Effect Of OCT4Expression On The Differentiation And Self-renewal Of Ⅰ-type Neuroblastoma Cells BE(2)-C

Background Neuroblastoma is the most common solid extracranial malignancy of childhood, arising during fetal or early postnatal life from sympathetic cells derived from the neural crest. The overall incidence of neuroblastoma is1case per100,000children. Neuroblastoma is an extremely heterogeneous disease; tumors can spontaneously regress or mature, even without therapy, or display a very aggressive, malignant phenotype that is poorly responsive to current intensive, multimodal therapy. Three major cell types have been identified in neuroblastoma cell lines, which were designated as N-(neroblastic), S-(substrate-adherenet), and I-type (intermediate) neuroblastoma cells. I-type cells are multipotent and differentiate to either N-or S-type cells when induced by specific agents or spontaneously; can express cell markers of both N-and S-type cells; exhibit significantly higher clonogenic activity in soft agar culture and tumorigenic potential in immunodeficient mice. Hence, I-type cells are considered as a population of neuroblastoma stem cells or malignant neural crest stem cells. The hypothesis of I-type cells representing neuroblastoma stem cells may responsible for the heterogeneity of neuroblastoma. OCT4maintains self-renewal and blocks cell differentiation in embryonic stem cells and is expressed by some cancer stem cells.AimObserve the self-renewal and differentiation features of in I-type neuroblastoma cells BE (2)-C with down and up regulation of the expression of OCT4.Materials and Methods1. The human neuroblastoma cell lines BE(2)-C were grown in DMEM with10%FBS. For differentiation assays, RA and BrdUrd were dissolved in DMSO and added into the DMEM to be a final working solution of10μM. BE(2)-C cells were treated for1-2weeks as subcultured. Observe and measure the morphological changes of the cells and extract total RNA and protein. Western blot and real time RT-PCR were facilitated to measure the expression level of OCT4.2. Lentiviral RNAi vector was used for down regulation of OCT4expression in BE(2)-C. Cell growth rates were measured by using Cell Counting Kit-8and Soft agar colony formation assay was used to determine the self renewal of BE(2)-C. Western blot and real time RT-PCR analysis were used to detect the lineage differentiation marker(N-type:Peripherin and NF68;S-type:Vimentin and S100).3. FUGW/OCT4was used to overexpress human OCT4in BE(2)-C cells. Cell growth rates were measured by using Cell Counting Kit-8and Soft agar colony formation assay was used to determine the self renewal of BE(2)-C. Western blot and real time RT-PCR analysis were used to detect the lineage differentiation marker(N-type:Peripherin and NF68;S-type:Vimentin and S100).4. Use gene expression chip to screen and identify the potential genes getting envoled in the regulation of pathways relating to OCT4.Result1. Following treatment of BrdUrd, in addition to the expected S-type morphological alteration, cells showed a progressive reduction of OCT4expression levels in both protein and RNA levels, and there was no related change in induced N-type cells.2. A reduction in cell growth rate was observed in cells with down-regulated OCT4than the controls measured by CCK-8absorption. BE(2)-C cells with down-regulated OCT4showed morphological alteration of S type cells and the soft agar assay indicated colony formation of cells was unable to form colonies on soft agar. Compared to cells infected with normal control vector by western blot analysis, BE (2)-C/OCT4interfered cells showed an apparent decrease in the expression of N-type cell markers, peripherin and NF68, which was accompanied by an increase in the expression of the S-type cell markers, vimentin and S100(Fig.5B-D). The corresponding N-or S-type marker changes were also confirmed by quantitative RT-PCR analysis, which showed similar up-regulation of vimentin and S100and down-regulation of peripherin and NF68.3. OCT4-overexpressing BE (2)-C cells grew faster and colony formation of cells with down-regulated OCT4increased using the soft agar assay. However, protein levels of lineage differentiation marker of N-type (peripherin, NF68) and S-type (vimentin, S100) remained relatively unaltered in the OCT4up-regulated cells as in the control cells 4. The genes of RET, RGS8, FIGF, FRRS1, SPRY4, FAM72D、DLGAP5、NEK2、 ALDOC、HMGCS1have significant expression changes as down-regulation of OCT4in BE(2)-C.Conclusion1. I-type neuroblastoma cells BE(2)-C can exhibit induced bidirectional differentiation when treated with different lineage induce reagent. OCT4expression level reduced as BE(2)-C cells were induced to differentiate into S-type cells with BrdUrd. This discovery indicated S-type cell is a relatively benign one within the neuroblastoma.2. As reduced expression level of OCT4, BE(2)-C cells differentiated off the stem cell type to a relatively benign S-type cells, abrogate the ability of self-renewal, suggest OCT4is essential to retain the stemness of I-type cells and can be designated as a target for treatment.3. As increased expression level of OCT4, BE(2)-C cells obtain higher growth rates and clonogenic self-renewal activity. These results imply that OCT4is related to the malignancy of neuroblastoma and could be used as a predictive marker for tumor progress.