Expression And Significance Of Oct4 In Gliomas

Glioma is the most common primary brain tumor.It is characterized by scattering,infiltration,and extreme malignancy,and is the leading cause of tumor related death in the central nervous system.Although major advances have been made in surgery and adjuvant therapy for gliomas,the mortality remains high.Until now, the cellular origin of gliomas remains uncertain.Recent discovery of neural stem cells (NSCs) and the identification of a small group of cells responsible for tumor formation and maintenance in leukemia and breast carcinomas have generated a wide interest on the cancer stem cell theory of carcinogenesis.It has been hypothesized that not all but only a minority of cells in the tumor,termed tumor stem cells,possesses the ability to proliferate and self-renew extensively to maintain the growth of the tumor.In this connection,several studies have isolated a small population of tumor stem cells from different types of brain tumors,termed brain tumor stem cells (BTSCs).These cells are endowed with a high self-renewal and proliferation capacity and grow as floating tumor spheres in the serum-free medium similar to that used to isolate NSCs.They express the NSC markers CD133 and nestin and can differentiate into neurons and glial cells.However,the molecular mechanisms involved in the formation and maintenance of BTSCs have remained elusive.Recent hypothesis proposed that cancer stem cells were derived either from normal stem cells with dysregulation of self-renewal process,or from the progenitors or differentiated cells obtaining the stem cell property of self-renewal.Therefore, aberrant activation of self-renewal is likely a key event in tumorigenesis and it becomes very important to examine the expression and involvement of stem cell's self-renewal regulation genes in carcinogenesis.Oct4(octamer transcription factor 4),also known as Oct3 or POU5f1,is a member of the POU family of transcription factors.It is one of the most important factors which regulate the self-renewal and differentiation of embryonic stem(ES) cells.Expression of Oct4 is restricted to totipotent embryonic cells,including inner cell mass,ectoderm of the gastrula,and primordial germ cells and their counterpart in vitro culture,such as ES cells and embryonic germ cells.Expression of this gene is down-regulated when these cells initiate differentiation;no expression has been detected in any differentiated cells.Knocking out the Oct4 gene in mice caused early lethality due to the differentiation of inner cell mass into trophoderm,indicating the critical function of Oct4 for self-renewal and pluripotency of ES cells.Recently,it has been proposed that Oct4 acts as an oncogenic factor.It has been suggested as one of four major factors that render the nuclear reprogramming of somatic cells into induced pluripotent stem cells.It was reported that Oct4 expressed not only in embryonic cells but also in adult stem cells.And the adult stem cells which express Oct4 are the target for carcinogenesis initiation.Continuous Oct4 expression in epithelial tissues is observed to lead to dysplastic disorders by inhibiting progenitor cells differentiation in a manner similar to that in embryonic cells.Oct4 has also been reported to be an oncogenic fate determinant.High levels of Oct4 increase the malignant potential of ES-derived tumors whereas inactivation of Oct4 induces a regression of the malignant component.Although expression of Oct4 has been documented in some cancers and cancer cell lines recently,little is know about the expression in the gliomas.Here,we detected the expression of Oct4 in human gliomas using reverse transcription polymerase chain reaction(RT-PCR),Western Blot analysis and immunohistochemistry,and explored the correlation between the expression levels of Oct4 and tumor grades.Using plasmid transfection and RNA interference(RNAi) technology,we further analyzed the roles of Oct4 in glioma tumorigenesis and the possible mechanisms.PART ONE Expression of Oct4 in human gliomas and the correlation between the expression levels and tumor grades.To dectect the expression of Oct4,41 clinical speciments were obtained from patients diagnosed with primary gliomas.All the tumor specimens were corroborated in final pathology and classified according to the WHO classification.There were 14 low-(WHO gradeâ…¡) and 27 high-grade(WHO gradeâ…¢,â…£) tumors.The adult normal brain tissues as normal controls were obtained from 5 trauma patients.In this part,we extracted total RNA and total protein from 41 glioma specimens and 5 normal brain tissues,and then evaluated the mRNA and protein levels of Oct4 using semi-quantitative RT-PCR and Western Blot analysis,respectively.The protein distribution of Oct4 was also evaluated in the different grades of glioma tissues using immunohistochemistry.The results of RT-PCR showed that Oct4 mRNA was expressed in 41 tumor samples and the expression levels increased with the increasing tumor grades.However,no obvious expression was detected in the 5 normal brain samples.Western blot analysis confirmed the results.The results of immunohistochemistry showed Oct4 expression was localized in the nucleus of tumor cells and the nuclei of most high-grade glioma cells were more intensely stained with Oct4 when compared with those in low-grade tumors that exhibited a weak staining. These results indicated that Oct4 is