-differentiation to achieve a stem cell-like state. Tumor stem cells are characterized by high proliferation ability, high plasticity, expression of multi-drug resistance proteins, and the ability to seed new tumors. Octamer-binding transcription factor 4 (Oct-4) and its activation targets are overexpressed in the tumor stem cells of various types of tumors, and this expression is associated with the pathogenesis, development, and poor prognosis of tumors. The primary objective of this study was to test if a stably transfected with Oct-4 gene cell line, RL95-2/Oct-4, has the characteristics of tumor stem cells. Materials and methodsHuman endometrial carcinoma cells (RL95-2) were transfected with a plasmid carrying genes for Oct-4 and green fluorescent protein (GFP). The stably transfected cells, RL95-2/Oct-4, were selected using G418 and observed to express the GFP reporter gene under the control of the Oct-4 promoter. GFP expression levels of RL95-2/Oct-4 cells were measured using flow cytometry. The proliferation potential of cells was determined according to cumulative population doubling and colony-formation efficiency. Gene expression was analyzed using reverse transcription-polymerase chain reaction. ResultsRL95-2/Oct-4 cells not only exhibited increased expression of the three most important stem cell genes, Oct-4, Nanog, and Sox2, but also had increased expression of the endometrial tumor stem cell genes CD133 and ALDH1. Furthermore, enhanced expression of these genes in the RL95-2/Oct-4 cells was associated with higher colony-forming ability and growth rate than in parental RL95-2 cells. We also observed that cisplatin induced less cell death in RL95-2/Oct-4 cells than in RL95-2 cells, indicating that RL95-2/Oct-4 cells were more resistant to chemotherapeutic agents. ConclusionThe study findings contribute to investigate the effects of Oct-4 on tumor stem cell origins.

Circ_rac GTPase–Activating Protein 1 Facilitates Stemness and Metastasis of Non–Small Cell Lung Cancer via Polypyrimidine Tract–Binding Protein 1 Recruitment to Promote Sirtuin-3–Mediated Replication Timing Regulatory Factor 1 Deacetylation

Circular RNAs have been identified as diagnostic and therapeutic targets for various tumors. The expression of circ_rac GTPase–activating protein 1 (circRACGAP1) is reported to drive the development of non–small cell lung cancer (NSCLC). This study further explored the potential mechanism of circRACGAP1-mediated development of NSCLC. The circRACGAP1 level was detected by quantitative RT-PCR. Sphere formation, CD133-positive cell percentage, and expression of octamer-binding transcription factor 4, Sox2, Nanog, and CD133 were detected to evaluate stemness of NSCLC. Migration and invasion were determined using wound healing and transwell assays. Protein expression was measured using Western blotting. The molecular mechanism was evaluated using RNA pull-down, RNA immunoprecipitation, and coimmunoprecipitation assays. In vivo tumor growth and metastasis were determined in nude mice. circRACGAP1 was highly expressed in NSCLC and was associated with stemness marker Sox2 expression. The stemness, metastasis, and epithelial mesenchymal transformation were repressed in circRACGAP1-depleted NSCLC cells. Mechanistically, circRACGAP1 recruited RNA-binding protein polypyrimidine tract–binding protein 1 to enhance the stability and expression of sirtuin-3 (SIRT3), which subsequently led to replication timing regulatory factor 1 (RIF1) deacetylation and activation of the Wnt/β-catenin pathway. circRACGAP1 overexpression counteracted SIRT3 or RIF1 knockdown–mediated inhibition in stemness and metastasis of NSCLC cells. The in vivo tumor growth and metastasis were repressed by circRACGAP1 depletion. Patients with NSCLC with a higher serum exosomal circRACGAP1 level had a lower overall survival rate. In conclusion, circRACGAP1 facilitated stemness and metastasis of NSCLC cells through the recruitment of polypyrimidine tract–binding protein 1 to promote SIRT3-mediated RIF1 deacetylation. Our results uncover a novel regulatory mechanism of circRACGAP1 in NSCLC and identify circRACGAP1 as a promising therapeutic target.

