Fig 2: YB1 regulates miR‐205/200b‒ZEB1 axis by inhibiting miRNA expression. A‐B. The expression levels of miR‐205 and miR‐200b were analyzed in HCCLM3 and MHCC97H cells stably expressing YB1 shRNAs (A) as well as SMMC7721 and BEL7402 cells stably overexpressing YB1 (B) by qPCR analysis. U6 was used as an internal control for qPCR. C. A schematic representation of the luciferase reporter constructs with ZEB1 or SIP1 3′UTR (left panel). The complementary binding sites among ZEB1 3′UTR luciferase reporter, miR‐205‐5p, and miR‐200b‐3p (right panel). D. The regulation of YB1 on the miR‐205/200b‐ZEB1 axis was identified by luciferase reporter assays, which were performed by cotransfecting ZEB1 3′UTR luciferase reporter with pre‐miR‐205 or pre‐miR‐200b expressing plasmid, YB1 expressing plasmid, and specific miR‐205 or miR‐200b inhibitors into HEK293T cells. E. YB1 regulated miR‐205/200b‐ZEB1 axis in a dose‐dependent manner. Luciferase activity assays were performed by cotransfecting ZEB1 3′UTR luciferase reporter with YB1 expressing plasmid and pre‐miR‐205 (left panel) or pre‐miR‐200b (right panel) expressing plasmid into HEK293T cells. The most representative results of three independent biological experiments were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.001

Fig 3: YB1 inhibits miR‐205/200b expression in a Snail‐independent manner. A. The expression levels of pre‐ and mature miR‐205 or miR‐200b were detected by qPCR analysis in YB1 shRNA knockdown MHCC97H cells transiently transfected with si‐Snail. B. The expression levels of pre‐and mature miR‐205 or miR‐200b were detected by qPCR analysis in YB1 shRNA knockdown HCCLM3 cells transiently transfected with si‐Snail. C. The expression levels of pre‐ and mature miR‐205 or miR‐200b were detected by qPCR analysis in YB1 knockdown HB611 cells transiently transfected with si‐Snail. U6 was used as an internal control for real time PCR. Vinculin was used as a loading control. The most representative results of three independent biological replicates were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.001

Fig 4: YB1 interacts with TUTs and influences miR‐205/200b expression. A. Pull‐down experiments were performed with S‐protein beads (upper panel) or Flag‐beads (lower panel) in HEK293T cells cotransfected with exogenous TUT4 and YB1. B. Pull‐down experiments were performed with S‐protein beads (left panel) or Flag‐beads (right panel) in HEK293T cells cotransfected with exogenous TUT1 and YB1. C. Pull‐down experiments were performed in HEK293T cells transiently transfected exogenous TUT1, and immunoblotted with antibodies against YB1 and Flag. D. The coimmunoprecipitation assays were performed by endogenous YB1 (upper panel) or TUT4 (lower panel) in HCC cell lines, and immunoblotted with antibodies against YB1, Akt1, and TUT4 respectively. E‐F. Pull‐down experiments were performed in HEK293T cells cotransfected with SFB‐tagged full‐length or truncated YB1 constructs, and Flag‐tagged TUT4 (E) or Flag‐tagged TUT1 (F). G‐H. RNA independent immunoprecipitations were performed in HEK293T cells cotransfected with exogenous YB1 and TUT4 (G) or TUT1 (H). The cell lysates were treated with RNase A (1 μg/μL) and pulled down by S‐protein beads, finally determined by immunoblotting analysis

Fig 5: A schematic model for YB1 regulation on miR‐205/200b‒ZEB1 axis by inhibiting microRNA maturation in hepatocellular carcinoma. This study reveals a novel mechanism that YB1 triggers cell invasion and cancer metastasis by regulating miR‐205/200b‒ZEB1 axis in a Snail‐independent manner. YB1 inhibits miR‐205/200b maturation through interacting with microprocessor DGCR8 and Dicer as well as precursor miR‐205/200b. The downregulation of miR‐205/200b enhances ZEB1 expression, leading to increased cell migration and invasion in HCC cells. Furthermore, YB1 regulates miR‐205/200b expression in concert with terminal uridylyltransferases (TUT4/TUT1). YB1 may restrain the activation of TUTs on group II miRNAs biogenesis via competitively binding with pre‐miRNAs