highly expressed in gliomas and the expression levels were increased with the malignant grade of tumors.In addition,we examined Oct4 expression in human glioma cell lines U87 and U251 by immunofluorescence.The results showed that U87 and U251 were positive for Oct4 staining and the expression was mainly localized in the nuclei of these cells.Based on the expression pattern and the critical roles of Oct4 in maintaining the stem cell state,we speculated that stem cell like tumor cells exist in the neoplasm;and Oct4 might be a marker of the stem cell like tumor cells and play a critical role in the formation and maintenance of these cells.PART TWO The roles of Oct4 in the proliferation and colony formation of glioma cellsAlthough many tumors and tumor cell lines are observed to express Oct4,very little is known about its potential function in cancers.To determine the roles of Oct4 in the carcinogenesis of gliomas,we cultured the classic glioma cell line C6 as the glioma model in vitro and tests were carried out with up- and down-regulation of Oct4 in C6 cells.C6 cells was transfected with Oct4 expression plasmid to up-regulate Oct4 expression,and transfected with Oct4 small interference RNA(siRNA) to down-regulate endogenous Oct4 expression.The results of RT-PCR and Western blot confirmed that Oct4 was successfully regulated at both mRNA and protein levels.Then we studied the proliferation and colony formation of tumor cells under Oct4 up-regulation and down-regulation respectively.The cell proliferation was measured by MTT assay.The results showed that the proliferation capacity and colony formation rate of C6 cells with up-regulation of Oct4 were higher than wild type C6 cells;however,C6 cells with down-regulation of Oct4 had the opposite results.This suggested that Oct4 promoted the proliferation and colony formation of glioma cells.It was suggested that tumor stem cells were transformed from their counterpart normal stem cells.So we detected Oct4 expression with RT-PCR and Western blot analysis in C6 cells and rat adult normal NSCs.Rat astrocytes and neurons,as well as rat normal brain tissues were used as controls.The results showed that Oct4 was only expressed in C6 cells and NSCs;and the expression level of Oct4 was higher in C6 cells than that in NSCs.Immunostaining analysis revealed that Oct4 protein was localized within the nuclei of C6 cells and NSCs.Similar expression pattern of Oct4 between C6 cells and NSCs implicated that NSCs were the more likely targets for tumor transformation and over-expression of Oct4 might contribute to the transformation.PART THREE The mechanism study of Oct4's roles in the proliferation and differentiation of glioma cellsTo ascertain the mechanisms of Oct4's roles in the proliferation and differentiation of glioma cells,tests were carried out with the same up- and down-regulation procedure of Oct4 described in Part Two.Previous study has reported that Stat3(signal transducer and activator of transcription 3) is constitutively activated in human gliomas and promotes tumor cells proliferation.Activation of Stat3 is via phosphorylation of Tyr705.In order to investigate if the mechanism of Oct4's function is linked to and mediated through Star3,the mRNA and protein levels of Star3 were evaluated in the up-and down-regulation procedure of Oct4 respectively.The results showed that Oct4 regulated Stat3 phosphorylation and had no effect on the total protein levels of Star3. We next examined wether Stat3 afffected the expression of Oct4.C6 cells transfected with Star3 expression vector showed elevated protein levels of total Stat3 and phospho-Stat3,and grew faster than wild type C6 cells,but Oct4 protein levels showed no significant changes.These results indicated that activation of Star3 might be one of the key downstream events of Oct4 and Oct4 promoted the proliferation and self-renewal of tumor cells via Stat3 pathway during the carcinogenesis of glioma and BTSCs formation.Many studies have reported that Oct4 could inhibit the differentiation of ES cells, so we speculated that Oct4 could inhibit the differentiation of tumor cells.Nestin and Glial Fibrillary Acidic Protein(GFAP) were the markers relating to the differentiated levels of glioma cells.The results showed that in Oct4 up-regulated cells,the expression of Nestin was increased but that of GFAP was decreased;however,in Oct4 down-regulated cells it had the opposite results.This suggested that Oct4 might expand and maintain stem cells in cancers by inhibiting their differentiation.In our work,we reported for the first time that Oct4 was highly expressed in gliomas,and furthermore,the expression level was parallel with the malignant grade of tumors.This indicated that Oct4 might be served as an important index for diagnosis of gliomas and evaluation of treatment as well.In in vitro cell cultures,Oct4 was only expressed in U87,U251 and C6 glioma cells as well as rat NSCs but not in rat neurons and astrocytes.Using C6 cells as glioma model,we found that Oct4 could promote tumor cell proliferation and colony formation.Further analysis revealed that Oct4 could up-regulate phosphorylation of Stat3 to promote tumor cells proliferation and self-renewal.In addition,Oct4 could inhibit the differentiation of glioma cells. The results indicated that Oct4 might be a tumor stem cells related gene and this would provide a new clue for the study of tumor stem cell theory.