Protein Arginine Methyltransferase 5 Is Necessary for Embryonic Development in Medaka Oryzias latipes

Protein arginine methyltransferase 5 (Prmt5), conserved from yeast to humans, catalyzes arginine’s dimethylation in proteins. Prmt5 is necessary for embryonic development in mice because it maintains embryonic stem cells. However, the embryos of zebrafish (Danio rerio) remain viable with a deficiency in germ cells and sexual development after the knockout of prmt5. Therefore, it was considered whether prmt5 is dispensable during embryogenesis in fish. Medaka (Oryzias latipes), another model fish organism, was used in this experiment. The medaka prmt5 was mutated with Transcription Activator-Like Effector Nucleases (TALEN) causing the premature stopping of transcription. None of the homozygous prmt5 mutant fish were viable, only the heterozygous offspring survived. Quantitative reverse transcription-polymerase chain reaction (qPCR) results showed a significant decrease in octamer-binding transcription factor 4 (oct4), homeobox transcription factor nanog (nanog), vasa, B-cell CLL/lymphoma 2 (bcl2), and the ratio of bcl2 to bax (bcl2 associated x), and a significant increase in caspase3 and caspase8 in the embryos of the heterozygous prmt5 mutant compared with that of the wild type. The results showed that the mutation of prmt5 caused down-regulation of the genes functioning in stemness and up-regulation of the genes in the cascade of cell death. These results suggested that prmt5 is necessary for embryogenesis via maintaining stemness and repressing apoptosis in medaka.

SENP1 induced protein deSUMO modification increased the chemotherapy sensitivity of endometrial cancer side population cells

Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) μg/ml to (11.55±3.12) μg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.

Effects of Long Non-coding Ribonucleic Acid Plasmacytoma Variant Translocation 1 Regulating Octamer Binding Transcription Factor 4 Expression on Hepatocellular Carcinoma Cells

The researchers aimed to evaluate the effects of long non-coding ribonucleic acid plasmacytoma variant translocation 1 (lncRNA PVT1) regulating octamer binding transcription factor 4 (OCT4) expression on hepatocellular carcinoma (HCC) cells. It was discovered that in contrast to the blank group, si-PVT1 group had lower PVT1 and OCT4 expressions, number of invading cells and wound healing rate, and higher radiosensitivity (P&lt;0.05). OCT4 served as a gene targeted by lncRNA PVT1. The PVT1 group exhibited declined luciferase activity compared with the NC group after transfection with wild-type OCT4 (P&lt;0.05). In comparison with those of the sh-NC group, the number of invading cells and wound healing rate dropped in the sh-OCT4 group (P&lt;0.05). At the same radiation dose, a lower survival fraction than that in the sh-NC group was observed in the sh-OCT4 group (P&lt;0.05). Silencing lncRNA PVT1 significantly suppresses HCC cell invasion as well as migration and elevates their radiosensitivity, probably by down-regulating OCT4 expression.

Metformin as a potential agent for modulating the faulty endometriotic mesenchymal stem cells: A case-control study.

According to stem cell theory, it seems that the proliferation/differentiation imbalance in endometrial mesenchymal stem cells (enMSCs) is the leading cause of endometriosis, so targeting them to modulate stemness-relevant factors seems to be a wise choice for endometriosis treatment. We aimed to investigate the effects of metformin on stemness properties of enMSCs by evaluating the expression profile of stemness-related genes and microRNAs (miRNAs). In this case-control study, MSCs were isolated from the eutopic endometrium of 3 endometriotic and 3 healthy women. After their characterization and culture, they were treated with 0.1, 1, and 10 mM metformin for 72 hr. Finally, the expression of octamer-binding transcription factor (OCT) 4A, OCT4B, OCT4B1, sex determining region Y-Box transcription factor 2, nanog homeobox, microRNA-200b, microRNA-145, and lethal-7b were analyzed by quantitative reverse transcription-polymerase chain reaction. Metformin modulated the expression of stemness-related genes and miRNAs, OCT4A, OCT4B, OCT4B1, sex determining region Y-Box transcription factor 2, nanog homeobox, microRNA-200b, microRNA-145, and lethal-7b in enMSCs, especially at 1 and 10 mM concentration. Notably, metformin had a paradoxical effect on normal enMSCs. We showed that metformin could modulate the expression of deregulated genes and miRNAs in faulty enMSCs, and restore their skewed self-renewal/differentiation balance, so it might be a promising drug for endometriosis treatment. The paradoxical effect of metformin on enMSCs and normal enMSCs might be because of their different metabolic patterns, so it requires further investigation to illustrate.

