Figure 8

Functional validation of neuronatin ( NNAT ). (a) Gene network through interaction analysis of the genes from the MLS CpG cluster. Connections represent known protein-wise interactions. Indicated are genes that show differential DNA methylation and upregulation (red) or downregulation (green) in MLS compared to the other CpG clusters. Numbers on the solid lines represent correlation of gene expression (Kendall’s tau) within the MLS samples and across the remaining sarcoma samples. Numbers after the gene name are the gene expression fold change compared to the normal fat samples and the remaining sarcoma samples. (b) Correlation between NNAT expression and DNA methylation of CpG cg22298088 for the whole sarcoma collection. The numbers of the sample clusters are shown. The solid line represents the correlation trend (Kendall’s tau -0.303; P = 0.001). (c) NNAT expression for the sarcoma collection together with binarized methylation status (P = 1 × 10^–5; expression fold change 24.7; methylation fold change 9.3). (d) Position of seven CpGs in the direct neighborhood of cg12862537 and cg22298088. The methylation levels were analyzed in fat tissue, three liposarcoma subtypes and two MLS cell lines. (e) Mean level of DNA methylation of all eight CpGs shown in (d) for fat tissue and the three liposarcoma subtypes and (f) validation of NNAT expression (red transcript in (d)). (g) Re-expression of NNAT in MLS cell lines MLS402 and MLS1765 following 5-aza-dC treatment. (h) Western blot and quantitative PCR analysis after stable NNAT re-expression in MLS1765. Recovery of NNAT caused (i) decreased migration revealed by wound-healing assay after 24 h compared to the empty vector cell line (an illustrative example from three independent experiments is shown) and (j) diminished cell proliferation determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Error bars represent standard error of the mean (t-test, * indicates P ≤ 0.05, ** indicates P ≤ 0.01). 5-aza-dC, 5-aza-2-deoxycytidine; Chr: chromosome; Ctrl, control; DDLS, dedifferentiated liposarcoma; EV, empty vector; LMS, leiomyosarcoma; MFS, myxofibrosarcoma; MLS, myxoid liposarcoma; MPNST, malignant peripheral nerve sheath tumor; PLS, pleomorphic liposarcoma; nd, not determined; SS, synovial sarcoma; UPS, undifferentiated pleomorphic sarcoma.