| Backgrounds and objectiveNeuroblastoma (NB) is the most common extracranial solid pediatric tumor and accounts for10%of childhood cancers. Although major advances have been made in surgery and chemotherapy of NB, the morbidity and mortality remain high. So far, the molecular mechanisms responsible for the pathogenesis of NB remain elusive, thus researches on the genisis and progress of NB are of great importance. NB is an embryonic tumor, and the inhibition of embryonal neural crest cell differentiation is the main reason of the genisis of NB and major risk factor of its manignancy and prognosis. Sox2is a transcription factor expressed in both embryonic and adult stem cells. It plays a pivotal role in the maintenance of self-renewal and differentiation potential. In this study, we explore the expression of Sox2in human NB and the relationship between the expression levels of Sox2and various clinicopathological parameters. Meanwhile, the potential functions of Sox2on the genesis and progress of NB are also explored in this study.Materials and Methods1. The research of Sox2expression in human NB:RT-PCR, Real-time PCR, Western blot analysis and immunohistochemical staining were employed to detect the expression of Sox2in human NB, and the relationship between Sox2expression and clinical data was assessed.2. The effects of Sox2on the biological characteristics of neuroblastoma stem cell:RT-PCR was performed to detect the expression of Sox2in human neuroblastoma BE(2)-C cell. Sox2-overexpressed and mRNA knockdown stem cell lines were established by infecting BE(2)-C cell via lentivirus vectors. Cell counting kit-8(CCK-8) assay and flow cytometry were applied to test the effect of Sox2on the proliferative ability. Western blot analysis was used to detect the marker proteins after drug interference in order to analyze the effect of Sox2on differentiation properties. Plate colony-forming assay and tumorigenicity assay were performed to analyze the effect of Sox2on the malignant potential.3. Gene chip detect the gene expression profiling changes in down-regulating Sox2neuroblastoma stem cell:The gene expression profiles of BE(2)-C-shRNA and BE(2)-C cells were investigated with Genechip Human Gene1.OST. Differentially expressed genes were analyzed by data analyses. Real-time PCR was performed to verify the gene chip resultsResults1. The research of Sox2expression in human NB:Sox2mRNA was detected in NB tissues by RT-PCR. The immunohistochemical staining showed that Sox2was primarily localized in the nuclei of NB cells, not observed in paracancerous cells. Using Real-time PCR, we found that Sox2mRNA relative expression levels was significantly higher in tumor tissues than in the adjacent non-cancerous tissues (P<0.05), and the relative expression levels in stage Ⅲ and Ⅳ NB were higher compared with those in stage Ⅰ and Ⅱ NB (P<0.05). Sox2expression was significantly decreased in the chemotherapy subgroup as compared with that of the non-chemotherapy subgroup in stage Ⅲ and Ⅳ tumors (P<0.05). Western blot analysis confirmed the results at the protein level.2. The effects of Sox2on the biological characteristics of neuroblastoma stem cell:Sox2mRNA was detected in human neuroblastoma BE(2)-C cell by RT-PCR. Sox2-overexpressed[BE(2)-C-Sox2] and mRNA knockdown[BE(2)-C-shRNA] stem cell lines were successfully established by infecting BE(2)-C cell via lentivirus vectors. BE(2)-C-Sox2cell showed relatively higher cell proliferative number than BE(2)-C cell (P<0.05) in CCK-8assay. The percentage of cells in S phase and PLI index in BE(2)-C-Sox2group was higher than BE(2)-C group (P<0.05) in flow cytometry assay. BE(2)-C-Sox2cell showed higher colony-forming number and efficiency (P<0.05), speed of tumorigenesis and volumes of tumors in vivo than BE(2)-C and BE(2)-C-shRNA cells (P<0.05), and exhibited decreased expression levels of other type cell marker proteins than BE(2)-C and BE(2)-C-shRNA cells (P<0.05). Conversely, down-regulation of Sox2showed an inverse result.3. Gene chip detect the gene expression profiling changes in down-regulating Sox2neuroblastoma stem cell:596differentially expressed genes were screened by Genechip Human Gene1.0ST. The significantly differentially expressed genes were FMN1, GFRA2, HIST1H2BK, CARTPT, AHNAK2, PLP2, TSPAN8, TIMP2, EPO, PRR11and etc. Real-time PCR verified the gene chip results.Conclusions1. Sox2is expressed in human neuroblastoma and neuroblastoma stem cell, and its expression correlates to the clinical stage of NB, but not other clinicopathological parameters including patient gender and age, tumor size, location and histological classification. Moreover, Sox2expression can be inhibited by chemotherapy.2. Sox2plays a key role in the abilities of proliferation, differentiation, colony-forming and tumorigenesis of neuroblastoma.3. Differentially expressed genes via gene chip in down-regulating Sox2neuroblastoma stem cell are FMN1, GFRA2, HIST1H2BK, CARTPT, AHNAK2, PLP2, TSPAN8, TIMP2, EPO, PRR11and etc. Further analyses and research are needed to explore the data. |
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