Two Retrotransposon Elements in Intron of Porcine BMPR1B Is Associated with Phenotypic Variation.

It has been established that through binding to bone morphogenetic proteins (BMPs), bone morphogenetic protein receptor I B (BMPR1B) can mediate transforming growth factor β (TGF-β) signal transduction, and is involved in the regulation of several biological processes, such as bone and muscle formation and homeostasis, as well as folliculogenesis. Also known as FecB, BMPR1B has been reported as the major gene for sheep prolificacy. A number of previous studies have analyzed the relationship between single nucleotide polymorphisms (SNPs) in this gene and its related performance. In recent years, with the illustration of the effect of retrotransposon insertion on the expression of the proximal genes or phenotypic variation, retrotransposon insertion polymorphisms (RIPs) have been used as a novel type of molecular marker in the evaluation of evolution, population structure and breeding of plant and domestic animals. In this study, the RIPs in porcine BMPR1B gene were excavated, and thereafter verified using a comparative genome and polymerase chain reaction (PCR). The potential effects of phenotype, gene expression and functions related to RIPs were also explored. The results showed that 13 distinct RIPs were identified in introns of porcine BMPR1B. Among these, only BMPR1B-SINE-RIP9 and BMPR1B-LINE-RIP13 displayed a close relationship with the growth traits of Large White pigs. Moreover, the total number of BMPR1B-SINE+/+-RIP9 individuals born was found to be significantly higher than that of SINE−/− (p < 0.05). These two RIPs showed an obvious distribution pattern among Chinese indigenous breeds and Western commercial breeds. The expression of BMPR1B in ovaries of adult BMPR1B-SINE+/+-RIP9 Sushan pigs was found to be significantly higher in comparison to those of BMPR1B-SINE−/−-RIP9 (p < 0.05). SINE insertion of BMPR1B-SINE-RIP9 and LINE insertion of BMPR1B-LINE-RIP13 were observed to significantly increase the activity of Octamer binding transcription factor 4 (OCT4) minipromoter in CHO and C2C12 cells (p < 0.01). Therefore, these two RIPs could serve as useful molecular markers for modulating the growth or reproductive traits in assisted selection of pig breeding, while the mechanisms of the insertion function should be studied further.

Oct4 promoted proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in colon cancer cells by activating the SCF/c-Kit signaling pathway

ABSTRACT Octamer-binding transcription factor 4 (Oct4) is closely related to the occurrence and development of cancer. In the present study, we paid a special interest in exploring the effect of Oct4 on colon cancer (CC) proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) and its molecular mechanism. Immunohistochemistry (IHC) was used to detect the expression level of Oct4 in colon tissue of patients with colon cancer. Oct4 overexpression vector pcDNA-Oct4 was used to stably express Oct4 in human colon cancer cells HT29 and SW480. Cell counting kit-8 (CCK-8) assay was used to detect the cell proliferation. The invasion and migration abilities were observed by transwell and wound healing assays. The expression of EMT relate genes were observed by Western blot. We found that Oct4 was up-regulated in human colon cancer tissues than that in paracancerous tissues. The proliferation, migration, and invasion of HT29 and SW480 cells was significantly induced by transfection of pcDNA-Oct4. Furthermore, Oct4 overexpression enhanced EMT of CC cells, characterized by the increased expression of vimentin, Twist, and Snail, as well as decreased expression of E-cadherin. Mechanistically, Oct4 overexpression activated stem cell factor (SCF)/c-Kit signaling pathway in CC cells, and the SCF/c-Kit signaling inhibitor imatinib reversed pro-oncogenic effects of Oct4. These finding provide an insight into the potential of Oct4 for CC diagnosis and therapy.

