Significance was defined as P < 0.05. Results Differential expression of DKK-1 mRNA and protein in various cell lines We first sought to identify the differential expression of the DKK-1 gene in 12 glioblastoma cell lines, find more Medulloblastoma cells, low-grade glioma cells, and human astrocytes as a control using semi-quantitative
RT-PCR analysis (Figure 1). In glioblastoma cell lines UW-28, SKI-N2, and SF295, DKK-1 mRNA expression was relatively lower as compared with other glioblastoma cells. Concentration of DKK-1 protein was also determined by ELISA in culture medium and cell lysate of these 14 cell lines (Table 1). U251 cells have the highest levels of DKK-1 expression in both of the culture medium BAY 80-6946 and cell lysate, while glioblastoma cell lines SKMG-4 and UW-28 have the lowest DKK-1 levels in the culture medium and cell lysate, respectively. Following normalization and statistical analysis of fluorescence intensity data by t test, we identified that the difference of DKK-1
protein expression was significant BTK inhibitor between the culture medium and cell lysate in 12 glioblastoma cell lines (p < 0.05), consistent with the fact that DKK-1 was a secreted peptide shown previously to influence cell growth, differentiation and apoptosis by inhibiting Wnt signaling . It should also be noted that the very low expression level of DKK-1 mRNA was not in concordance with the higher level Leukotriene-A4 hydrolase of its
protein in SKI-N2 cells. Expression of DKK-1 mRNA and protein was undetectable in medulloblastoma cells, low-grade glioma cells, and human astrocytes. Thus, DKK-1 can serve as a marker for diagnosis of glioma through detecting the expression of the protein and mRNA of DKK-1. Figure 1 Expression of DKK-1 mRNA in glioblastoma cell lines was higher than that in control by using semi-quantitative RT-PCR. Table 1 Levels of DKK-1 expression were detected in the culture medium and cell lysate of all 14 cancer cell lines by ELISA Cancer cell lines and control Concentration of DKK1 (pg/ml) Normal cell s Human astrocytes 0 0 Low-grade glioma cell line SHG-44 0 0 Medulloblastoma cell line D341 0 0 Glioblastoma cell lines Culture medium* Cell lysate** U251 18238 4917 SF767 5760 729 T98G 1558 258 UW-28 2390 45 MGR1 1089 151 MGR2 3826 434 MGR3 3901 375 SKI-N2 766 260 SKMG-1 6691 2192 SKMG-4 301 72 UWR7 5290 910 SF295 8628 1780 * and ** indicate the respective concentration of DKK-1 protein tested in the culture medium and cell lysate. DKK-1 expression in tumors and normal tissues To identify the association of DKK-1 expression with pathologic tumor classification, we did DKK-1 expression profile analysis in patients at various clinical stages of glioma and in healthy controls.