Tumorigenicity And Pluripotency Of Tumor Stem Cells Were Regulated By Oct4

In the last few years, a growing body of evidence has been reported supporting the notion that tumors are organized in a hierarchy of heterogeneous cell populations with different biologic properties and that the capability to sustain tumor formation and growth exclusively resides in a small proportion of cells called tumor stem cells, although heterogeneous genetic alterations have been reported, continued identification of signature gene alterations in hepatocarcinoma tumor stem cells will provide a conceptual framework to guide future analyses of this complex disease and the development of strategies for its early detection and effective treatment. Oct4has been implicated in the self-renewal and pluripotency of human embryonal stem cells. Transcription of Oct4was recently found be associated with an undifferentiated phenotype and tumors, suggesting the possibility of a role for Oct4in the sternness maintenance of tumor stem cells.Objectives:To explore the role of Oct4in regulating tumorigenicity and pluripotency of tumor stem cells and the related mechanism.Methods:We examined Oct4expression in stem-like cells T3A-A3derived from hepatocarcinoma, the hepatocarcinoma cell lines BEL-7402, SMMC-7721and HepG2, and the immortalized liver sinusoidal endothelial cells LSEC by RT-PCR and western blotting. Infection of pLVTHM-shOct4lentivirus was use to knockdown the Oct4levels in T3A-A3cells. The cell proliferation inhibition effect of shOct4on T3A-A3cells was examined by clonogenic formation assay and cell cycle analysis. Cell senescence was detected by senescence-associated β-galactosidase staining and p16fluorescence staining. Self-renewal ability of T3A-A3was evaluated by tumorsphere-formation assay.On the other hand, Oct4was ectopic expressed in HepG2cells by pWPTS-Oct4 lentivirus infection. MTT assay was used to investigate the cell proliferation of HepG2cells infected with a lentivirus harboring pWPTS-Oct4or pWPTS-GFP.In addition, in vivo tumor growth experiments and tumorigenicity experiments in nude mice were used to determine the role of Oct4in regulating tumor growth rate and tumorigenicity of T3A-A3cells or HepG2cells. Immunohistochemistry was use to examine the expression of Oct4and PCNA in xenograft tumors.In order to investigate the role of Oct4in regulating multiple differentiation potential of T3A-A3cells, human melanoma cell line SK-MEL-1and human lymphoma cell line Daudi were selected to prepare the tumor inducing conditioned mediums, the tissue conditioned medium were made from tissue homogenate of tumors formed by both cell transplanted in NOD/SCED mice. T3A-A3cells infected with shOct4or shNC were cultured with melanoma/lymphoma tissue conditioned mediums for21days respectively, then the expression of melanoma specific molecule (gp100) and lymphoma correlative molecule (CD10) were detected by RT-PCR, western blotting and immunofluorescence staining.Results:T3A-A3cells have higher Oct4levels compared to other cell lines, and Oct4deficiency is correlated with a decrease in tumorigenicity. Oct4knockdown in T3A-A3cells by lentivirus infection induced G1arrest in cell cycling and significantly inhibited clonogenic cell expansion in vitro and xenograft tumor growth in vivo. Moreover, Oct4knockdown diminished tumorsphere growth in vitro and tumor formation in vivo in T3A-A3cells. On the other hand, Oct4ectopic expression in HepG2cells results in increasing cell growth in vitro and tumorigenesis in vivo. More significantly, Oct4knockdown also inhibit the ability of T3A-A3cells to differentiate toward different types of cancer cells when induced with corresponding type of tumor tissue medium.Conclusions:We conclude that Oct4is a crucial regulator of tumorigenicity and pluripotency in tumor stem cells and plays important roles in tumor initiation, progression and cell fate determination. Knockdown of Oct4may hold significant promise as a novel molecular therapy for human tumors. As the third leading cause of death from cancer, HCC accounts for85-90%of all primary liver cancers and ranks as the fifth most prevalent malignancy worldwide. Despite great advances in the treatment of the disease, relapse and metastasis are frequently observed in the clinic, and the5-year survival rate remains quite low among patients with HCC. MicroRNAs (miRNAs) are implicated in cancer initiation and progression by their ability to affect the expression of genes and proteins that regulate cell proliferation and death. Recent studies found that p53directly regulates miR-145transcription. Sox2, Oct4, and Klf4are among the target genes regulated by miR-145, suggesting that miR-145possibly plays a role in the maintenance and survival of tumor stem cells.Objectives:To explore the role of miR-145in regulating tumorigenicity of tumor stem cells T3A-A3and the related mechanisms.Methods:We examined miR-145expression in tumor stem cells T3A-A3, the hepatocarcinoma cell line BEL-7402, and the immortalized liver sinusoidal endothelial LSEC cells by real time PCR. miR-145was restored in T3A-A3cells by the infection of Lenti-miR-145lentivirus. Cell proliferation inhibition effect of miR-145on T3A-A3cells was examined by clonogenic formation assay and cell cycle analysis. Cell senescence was detected by senescence-associated β-galactosidase staining and p16fluorescence staining. Self-renewal ability of T3A-A3cells was evaluated by tumorsphere-formation assay.In addition, in vivo tumor growth experiments and tumorigenicity experiments in nude mice were used to determine the role of miR-145in regulating tumor growth and tumorigenicity of T3A-A3cells. The expression levels of Oct4and PCNA in xenograft tumor were examined by immunohistochemistry. In order to investigate the role miR-145on the expression of Oct4, RT-PCR and western blotting were used to detect the expression of Oct4after miR-145restoration in T3A-A3cells. Then Oct4expression vector was introduced into the Lenti-miR-145culture by pWPTS-Oct4infection, and the cell proliferation in vitro was evaluated by MTT assay, and tumor growth rates were determined by xenograft tumor assay.Results:T3A-A3cells have lower miR-145levels compared to other cell lines, and miR-145deficiency is correlated with an increase in tumor development. miR-145restoration in T3A-A3cells by lentivirus infection induced senescence-like G1arrest in cell cycling, and significantly inhibited clonogenic cell expansion in vitro and xenograft tumor growth in vivo. Moreover, miR-145restoration diminished tumorsphere growth in vitro and tumor formation in vivo in T3A-A3cells. The increase in miR-145levels paralleled the decrease in Oct4protein levels. The inhibition effect of Lenti-miR-145on T3A-A3cells can be partly abrogated by pWPTS-Oct4both in vitro and in vivo.Conclusions:Collectively, our data indicate that miR-145may play an important role in maintaining the tumorigenicity of tumor stem cells T3A-A3, potentially via direct modulation of the downstream target Oct4.