Dietary zinc deficient condition increases the Bisphenol A toxicity in diabetic rat testes.

Bisphenol A (BPA) is a widely used endocrine disrupter that causes male reproductive dysfunction in humans and rodents. Diabetes-induced hyperglycemia alters spermatogenesis and antioxidant status, which negatively impacts male fertility in adults. Zinc (Zn) deficiency is a global health concern maintaining the testicular structure and functions in developing gonads. The present experiment was designed to investigate the role of Zn deficiency on BPA-induced germ cell and male gonadal toxicity in diabetic conditions. Rats were randomly divided into eight different groups - control (normal feed and water), BPA (10mg/kg/day), ZDD (fed with a Zn-deficient diet), DIA (diabetic), BPA+ZDD, BPA+DIA, ZDD+DIA and BPA+ZDD+DIA for four weeks. Animals' body and organ weight, sperm count, motility and sperm morphology were examined; testes and epididymis histopathology were investigated. Testicular DNA damage and sperm apoptosis were evaluated by halo and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays respectively. Testicular catalase and octamer-binding transcription factor 4 (OCT4) expressions were evaluated by western blot analysis. The present results demonstrated that dietary Zn-deficient condition significantly increased the BPA-induced testicular, epididymal and sperm toxicity in diabetic rats due to hypogonadism, increased sperm abnormalities, epididymis, testicular structure and DNA damages, sperm apoptosis as well as decreased testicular catalase and OCT4 expressions. The present results revealed that dietary Zn-deficient condition exacerbated the BPA-induced testicular and epididymal toxicity as well as perturbed the general male reproductive health in diabetic rats.

The Transcription Factor Otc4A Stimulates the Proliferation, Invasion, and Stemness of Colorectal Cancer Cells by Inhibiting the Regulation of miR-7-5p on TLR4.

Background To investigate the effects and mechanism of octamer-binding transcription factor 4 (Otc4A) on proliferation, invasion, and stemness of colorectal cancer (CRC) cells. Methods Firstly, normal fetal human cells (FHC, colon epithelial cells) and HT29 cells (CRC cells) were cultured. The expression levels of Otc4A, miR-7-5p, and TLR4 in cells were then detected by qRT-PCR. CCK-8 was adopted to measure cell proliferation rate after Otc4A, miR-7-5p, and TLR4, respectively, were either knocked out or overexpressed in HT29 cells. Later, the cell viability was detected by cell cloning assay; cell invasion by transwell; cell sphere-forming ability by sphere-formation assay; protein expression level of Otc4A, p65, p-p65, and TLR4 by western blot; and the targeting relationships between miR-7-5p and Otc4A as well as miR-7-5p and TLR4 by dual-luciferase reporter assay. Finally, chromatin immunoprecipitation was applied to verify the interaction between Otc4A and miR-7-5p. Results In HT29 cells, Otc4A expression was significantly increased. Additionally, the knockdown of Otc4A prevented HT29 cells from proliferating, migrating, forming spheres, and activating NF–B signaling. Otc4A could negatively regulate miR-7-5p, and miR-7-5p could target TLR4 expression. Besides, a negative correlation was found between Otc4A and miR-7-5p. Finally, the knockdown of miR-7-5p or overexpression of TLR4 could significantly reverse the effect of the knockdown of Otc4A on HT29 cells. Conclusion The transcription factor Otc4A can regulate the level of TLR4 by inhibiting the expression of miR-7-5p and then promote the proliferation and invasion of CRC cell HT29 as well as enhance cell stemness.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

Pluripotency of periodontal ligament and dental pulp cells is induced by intercellular communication via the CDX2/Oct-4/Sox2 pathway.

