Figure 2.

YAP1 activation initiates hepatoblastoma through up-regulating methyltransferase G9a. (A) Metabolomic analysis revealed that S-adenosylmethionine (SAM) was down-regulated while S-adenosyl-L-homocysteine (SAH) was up-regulated in YAP1-activated HB organoids. (B) ADOX treatment for 7 days specifically inhibited the growth of YAP1-activated HB organoids. Data were presented as means ± SD (n = 4). Scale bar, 1 mm. (C) BIX 01294 treatment for 7 days specifically inhibited the growth of YAP1-activated HB organoids. Data were presented as means ± SD (n = 4). (D) The data from HBprem DataBase showed that G9a was up-regulated in HB at protein level when compared to normal tissue. Data were presented as means ± SD (n = 5). (E) Western blot determined the expression of G9a in two lines of organoids transfected with GFP or YAP1^5SA. (F) qRT-PCR analysis of EHMT2 expression in organoids transfected with GFP or YAP1^5SA. H3 was used as an internal control. Data were presented as means ± SD (n = 3). (G) Wild-type G9a but not its enzyme-dead mutant ($\mathrm{\Delta }$ SET) drove HB oncogenesis. Data were presented as means ± SD (n = 4). Scale bar, 1 mm. (H) Knockdown of EHMT2 by shRNAs inhibited the growth of YAP1-activated HB organoids. E2-CDS: EHMT2 shRNA targeting CDS; E2-3′U: EHMT2 shRNA targeting 3′UTR. Data were presented as means ± SD (n = 4). (I) Overexpression of G9a restored the growth of YAP1-activated HB organoids transfected with EHMT2 shRNA targeting 3′UTR. Data were presented as means ± SD (n = 4). (J) qRT-PCR analysis revealed that EHMT2 knockdown down-regulated HB signature genes in YAP1-activated HB organoids. H3 was used as an internal control. Data were presented as means ± SD (n = 3). (K) Human fetal liver organoids transfected with YAP1^5SA for 40 days to achieve HB tumorigenesis. These HB organoids were then treated with ADOX for 12 days. Data were presented as means ± SD (n = 4). (L) NSG mice were transplanted with YAP1-activated HB organoids to obtain liver tumors. Then, these mice were then received daily treatment of BIX 01294 (5 mg/kg) for 10 days. Data were presented as means ± SD (n = 5). * Indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001