Overexpression of WT or mutant LIN28B increases the penetrance of MYCN-induced neuroblastoma. (A and B) Schematic of the WT (A, LIN28B_WT) and mutant (B, LIN28B_MU) human LIN28B proteins. CSD; NoLS, nucleolar localization signal; CCHC zinc-finger domain; NLS, nuclear localization signal. Five point mutations (arrowheads) were created in the LIN28B_MU. (C–H) Lack of detectable EGFP expression in the IRG of a 17-wpf EGFP transgenic fish (C) and 39-wpf LIN28B_WT (D) and LIN28B_MU (E) fish; and EGFP expression in the tumors (arrows) arising from the IRG of 17-wpf EGFP;MYCN (F), LIN28B_WT;MYCN (G) and LIN28B_MU;MYCN (H) fish. (Scale bar, 1 mm.) (I) Kaplan–Meier analysis of cumulative frequency of neuroblastoma in transgenic lines. Statistical analysis was performed using the logrank test. (J and K) The relative expression of zebrafish let-7b (J) and let-7f (K) miRNAs in EGFP;MYCN (M), LIN28B_WT;MYCN (WT), and LIN28B_MU;MYCN (MU) tumors by qRT-PCR. Five independent tumors from 6-mo-old fish were analyzed for each group. Each dot represents the expression levels of the indicated gene normalized to U6 snRNA. Horizontal bars indicate means ± SD. Statistical analysis was performed using the two-tailed unpaired t test. ***P < 0.001.
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