BackgroundIntercellular communication in the environments of mature or aged cells can restore and regenerate their function and promote the expression of pluripotency markers. The regeneration of dental tissue is stimulated by periodontal ligament cells (PDLCs) and dental pulp cells (DPCs). However, the communication networks between the cells and their microenvironments are poorly understood.MethodsIn this study, gene expression was analyzed by polymerase chain reaction, and chromatin immunoprecipitation assays, dual-luciferase assays, and electrophoretic mobility shift assays were used to analyze the signaling pathways associated with pluripotency after the knockdown or overexpression of caudal-type homeobox transcription factor 2 (CDX2).ResultsElevated levels of SRY-box transcription factor 2 (Sox2) and octamer-binding transcription factor 4 (Oct-4) were observed in the co-culture system, while the levels of CDX2 were significantly reduced. The overexpression of CDX2 promoted cell apoptosis and reduced the synthesis stage of the cell cycle. CDX2 was shown to bind directly to the promoter regions of Sox2 and Oct-4. The silencing of CDX2 promoted calcium deposition, adipogenic differentiation, and elevated alkaline phosphatase (ALP) activity in the DPCs.ConclusionsThese findings demonstrate the enhancement of DPC and PDLC pluripotency by intercellular communication. CDX2 plays a significant part in the regulation of DPC and PDLC pluripotency via its regulation of Oct-4 and Sox2 expression.

Effects of different concentrations of reversine to enhance conversion of dedifferentiated fat cells into mature cardiomyocytes

Background: There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn’t fully understood. Methods: We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results: ANOVA with Tukey’s post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p &lt; 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p = 0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p = 0.001) increased significantly from day 7 until day 21. Conclusions: Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to stem cell-ness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Assessment of the association of OCT3/4 with GLUT1 and CD105 in oral squamous cell carcinoma using dual immunohistochemistry

BackgroundOral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral and maxillofacial region. This study aimed to investigate the role of cancer stem cells (CSCs) in angiogenesis and hypoxic response in OSCC.MethodsThis retrospective observational study evaluated 56 cases of OSCC using dual immunohistochemistry. Octamer-binding transcription factor 3/4 (OCT3/4) marker was used to evaluate CSC activity. Glucose transporter 1 (GLUT1) marker was used to evaluate the hypoxic response and angiogenesis, while endoglin (CD105) was used to evaluate the late stage of angiogenesis and blood vessel formation.ResultsCo-expression of OCT3/4 and GLUT1 was noted in 11 of 12 patients with grade III OSCC. However, we did not observe co-expression of these markers in 13 of 22 patients with grade I OSCC. Although we observed a significant correlation between co-expression of GLUT1 and OCT3/4 and tumor grade, there was no significant correlation between co-expression of OCT3/4 and CD105 and different grades of OSCC.ConclusionsCSCs could play important roles in the initial stages of hypoxic response and angiogenesis. Our result reported that in higher grades of OSCC, GLUT1 as a first response to hypoxic situations might be a result of CSCs. Further studies are required to discover other biomarkers, their roles, and associated pathways of CSCs in OSCC.

Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor

BackgroundThe number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had.MethodsOCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed.ResultsOCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4.ConclusionsUsing a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications.

HPV16 E6-Activated OCT4 Promotes Cervical Cancer Progression by Suppressing p53 Expression via Co-Repressor NCOR1.

