Fig 2: 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 3: YB1 promotes cell invasion and tumor progression by regulating the miR-205/200b?ZEB1 axis. A. Transwell matrigel invasion assays were performed in YB1 shRNA knockdown MHCC97H cells transiently transfected with random control, specific miR-205-5p or miR-200b-3p inhibitors. Scale bar: 100 µm. B. Representative images of murine lung metastatic tumors derived from MHCC97H cells stably expressing YB1 shRNA or shControl by tail veil injection. C. qPCR analysis of miR-205 and miR-200b expression levels in lung metastatic tumors derived from MHCC97H cells stably expressing YB1 shRNA or shControl. D-E. The expression levels of YB1, ZEB1, E-cadherin, and N-cadherin proteins were determined by immunohistochemistry (D; magnification, ×400; scale bar, 50 µm) and immunoblotting analyses (E) in lung metastatic tumors derived from MHCC97H cells stably expressing YB1 shRNA or shControl. F. Box plots comparing YB1 expression levels in 50 matched normal and HCC tissues from the TCGA database. The boxes showed the median and the interquartile ranges and the whiskers indicate the minimum and maximum values. G. Kaplan-Meier curves about the overall survival time of the HCC patients, stratified by YB1 expression levels where high and low groups referred to patients with the highest 40% (n = 148) or lowest 40% (n = 148) YB1 mRNA expression levels among all 371 HCC patients. Data were obtained from TCGA database. Statistical significance was determined by the log-rank test. H. Boxplots comparing YB1 mRNA expression levels in the TCGA-HCC patients from different stages (stage I to III). P values were calculated by Wilcox-test (two-tails, unpaired). I. Scatterplots showing the positive correlation between YB1 and ZEB1 mRNA expressions in liver tissues (n = 175) from GTEx database. The correlation was determined by Spearman's test. U6 was used as an internal control for real-time PCR. Data were presented as mean ± SD. *P < 0.05, **P < 0.001

Fig 4: YB1 binds to pre-miR-205/200b and inhibits miRNA maturation. A-B. qPCR analysis of pri-, pre- and mature miR-205 or miR-200b expression levels in HCCLM3 cells stably expressing YB1 shRNAs (A) as well as in SMMC7721 cells stably overexpressing YB1 (B). C. RIP-qPCR analysis of pre-miR-200b (upper panel) and pre-miR-205 (lower panel) enriched by SFB-tagged YB1 in HEK293T cells. D. Schematic representation of the wild-type (black) and the mutant (red) loop sequences of pre-miR-205. Mature miR-205-5p sequence is shown in blue. Arrows indicate Drosha processing sites. E. RIP-qPCR analysis of the wild-type and the mutant pre-miR-205 enriched by SFB-tagged YB1 in HEK293T cells. F. RNA pull-down assays were performed in vitro by incubating the biotin-labeled wild-type and the mutant pre-miR-205 (left panel) or pre-miR-200b (middle panel) with cell lysates from YB1 transiently transfected HEK293T cells. The biotin-labeled pre-miRNAs were identified by electrophoresis (right panel). All pre-miRNAs were transcribed in vitro with an SP6 promoter except that the mutant pre-miR-200b was transcribed by a T7 promoter. The most representative results of three independent biological experiments were shown. U6 was used as an internal control for real-time PCR. Data were presented as mean ± SD. *P < 0.05, **P < 0.001

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