Journal: Cancer cell
Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis
doi: 10.1016/j.ccell.2019.12.007
Figure Lengend Snippet: (A) Pie chart of the fraction of circRNAs detected in the indicated number of melanocyte or melanoma STC samples. (B) MDS plot of melanocytes (n=4) and STCs (n=10) defined by circRNA expression. (C) Hierarchically-clustered heat map of circRNAs differentially expressed in melanoma STCs (n=10) vs. melanocytes (n=4) (EdgeR, logFC >1, FDR <0.05). (D) Average correlation coefficients (across samples, Pearson, r) between circular and linear RNAs, grouped by the number of samples in which the circRNA was detected. Mean ± SD per sample group are presented. (E) Cumulative distribution plot of the average circular/linear ratios across samples. (F) Backsplice read counts for CDR1as in melanocytes and individual melanoma STCs. Count or mean count ± SD. (G) FPKM or mean FPKM ± SD of CDR1as from cultured melanocytes (n = 6), and melanoma STCs (n = 13) and cell lines (n = 7). ND = not detected. Red hashed line set to mean FPKM of melanocyte samples. Color coding of cell lines indicating high (red circle), moderate (yellow circle) or low/absent (blue circle) CDR1as expression in subsequently used cell models. (H) Median normalized CDR1as expression (RT-qPCR) in cultured melanocytes (n = 3) and melanoma cell lines (n = 1 per cell line). I, Representative images of single molecule in situ hybridization (smISH) for CDR1as or PPIB (control) in high- and low-expressing melanoma cells. Scale bar is 100 μm. (J) Median normalized expression (log2) of CDR1as by RT-qPCR of melanoma patient samples, grouped by primary (n = 53) and metastatic (n = 52) samples. Primary samples grouped by Breslow thickness (<1 mm or >1 mm). Metastatic samples grouped as in-transit (ITM), lymph node (LN), or distal metastases (DM). (K-M) Median normalized CDR1as expression (RT-qPCR, log2) of primary vs. metastatic samples (K), stage I vs. II samples (L), and ulceration status (primary only) (M). Box plots depict median, 25th and 75th percentile, min-max whiskers. Statistical analyses by two-tailed student’s t test. * p<0.05, ** p<0.01, *** p<0.001 (N-P) Dot plots of median normalized CDR1as expression (log2) from primary melanoma patient samples with Breslow thickness (N), mitoses per mm2 (O), and age at diagnosis (P). Pearson correlation coefficients (r) and p values are indicated. (Q and R) Kaplan-Meier curves of primary melanoma patients stratified by CDR1as expression (Bottom 25% (n = 13) vs. Top 75% (n = 40)) for metastasis-free survival (Q) or overall melanoma-specific survival (MSS) (R). Statistical analyses by Log Rank test.
Article Snippet: Single Molecule in situ hybridization Target probe sequences, preamplifier, amplifier, wash buffer and target retrieval buffers are proprietary (Advanced Cell Diagnostics, CA).
Techniques: Expressing, Cell Culture, Quantitative RT-PCR, In Situ Hybridization, Control, Two Tailed Test, Biomarker Discovery