Human papillomaviruses (HPV), mainly HPV16 and HPV18, of high-risk classification are involved in cervical cancer carcinogenesis and progression. Octamer-binding transcription factor 4 (OCT4) is a key transcription factor that is increased in various cancer types. Cervical cancer patients with higher levels of OCT4 had worse survival rates. However, the definite mechanisms underlying its function in the development of cervical cancer still remain to be explicated. Here, our study demonstrated that OCT4 expression was slightly increased in cervical cancer tissues than in precancerous ones. However, OCT4 was significantly upregulated in HPV16-positive tissues, in contrast to the expression profiling for p53. Moreover, knockdown of HPV16 E6 in SiHa cells suppressed the expression of OCT4 with impaired activities of cell proliferation, migration, and invasion, while it recovered the expression of p53. Overexpression of OCT4 and p53 exerted opposite roles on cell proliferation, migration, invasion, and colony formation of cervical cancer cells. More importantly, the enforced expression of OCT4 augmented p53-inhibited cell migration, invasion, and colony formation in human cervical cancer by promoting EMT. Finally, we identified that OCT4 could bind to the p53 promoter region to repress p53 expression by recruiting co-repressor NCOR1 using luciferase, ChIP, and co-IP experiments. We further illustrated that OCT4 not only increased the lung metastasis of cervical cancer but also effectively reversed p53-inhibited lung metastasis. In conclusion, our results suggested that HPV16 E6 activated the expression of OCT4 and subsequently crippled the transcription of p53 via co-repressor NCOR1, which contributed to cervical cancer progression.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Identification of Stem Cell Related Gene Expression from the Osteosarcoma Cell Core Side

Osteosarcoma is the most frequent primary malignant bone tumor with higher incidences in children and adolescents. Despite clinical evolutions, patients with osteosacoma have had a poor prognosis. There has been increasing evidence that cancer is a stem cell disease. This study sought to isolate and characterize cancer stem cells from human osteosarcoma with relevant literature reviews. Here we show that the emerging evidence suggests osteosarcoma should be regarded as a differentiation disease such as stem cell disease. Two human osteosarcoma cell lines were cultured in non-adherent culture conditions as sarcospheres. Sarcospheres were observed using histomorphology and alkaline phosphatase (ALP) staining. Expression of the embryonic stem cell marker was analyzed with use of reverse transcriptase-PCR. Sarcospheres could be reproduced consistently throughout multiple passages and produced adherent osteosarcoma cell cultures. Expression of stem cell-associated genes such as those encoding Nanog, octamer-binding transcription factor 3/4, sex determining region Y box 2 , c-Myc and ALP indicated pluripotent stem-like cells. These results support the extension of the cancer stem cell theory to include osteosarcoma. Understanding the cancer stem cell derived from human osteosarcoma could lead to the evolution of diagnosis and treatment for osteosarcoma patients.

Abstract 5125: Biological and clinical value of OCT4A/SPP1C axis in endometrial and lung adenocarcinoma

Abstract Background: It is largely unclear whether octamer-binding transcription factor 4A (OCT4A, OCT3/4A) functions physiologically or pathologically in adult somatic tissues and related cancers. We have recently identified OCT4A translation in a human cancer cell subpopulation that might exacerbate cell migration and invasion. Here, we focus on biological and clinical features of OCT4A and its relationship with secreted phosphoprotein 1(SPP1), which is strongly related to the malignant progression of numerous tumors. Methods: RT-PCR or qPCR analysis was used to identify full-length spliced forms of OCT4A and SPP1 transcripts in non-tumor and tumor human cells. An OCT4A-FLAG-tagged genomic transgene identified OCT4A-positive cancer cells. Cell-ablation of OCT4A-positive cells using promoter-driven diphtheria toxin A was used to examine the potential role of OCT4A in lung and endometrial adenocarcinoma cells. OCT4A and SPP1 transcripts in early-stage lung adenocarcinoma (stage I, fifty-eight cases) were analyzed and compared with pathohistological and prognostic features. Results: We found that OCT4A and SPP1C are co-expressed in highly aggressive human lung and endometrial adenocarcinoma cell lines but not in mesothelioma or non-tumor cell lines. Ablation of OCT4A-positive cells significantly decreased cancer-cell migration and SPP1C mRNA levels. The OCT4A/SPP1C axis was found in primary, early-stage, lung adenocarcinomas, and all four cases of recurrence and/or metastasis out of 58 cases were included in the OCT4A and SPP1C positive group. Conclusions: Co-expression of OCT4A and SPP1C may correlate with human cancer cell migration and the OCT4A/SPP1C axis may have a prognostic value in high-risk, early-stage lung adenocarcinomas.